Enhanced amplifiability of minute fixative-treated tissue samples, minute stained cytology samples, and other minute sources of DNA
The specification provides materials and methods for enhancing the amplifiability of nucleic acids that are present in minute amounts. Preferably, the materials and methods are utilized in combination with pathology techniques to amplify nucleic acids obtained from fixative and/or stained tissue or cell samples. The amplified nucleic acids can then be utilized for rendering a diagnosis or prognosis to the subject from which the tissue or cellular sample was obtained.
This application claims benefit of U.S. Provisional Applications Nos. 60/620,926 filed Oct. 22, 2004; 60/631,240 filed Nov. 29, 2004; 60/644,568 filed Jan. 19, 2005; 60/679,969 filed May 12, 2005; and 60/679,968 filed May 12, 2005, all of which are herein incorporated in their entirety for all purposes.
FIELD OF THE INVENTIONThe application relates to a method, compositions, and apparatus for improving nucleic acid amplifiability of minute quantities of DNA subjected to chemical tissue fixation and/or microscopic slide staining or both, or which may exist in minute quantities in a biological sample.
BACKGROUNDSpecimen diagnosis is an essential part of medical practice upon which disease is classified and treatment is based. This is especially true for cancer and related disease diagnosis where pathology diagnosis remains an absolute requirement for patient management. Regardless of how certain clinical examination, imaging studies, or indirect laboratory testing may be, it is an absolute requirement that cancer or pre-cancer diagnosis be confirmed by specimen evaluation. This is the role of pathology, and there are major medico-legal implications for this activity if errors or inadequacies arise.
Current medical practice for cancer and related conditions has tended towards greater reliance on diagnostic biopsies to achieve diagnosis and plan therapy. In the past, surgery was often performed first providing relatively large sized specimens for initial diagnosis. This approach is used less often now, reserving the option for preoperative chemotherapy and/or radiation as a means to better treat certain forms of cancer. Also, the availability of preoperative diagnosis can influence the manner in which surgical treatment is performed (i.e., laparoscopic surgery versus open surgery). Thus pathologists have had to accept the reality of biopsy diagnosis to render a definitive diagnosis for cancer treatment or determination of precancerous conditions.
Diagnostic biopsy techniques are of several different types. Small-sized, core biopsies can be obtained or fine needle aspiration (FNA) techniques can be used to gather cellular material for cytologic diagnosis. At the same time, advances in imaging techniques now afford the opportunity to accurately targets small fluid collections from organs, such as the pancreas, by means of ultrasound-guided endoscopic fine needle aspiration biopsy (FNAB). This opens the door to early diagnosis of cancers that are poorly treated at this time due to significant delays in diagnosis.
Recent years has seen a massive explosion in the availability of molecular information through efforts such as the human genome project, which has succeeded in sequencing the human genome. While our understanding of cancer has increased, the application of this information to everyday clinical specimens has proceeded very slowly and with little comparative gain. The reason for this lies in the fact that biopsy specimens of cancer or precancerous lesions are too small in most cases to be adequately analyzed using research types of molecular analysis. More importantly, there remains an absolute requirement to handle the specimens according to established pathology criteria. As a result, all specimens must undergo cellular fixation and staining in order to maintain cellular morphologic integrity for traditional microscopic evaluation. Molecular analysis, if it is to have an impact on the way cancer is more effectively diagnosed and characterized, must operate on small, fixative-treated specimens. This is the impediment to effectively incorporating molecular analysis into pathology practice or being able to do fundamental research on tissue bank libraries of blocks of tissue taken from populations of patients over decades.
SUMMARYThe embodiments described herein serve to provide new methods, compositions, and apparatus for enhancing the nucleic acid amplifiability of fixative-treated and/or histologically stained microscopic sections. The methods can also assist the amplifiability of other forms of minute amounts of DNA.
One aspect provides for a kit for enhancing amplifiability of a nucleic acid from a sample with low nucleic acid concentration comprising: (a) a buffer comprising a nonionic detergent; (b) a proteinase; (c) XCl2, wherein X is magnesium or manganese; and (d) sucrose; and wherein XCl2 and sucrose when admixed with the sample for amplification have a final concentration of about 5 mM to about 10 mM XCl2 and about 5 gram percent to 15 gram percent sucrose.
The kit can be for use with any type of biological sample, including fixative-treated and/or stained cellular material, as well as liquid cytology and other biological fluid samples. The nucleic acid is DNA.
The nonionic detergents can be any nonionic detergent, but is preferably Nonidet P40 (NP-40), Tween, Triton X, or Nikkol. The nonionic detergent (or nonionic detergent combination) is present in the amount of about 0.1% to about 3.0% (with every 0.1% value there between), and more preferably from about 0.5% to about 2.0%. Preferably, the nonionic detergent is Nonidet P40, and preferably it is present in the amount of about 1.0%. The nonionic detergent can be used alone, or more preferably in combination with a salt (e.g., NaCl), Tris (or other buffer), and a chelating agent such as ethylenediaminetetraacetate (EDTA) or ethylene glycol-O-O′-bis(2-amino-ethyl)-N,N,N′,N′-tetraacetic acid (EGTA).
Another aspect of the kit is directed to comprising a proteinase. The proteinase can be any proteinase including but not limited to proteinase K, pronase, subtilisin, thermolysin, papain, as well as combinations or cocktails of proteinases. The proteinases are preferably present in the amount of about 0.1% to about 3.0% of the total volume (or any 0.1% value there between), and more preferably from about 0.5% to about 2.0%. For example, proteinase K which can be present in an amount of about 1.0 mg/mL to about 5.0 mg/mL (and every 0.1 mg/mL value there between). Preferably, proteinase K is present in the reaction mixture in the amount of about 2.0 mg/mL.
Another aspect of the kit contemplates a XCl2. The is XCl2 is preferably MgCl2 or MnCl2. The XCl2 is present in the amount of about 6.0 mM to about 10.0 mM (and any 0.1 mM value there between). Preferably, the XCl2 is MgCl2 and is present in the amount of about 8.0 mM.
Another aspect of the kit includes sucrose in the amplification mixture. Sucrose is preferably present from about 5.0 gram percent to about 15.0 gram percent, more preferably about 8.0 gram percent to about 13.0 gram percent, and more preferably about 12.0 gram percent sucrose. Values of every about 0.1 gram percent value in between 5.0 gram percent and 15.0 gram percent are also contemplated.
Yet another embodiment provided here is a method of enhancing amplifiability of DNA of samples with low desired DNA concentration comprising:
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- (a) combining a sample of cellular containing material and an aqueous buffer comprising sodium chloride, EDTA, Tris, and a nonionic detergent;
- (b) adding a proteinase to the buffer and the sample and incubating the buffer, the sample, and proteinase at a temperature suitable for proteinase digestion of protein;
- (c) deactivating the proteinase;
- (d) adding sucrose to the buffer of step (c) to a final concentration of about 5.0 gram percent to about 15.0 gram percent; and
- (e) adding XCl2 to the sample of step (c) to a final concentration of about 5.0 mM to about 10.0 mM.
1 μL or more of the amplification mixture containing the DNA of step (e) can then be amplified. The cell-containing sample can be from a fixative-treated and/or a stained sample. The sample can also be cell free, a liquid biological sample or a liquid cytology sample. The nonionic detergent can include but is not limited to Nonidet P40 (NP-40), Tween, Triton X, or Nikkol, or any combination thereof, wherein the nonionic detergent or detergent combination is present in the amount of about 0.1% to about 3.0% total volume (and every 0.1% within this range, e.g., about 1.5% to about 2.5%). More preferably, the range is between about 0.5% to about 2.0%. For example, the nonionic detergent can be NP-40 and can be present in the amount of about 1.0%.
Another aspect of the method contemplates the use of proteinases including but not limited to proteinase K, pronase, subtilisin, thermolysin, papain, or combinations thereof. The proteinase or combination of proteinases can be present in the amount of about 1.0 mg/mL to about 5.0 mg/mL or alternative from about 0.1% to about 3.0% total volume (and every 0.1% value there between, e.g., 1.5% to 2.5%). More preferably the range is from about 0.5% to about 2. %. For example, proteinase K can be present in the amount of about 2.0 mg/mL.
Yet another aspect of the method contemplates utilizing a XCl2 wherein the XCl2 is MgCl2 or MnCl2. Preferably the XCl2 is present in the amount of about 6.0 mM to about 10.0 mM (and every 0.1 mM value there between, e.g., 7.5 mM to 8.5 mM).
Another aspect of the method is the presence of sucrose, preferably the sucrose is present in the amount of about 5.0 gram percent to about 15.0 gram percent (and every 0.1 percent within that range, e.g., 11.5 gram percent to 12.5 gram percent). More preferably, the sucrose is present in the amount of 12 gram percent final volume.
Thus, in one aspect, the above method utilized proteinase K, which is present in the amount of about 2 mg/ml; sucrose which is present in the amount of about 12 gram percent; the nonionic detergent, Nonidet P-40, which is present in the amount of about 1%; and MgCl2 which is present in the amount of 8.0 mM.
The pH of the buffer and the amplification composition of any of the kits and materials described above and herein is about 7.0 to about 8.5 and every 0.1 value within that range.
BRIEF DESCRIPTION OF THE DRAWINGSIt is to be understood that both the following detailed description are exemplary and explanatory only, and are not restrictive of the material methods, devices, and kits. The accompanying drawings, which are incorporated herein by reference, and which constitute a part of this specification, illustrate certain embodiments and together with the detailed description and serve to explain the principles of the materials, methods, devices, and kits. The drawings are exemplary only, and should not be construed as limiting the materials, methods, devices and kits described herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Materials and kits for enhancing minute quantities of DNA from aspirates (e.g., liquid cytology samples or needle aspirations and brushings), from fixed and/or stained tissue samples and the like are provided which can be used in conjunction with analytical platforms and methods analyzing the samples. Examples of such analytical platform and methods of use are described herein which can be used in conjunction with the materials, methods, and kits disclosed, including an analytical platform that includes:
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- (1) microdissecting the cellular material into an enhancing buffer;
- (2) optimizing the ratio of buffer to microdissected material so that nucleic acid amplification can proceed in an optimal manner;
- (3) treating the crude lysate containing all the microdissected cellular material with a proteinase;
- (4) deactivating the proteinase;
- (5) centrifuging the crude lysate thereby creating a separation between the residual tissue elements present as a pellet and the liberated, amplifiable DNA present in the overlying supernatant;
- (6) extracting the DNA-containing supernatant optimally from the region of the supernatant immediately above the pelleted tissue;
- (7) formulating a nucleic acid amplification reaction having high magnesium concentration (or similar valency ions) and a high sucrose concentration.
In particular, a preferred analytical platform is designed to correlate with and significantly improve histopathologic and cytologic observation, thereby complementing and improving existing molecular pathology analysis using topographic genotyping as described in U.S. Pat. No. 6,340,563, which is incorporated herein in its entirety for all purposes.
1. Definitions and Acronyms
1.1 Definitions
It must be noted that as used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a patient” includes a plurality of patients, and so forth.
By “patient” or “subject” is meant to include any mammal including, but not limited to, bovines, primates, equines, porcines, caprines, ovines, felines, canines, and any rodent (e.g., rats, mice, hamsters, and guinea pigs). A preferred primate is a human. However, subjects may also be agricultural animals (e.g., chickens and other fowl) and domesticated animals.
By “anomaly” is meant a broad, encompassing term to indicate and disease related change in a lymphatic or hematopoetic cell or tissue of an organ. Thus, “anomaly” includes cancer, precancerous conditions such as dysplasia (e.g., a pre-cancerous breast neoplastic state, or colon polyps), and a non-neoplastic condition. The cancer includes, but is not limited to, any particular form of cancer, but encompasses all types and subtypes of malignancy. The pre-cancerous state includes proliferative lesions. The non-neoplastic condition may be inflammatory and adaptive state that may include features of cell proliferation, but needs to be clearly discriminated from neoplasia.
By “non-neoplastic condition” and “non-neoplastic abnormality” are meant a broad, encompassing term to indicate specimens from sites known not to contain neoplasia.
By “biological sample” or “specimen” is meant to include a frozen section, a biopsy of fresh tissue, or fixative-treated tissue. By “biological fluid sample” is meant to include, but is not limited to, breast lavage samples, ascites fluid samples, fine needle aspirates (FNAs) from a cyst, urine, blood, cerebrospinal fluid, and/or saliva. The sample can contain cells or may contain only free-floating DNA (non-nuclear DNA) in the fluid sample. A biological sample or specimen is meant to include any sample obtained from a subject. The sample may be an aspirate, a surgical biopsy, or a block of tissue previously obtained from a subject. Such blocks of tissue may be from a tissue bank or tissue repository. Tissue from repositories typically are paraffin embedded. The samples can be stained, inked, and/or fixative-treated cells and tissue. Also contemplated to be within the scope of the methods are samples of DNA from any source, biological or non-biological. The DNA in the sample may be, for example, synthetic or previously processed by other methods or procedures (i.e., a dried precipitate).
1.2 Acronyms
The following acronyms are used in this application and have the associated term, unless indicated otherwise in the specification.
CEL carboxyl ester lipase gene
CNS central nervous system
CT-PCR competitive template PCR
DNA deoxyribonucleic acid
FNA fine needle aspiration (or aspirate)
GCS glucocerebrosidase
LOH loss of heterozygosity
NP-40 Nonidet P-40
OD optical density
PCR polymerase chain reaction
qPCR quantitative polymerase chain reaction
SDS sodium dodecylsulfate
2. Conditions, Diseases and Tissues
The methods and compositions described herein can be used on any soft tissue. For example, the procedures can be used on any cell, but preferably is from a soft tissue such as tissue from any organ, skin, connective tissue, or nerve tissue. The methods and compositions also can be used to amplify DNA obtained from aspirated samples, obtained for example by needle biopsies.
The compositions and methods described can be used to determine any genetic condition. Preferably, the methods can be used to diagnose and or provide a prognosis for hyperplasia and other reactive causes of cell proliferation, pre-cancer states such as but not limited to dysplasia, and malignancy. Preferably, the compositions and methods are utilized to determine disease of any organ (e.g., brain, heart, lung, kidney, liver, pancreas or bile duct organ, stomach, intestine, colon, or other gastrointestinal organ), tissue (e.g., bone, skin, connective tissue) and nervous system. Preferably, the cancer or dysplasia is selected from the group consisting of a carcinoma, an epithelial malignancy, a sarcoma, a mesenchymal malignancy, a neuroepithelial cancer, and a central nervous system (CNS) cancer. Preferably, the pre-cancerous state is selected from the group consisting of wherein the pre-cancerous state is selected from the group consisting of epithelial pre-cancerous states including but restricted to mucinous cystadenoma, leukoplakia, serous cystadenoma, colon polyp, mesenchymal precancerous lesions, neuroglial precancerous lesions, lymphohematopoietic precancerous lesions, and precancerous lesions of other cellular types.
The cancers, which can be diagnosed using the materials and methods described include, but are not limited to, cancers of the head and neck (e.g., nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas), lung tumors (e.g., non-small cell and small cell lung tumors), neoplasms of the mediastinum, brain cancers, cancers of the gastrointestinal tract (e.g., colon, esophageal carcinoma, pancreatic carcinoma, gastric carcinoma hepatobiliary cancers, cancers of the small intestine, cancer of the rectum, and cancer of the anal region), genitourinary cancers (e.g., kidney cancer, bladder cancer, prostate cancer, cancers of the urethra and penis, and cancer of the testis), gynecologic cancers (e.g., cancers of the cervix, vagina, vulva, uterine body, ovaries, fallopian tube carcinoma, peritoneal carcinoma, and gestational trophoblastic diseases), breast cancer, cancer of the endocrine system (e.g., thyroid tumors, parathyroid tumors, adrenal tumors, pancreatic endocrine tumors, carcinoid tumors, carcinoid syndrome, and multiple endocrine neoplasias), sarcomas of the soft tissues and bone, benign and malignant mesothelioma, skin cancers, liver cancers, malignant melanoma (e.g., cutaneous melanoma and intraocular melanoma), neoplasms of the central nervous system, pediatric tumors (e.g., neurofibromatoses, neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma and peripheral neuroectodermal tumors, germ cell tumors, primary hepatic tumors, and malignant gonadal and extragonadal germ cell tumors), paraneoplastic syndromes, and solids cancers with unknown primary sites. By cancer or neoplasm is also meant to include metastatic disease and reoccurrence or relapse of a cancer(s). Also contemplated are virally induced neoplasms such as adenovirus, HIV-1, or human papilloma virus induced neoplasms (e.g., cervical cancer and Kaposi's sarcoma, and primary CNS lymphoma) as well as any secondary cancer appearing in lymph.
3. Method for Assaying Minute Quantities of DNA and Compositions Therefor
Generally, cellular specimens can be obtained from microdissected tissue removed from fixative-treated microscope sections or microdissected-stained cytology preparations. The tissue can also be derived from paraffin embedded blocks, for example from tissue repositories. The cellular specimens are placed into a convenient volume of buffer in order to create a crude lysate. Preferably, the buffer consists of NP-40 diluted to a final concentration of about 1.0%. The composition of the buffer can be as follows: 100 mM NaCl; 25 mM EDTA, pH 8.6; 10 mM Tris, pH 8.3; and 1.0% NP-40 prepared using deionized, filtered water (final composition pH can be anywhere within about 7.0 to about 8.6 and every 0.1 value in between that range). NP-40 is the preferred non-ionic detergent, however other non-ionic detergents can also be substituted. For example, Tween (e.g., Tween-20 or Tween-40), Triton X-100, and Nikkol may be substituted for NP-40. Detergents such as sodium dodecylsulfate (SDS) must be avoided, because its presence in the crude lysate will interfere with nucleic acid amplification.
Direct transfer of the microdissected material without formal DNA extraction is not an intuitively obvious action. Standard teaching in pathology puts forth that DNA should be extracted, and that the extraction process should isolate the DNA in a highly purified form. Avoidance of DNA extraction as a prelude to biochemical reactions that are DNA based is highly unusual and would be discouraged by conventional pathology teaching. Moreover, the PCR reaction to follow is one that utilizes a powerful enzyme capable of enormous amplification of DNA. The use of DNA that is not highly purified to trigger such a reaction would not be recommended under normal pathology standards.
One aspect of the methods described herein is not to overload the crude lysate with cellular material. Excessive cellular material will prevent effective nucleic acid amplification. Since microdissected samples can vary greatly in the amount of cellular material present, and it is not convenient to measure the amount of cellular material that is removed, the best means to gauge the desired quantity of cellular material is to identify a crude lysate that is mildly turbid. The degree of turbidity from which the DNA can preferably be amplified is one that is cloudy with particulate material, yet sufficiently clear such that one can see through the particulate suspension and visually identify objects through it. The sample should not be opaque and prevent light from getting through or prevent the ability to see through the suspension. Such a sample would be considered too heavy or too turbid for transfer of the DNA-containing material into the buffer. Moreover, it is better to be too light with material transfer (i.e., less turbidity) than to be too heavy, because the inclusion of excessive material will only serve to prevent effective nucleic acid amplification. Samples that have heavy turbidity are not optimal. For example, turbidity determination is usually performed in 200 microliter microcentrifuge tubes that are composed of virtually fully transparent polypropylene plastic. The usual volume of buffer into which the microdissected cellular material is placed is 25 μL. However, larger or smaller volumes of buffer can also be utilized. The tissue is deposited into the tub by placing the scalpel tip in the buffer and allowing the dissected cellular material to slide off the tip into the fluid. The scalpel may also be shaken to fully transfer the tissue into the fluid. Once deposited, the tube is gently shaken to evenly disperse the cellular material and the turbidity observed. The preferred turbidity is that which enables a visual inspection through the column of turbid fluid.
Blood is to be avoided, because red blood cells do not contain genomic DNA and only contributes material into the reaction without any effective genomic template for amplification.
Another aspect of the method is to examine the histopathologic appearance of the sample being placed into the buffer. If microscopic examination reveals a relatively high content of acellular material, such as collagen, then more cellular material can be used to prepare the crude lysate. If excessive cellular material has been added to the buffer such that it is too turbid, additional buffer can be added to achieve the desired degree of turbidity.
The lysate is then treated with a proteinase (e.g., proteinase K, pronase, subtilisin, thermolysin, or papain, or other proteinases, or combinations thereof). Preferably, the proteinase is proteinase K. Proteinase K (tritirachium alkaline proteinase) is then added to the crude lysate to achieve a final concentration of about 2 mg/mL. One or more proteinases are added to break down histone and other proteins that may be in contact with the DNA, thereby preventing DNA amplification. For example, a combination of proteinase K and pronase, or subtilisin and pronase, or proteinase K and thermolysin and papain can be combined such that a final amount of 2.0 mg/mL proteinase is present in the reaction mixture. Although different concentrations of proteinase K can be used, both greater and less than about 2.0 mg/mL, a preferred amount is about 2.0 mg/mL. Proteinase K or other proteinases or proteinase combinations can be substituted at equal concentrations. The range of effective protease action is ideal at about 2.0 mg/mL. However, the range of effective action can be very wide (about 0.2 mg/mL to about 20.0 mg/mL with every 0.1 mg/mL value in between this range). Also, combinations of proteinases can be used if desired considering the concentration of each as equivalent. The crude lysate is incubated at 37° C. for about two hours to overnight. Other temperatures and durations of incubation may be substituted. If a higher concentration of proteinase or proteinases is used then the duration of incubation can be shortened in a commensurate fashion.
At the completion of the proteinase digestion, the specimen is heated to about 100° C. for 5 minutes to inactivate any residual proteinase enzyme. The crude lysate may then be stored indefinitely at about −22° C. or lower until needed for the step of nucleic acid amplification. When nucleic amplification is to be performed on the sample, the sample is then defrosted.
The crude lysate (defrosted or fresh) is centrifuged 10,000 revolutions per minute in a table-top centrifuge to pellet the undigested material at the bottom of the tube. One microliter aliquots of the crude lysate are removed from the lysate immediately above the pellet. The lysate in this region is preferred for performing direct PCR amplification, although other portions of the lysate from the sample may also be utilized. The 1-μL samples should not however include the pellet itself, which contains tissue that will interfere with PCR amplification.
One microliter of the crude lysate is added to a 12.5 μL of nucleic acid amplification reaction buffer (e.g., a 10×PCR Buffer is composed of 500 mM potassium chloride, 100 mM Tris-HCl (pH 8.3 at room temperature), 15 mM magnesium chloride, and 0.01% (w/v) gelatin) (final composition pH can be anywhere within about 7.0 to about 8.6 and every 0.1 value in between that range). Oligonucleotide primers are added as desired. Primers are always present at an excess and equal concentration in conventional (symmetric) PCR amplification and, typically, are within the range of about 0.1 μM to about 1 μM (and any 0.1 μM in between). A stock mixture buffer is then used to add Taq polymerase, deoxyribonucleotides, and salts to the final amplification mix. In order to enhance amplification of the minute quantities of DNA, the magnesium concentration is significantly increased to a level of about 8 mM. While 8 mM magnesium chloride has proven to be optimal, effective performance can be seen within a range from about 6.0 to about 10.0 mM (and any 0.1 value in between this range). Values less than 6.0 and greater than 10.0 become less effective. Magnesium is added in order to improve hybridization of the probe to the template DNA. Manganese and alternative salt ions of equivalent valency may also be utilized (e.g., MnCl2). However, use of magnesium is preferred. Calcium cannot be substituted, because the enzyme is sensitive to its presence.
Another aspect of the amplification mix that enhances the amplifiability of minute quantities of DNA is an amplification mix having a final concentration of sucrose of about 12 g per 100 mL. While about 12 gram percent has been found to be optimal for use in the reaction, sucrose can be added in a range from about 5.0 gram percent to about 15.0 gram percent and any 0.1 gram percent value there between. The nature of how the sucrose concentration enhances amplification of the nucleic acid is not fully understood, but may occur by assisting the relaxation of double strands of DNA and in turn encouraging primer annealing. The use of sucrose is not intuitive. There is no reason to expect that the presence of sucrose in an enzymatic reaction will enhance efficacy.
Thus, for greatest amplification of DNA from a minute sample, the fixative-treated tissue is placed directly into a NP-40 containing buffer as described above, so as to form a crude lysate for direct nucleic acid amplification. Careful attention should be taken to ensure that the specimen is neither excessive nor insufficient in amount in the buffer. The final amount of sample in buffer does not have to be precise but within a certain range of turbidity. Preferred final turbidity is as discussed above. Then the crude lysate is treated with one or more proteinases for a certain period of time. The proteinase(s) is deactivated by boiling, after which the sample centrifuged. The fluid immediately above the pellet is withdrawn in about 1 μL aliquots for individual amplification reactions. The final amplification mixture contains significantly higher than normal MgCl2 and sucrose to both facilitate double strand relaxation and primer hybridization.
The materials and methods above can be used with an analytical platform. The analytical platform can include:
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- (1) techniques to separate any cells present in the fluid from the cell free fluid for separate molecular pathology analysis;
- (2) techniques to measure the quantity of DNA serving as means to infer the proliferative rate of neoplastic states;
- (3) techniques to measure the quality of DNA including the extent of its breakdown into progressively shorter pieces of nucleic acid serving as means to infer the proliferative rate of neoplastic states;
- (4) mutation analysis of minute specimens affording determination of genomic and specific gene copy number imbalance;
- (5) mutation analysis of minute specimens affording determination of point mutational change of nucleic acids in the sample;
- (6) mutation analysis of minute specimens affording determination of specific gene amplification and/or homozygous gene loss;
- (7) mutation analysis of minute specimens affording determination of microsatellite instability;
- (8) mutation analysis of minute specimens affording determination of gene rearrangement;
- (9) mutation analysis of minute specimens affording determination of genomic deletional expansion;
- (10) mutation analysis of minute specimens affording determination of altered DNA methylation status uniquely innovated to operate on fixative-treated tissue sections and cytology smears;
- (11) techniques to determine the time course of mutation accumulation providing a dynamic understanding of molecular change; and
- (12) quality control measures to monitor the reliability and representativeness of mutation detection consisting of recommendations for replicate analysis at both the specimen level (sample replication) and/or replication of the individual marker results (i.e., marker replication).
In particular, a preferred analytical platform has been designed to correlate with and improve histopathologic and cytologic observation, complementing and improving existing pathology practice. The methods, materials, and kits provided herein permit the objective diagnosis of invasive cancer, low- and high-grade forms of dysplasia, and neoplastic and non-neoplastic conditions. The methodology comprises detailed molecular analysis incorporating DNA quality and quantity, point mutational analysis of oncogenes and other genes and other forms of mutational damage correlated with cancer, broad spectrum of tumor suppressor genes, and other breast cancer associated genes linked to microsatellite loss of heterozygosity (LOH), and new approaches for accurate copy number determination of genes and oncogenes (copy number analysis). Another aspect provides methods to clearly discriminate cancer from non-cancer states, such as those produced by inflammation, infection, and trauma to a particular organ or tissue. Methods for diagnosing, determining prognosis of, and defining a course of treatment for cancer, high-grade dysplasia, pre-cancerous states, and non-neoplastic conditions are also provided.
An important component of the present methods, materials, and kits provided relates to the quality control assessment of small amounts of DNA that, due to paucity of diagnostic material, may produce both false positive and false negative results for mutation detection (C. R. Miller, et al., 2002 Genetics 160(1): 357-66; J. C. Dreesen, et al., 1996 J. Assist. Reprod. Genet. 13(2): 112-4). Standard pathology practice addresses this need by extracting purified DNA from specimens and measuring its content by optical density (OD) determination. Unfortunately, extracted DNA from fixative-treated, stained specimens is vanishingly low, e.g., below about 1 ng for fixative-treated, paraffin-embedded specimens measuring about 1 cm in size or less. Although quantitative polymerase chain reaction (qPCR) has been employed on fixative-treated tissue, qPCR assumes high quality starting nucleic acid content, which cannot be assumed when dealing with fixative-treated, stained specimens of limited size. Methods and devices that address the need for analytic validation and quality control are provided herein.
Non-neoplastic fluid collections have very low amounts of DNA present in the fluid, because the lining cells contain very few cells that replicate slowly. Low-grade, slow-growing, neoplastic anomalies would generate an associated fluid that contained relatively greater amounts of DNA reflecting the greater turnover of replicating lining cells. Finally, a malignant tumor is believed to possess greater amounts of DNA of high quality and intactness in fluid localized near the tumor than benign tumors and precancerous lesions.
To confirm this belief, a novel approach as set forth herein was created to quantitatively define the amount and integrity of DNA, and then demonstrate statistically significant thresholds that would separate non-neoplastic, indolent, and malignant states from each other. The combined applications of these concepts to pathology analysis of specimens and the specific methods used were uniquely created to address these needs.
The approach includes combining the direct quantitation of extracted DNA using established optical density (OD) measurement techniques, the qPCR determination of DNA concentration, and the competitive template PCR (CT-PCR) reaction involving defined genomic segments of, for example, glucocerebrosidase (GCS) gene and its pseudogene (other genes and their associated pseudogenes can be used alone or in combination with GCS and its pseudogene). The qPCR reaction is not used to measure DNA concentration, because that is accomplished by the first step, OD measurement. Rather, the OD concentration is used to standardize the qPCR reaction and other mutational analyses to a starting concentration of about 5 ng/μL, although other levels for normalization can be used with equal effectiveness. The variation in the qPCR reaction then serves as a measure of the degree of intactness of DNA since the highest qPCR values for DNA starting concentration will be seen when DNA has not undergone degradation. This use of the qPCR provides data on DNA integrity.
The CT-PCR reaction, performed in triplicate (or more) at varying concentrations, provides quantitative information on the quality of DNA with respect to degradation, and quantitative information on the representative amplifiability of this DNA. The CT-PCR reaction also provides a sensitive means to quantitatively characterize DNA degradation in a fashion that is essentially independent of the various phases of nucleic acid amplification (exponential phase, plateau phase), and is effective on minute specimen samples. By means of gene/pseudogene sequences that are nearly identical, but differ by varying lengths of base deletion, the effect of DNA degradation can be accurately characterized. This can be done, for example, using the GCS gene and its pseudogene or other gene/pseudogene combinations. By performing CT-PCR in triplicate at different concentrations of starting template, the effect of allelic dropout can be effectively controlled, and the degree of degradation can be accurately defined. Adequacy of amplifiable DNA is reflected by replicate, reliable amplification of both the shorter and longer sized amplicons. Low amounts of inadequate levels of amplifiable DNA for mutational analysis is represented by variability in the effectiveness of relative amplification of the longer sized amplicon. The use of CT-PCR as described provides the information needed to effectively analyze minute clinical tissue specimens subject to tissue fixation and staining.
Also described herein are methods for determining specific gene copy number also specially adapted for use with minute samples. The DNA sequence of a gene of interest and a comparator gene are searched for homology to direct the creation of oligonucleotide primers for duplex amplification. More importantly, the amplicons generated are also designed to be of approximate but not equal length, thereby enabling a stoichiometric relationship to be developed between them. If both genes are present at the same copy number, then the amplicons generated will be equal. If the gene-of-interest undergoes amplification, then its amplicon is in relatively greater excess than the comparator gene. Conversely, if the gene-of-interest undergoes homozygous loss, then the comparator amplicons will dominate. This unique approach is one that can provide quantitative information on specific gene copy number effective on virtually any type of specimen.
These innovations are combined with several standard procedures to produce a quantitative system for molecular analysis of clinical specimens for diagnostic and other purposes. The system is entirely complementary to standard pathology practice. It does not compete with current pathology practices, but rather advances them, eliminating the roadblock that currently exists to advancing patient medical care. Working within established pathology practices is important for obtaining standardization and approval of the techniques by practitioners and histopathology technicians. Creating methods that diverge from standard practices will be more difficult to gain acceptance in mainstream pathology laboratories.
3.1 Method of Quantifying DNA and DNA Quality Analysis
After purifying the DNA, optical density (OD) analysis is performed to quantify the DNA. One approach can be the Nanodrop technique, because it requires only one microliter to be sacrificed for the purpose of obtaining the DNA concentration. (Ding et al., 2004 J. Biochem. & Mol. Biol. 37(1): 1-10). Other techniques for quantifying DNA can also be utilized.
The higher the OD value indicates that a larger amount of DNA is present. The quantity of DNA extracted can vary. However, the higher the amount of DNA, the more likely a high-grade dysplasia or a malignancy is present. The exact level of the DNA measured in this step, and the significance to the biological process, is not universal but must be interpreted in the overall context of findings. It has been shown that samples with an OD value of about 2.0 ng/μL or less are considered to have insufficient DNA. Values in the range of about 2.0-10.0 ng/μL (and any 0.1 ng/μL value in between) indicate pre-cancerous lesions and/or low-grade indolent forms of neoplasia. Values over about 10.0 ng/μL are generally indicative of the presence of a malignancy. For example, a value of about 359 was observed with a sample from a pancreatic cyst, which indicated that the tumor was growing very actively within the cyst or ductal space. Thus, ranges of between 10.0 to 500 ng/μL or to 1,000 ng/μL are contemplated as representative of a malignancy. However, the upper limit in some instances could be even higher than 500 or 1,000. These conclusions are not definitive, but only suggestive and are to be used with the other data obtained by the other steps of the methods described herein to come to a definitive assessment.
The DNA can be quantified by OD measurement at wavelengths of 230, 260, and 280 nm. The 260/280 and 260/230 ratios should be 1.7-2.0, in keeping with extraction of purified DNA and for the purpose to exclude protein and other contaminants. Other suitable settings can also be substituted. Any technique that quantifies the DNA in the sample can be a suitable substitute to OD measurement or as discussed herein.
3.2 Determining DNA Quality for DNA Quantity and Quality Analysis
A variety of techniques are used to quantify DNA quality. These fall into two categories, quantitative polymerase chain reaction (qPCR) and competitive template polymerase chain reaction (CT-PCR). Each technique provides valuable information on DNA quality. Both techniques used together on specimens provide highly valuable information. The qPCR reaction can be performed using, for example, sybr green as the indicator in a suitable thermocycler capable of measuring fluorescence during the amplification process, as this is a simple and relatively inexpensive technique. Other techniques for qPCR determination using fluorescent labeled primers can also be substituted. Known quantitative controls and replicate analysis of samples can be used to standardize amplification reactions and is preferred. The use and configuration of controls and replicate analysis may be varied as determined by the user. For example, standardization of qPCR amplification of the first exon of the K-ras-2 gene may be used for this purpose. However, any PCR product from any gene or genomic segment may be used.
The DNA obtained from aspirates or biological fluid samples may be free-floating or free and adherent to the surface of cells or tissue constituents of the cyst. DNA possesses a physicochemical tendency to adhere to biological surfaces such as cell membranes and physical structures, such as glass or plastic. From these locations, the DNA can be extracted and analyzed. This DNA is derived from cells that line or are in contact with the region from which fluid is collected (e.g., aspirate or lavaged samples). The DNA present would then be representative of those cellular elements that constitute the fluid source. The free or surface-attached DNA is not visible by microscopic examination. However, it can be extracted and analyzed as a means to assess the etiology and character of a fluid collection. The same concepts hold true for fluid moving through a channel, such as a breast duct or pancreaticobiliary ductal system. The sample will generally contain both intact cells with internal nuclear DNA and free genomic DNA (non-nuclear DNA). Both internal nuclear DNA and a free-genomic DNA can be used to determine the characteristics of the lining cells. In addition to searching for mutations, the quantity and amplifiable quality of the DNA can serve as end points for the analysis of DNA.
3.3 Quantitative PCR Amplification
The first step in the qPCR process is to adjust the extracted sample DNA concentration to a value of about 5 ng/μL so that the absolute amount of DNA present in each reaction is the same. This will still allow the integrity to vary, which is the purpose of the analysis. About 5 ng/μL is preferred as it has been found to be a minimal value for robust amplification. However, other amounts may be used (e.g., about 10 ng/μL to 100 ng/μL or any integer value in between; more preferably about 1.0 ng/μL to about 10 ng/μL or any 0.1 ng/μL value in between). All other values may be substituted quite freely and is up to the discretion of the investigator.
The number of qPCR cycles may be used as a marker of DNA quality. The lower the number cycles required to reach a desired threshold is indicative of higher quality DNA. In general, if over 30 qPCR cycles are required, then the DNA quality is considered suboptimal due to, for example, allelic imbalance resulting from inadequate amounts of template DNA. Specifically, Ct values (i.e., threshold values for quantitative PCR product detection) over 30 cycles is considered evidence of poor quality of DNA, especially if the DNA quantity present is above about 2.0 ng/μL. Ct values of about 29-30 are considered borderline and representative of poor quality DNA. Cycle values of 29 or less are indicative of good quality DNA, and are generally characteristic of the breast anomaly being malignant. Additionally, values of 29 or less may indicate the presence of neoplasia. The lower the Ct value, the more likely neoplastic cell proliferation is malignant. These values may vary based on the conditions of the assay and amounts of DNA employed. Cancers that proliferate slowly, are well differentiated in growth pattern, and that are relatively less cellular may be expected to show borderline Ct values that are significantly higher than 29-30 cycles.
3.4 Competitive Template Nucleic Acid Amplification to Assess DNA Quality
DNA quality may be further assessed by performing competitive template PCR amplification for a unique pair of genes, (e.g., glucocerebrosidase gene and its pseudogene) at a particular point where the two genes have virtually identical sequences. In the case of GCS and its pseudogene, there is a 55 base pair deletion in the pseudogene. A second region in exon 1 of the glucocerebrosidase gene provides an equivalent opportunity to utilize a 19 base pair deletion in the same manner. This is not the only gene that can be used in this fashion. In fact, any pairing of gene or genomic segments of similar sequence, but different in length, can be substituted.
This PCR reaction creates two amplicons that are identical in sequence except for the deletional region. During the reaction, a competition exists between the two similar templates (but having different lengths). The degree of DNA degradation in the sample will be reflected by less effective amplification of the longer template as compared to the shorter template. This serves as a measure of DNA integrity. The amount of each product, short and long, may be quantitatively measured by capillary electrophoresis. Methods of performing the PCR reaction and electrophoresis are well known in the art. A non-neoplastic process shows prominent DNA degradation, while a malignancy is associated with the presence of abundant, good quality DNA. Reagents are added to the final sample to enhance DNA availability and to enhance the ability to amplify the DNA.
While the use of two highly similar amplification targets is recommended, any system that utilizes similar primers to amplify products of different lengths can be substituted. The genetic targets used should be selected to preferentially produce different sized products under similar amplification conditions in the biological samples. The closer to the value one (1.00) the ratio of amplified product to each product is, the better the quality of DNA (i.e., the more likely the tissue from which it was derived is malignant or hyperplastic).
The procedure for PCR amplification has been well described and variations on its performance will not impact the materials, methods and kits disclosed. As described in the Examples below, the recommended procedures of the manufacturers' for the PCR reagents are followed (e.g., GeneAmp kit, Applied Biosystems). However, other commercial and non-commercial systems for PCR amplification can be readily substituted. It is preferred that the PCR reaction is performed in a manner that is highly robust. “Robust,” in this context, indicates the reliable generation of abundant amplified DNA that accurately reflect the starting composition mixture of normal and mutated DNA derived from a particular specimen, especially when using minute samples such as dilute fluid specimens. Reagents such as dimethylsulfoxide or dextran sulfate can be added to the amplification reaction to enhance amplification. Other similar reagents can be substituted. Also, manipulations, such as nested PCR, can be performed to further enhance amplification. Other similar steps may be used, though they are not mandatory.
Based the concept that a longer sized PCR product is present in lesser amounts than a shorter sized product due to greater chance for strand breakage as a result of DNA degradation, a competitive duplex PCR reaction of highly similar DNA sequence, but differing in length was needed. This is accomplished by simply carrying out a short- and long-product PCR reaction in one container (e.g., test tube) on one source of DNA. Unfortunately, such duplex reactions are not equivalent, because they use different primers and generate radically different products. Moreover, the status of the PCR reaction must be carefully controlled, because amplification is not the same during the different phases of the reaction (i.e., exponential phase versus plateau phase). The methods described herein overcome this obstacle.
The human genome is searched for gene/pseudogene pairings that possessed the identical genomic sequence except for a segment present in one that was deleted in the other. The only difference in precise genomic sequence is the deleted segment, which can vary in length. The greater the length of the deletion, the greater the ability is to detect differences related to DNA degradation. Importantly, the primer hybridization sites for PCR are identical, and the relative amounts of product made are essentially independent of the status of the PCR reaction itself. For example, the glucocerebrosidase gene (GenBank D13286) and its pseudogene (GenBank D13287), and the human carboxyl ester lipase (CEL) gene (GenBank M94579) and its pseudogene (GenBank M94580) can be used. Other similar gene/pseudogene examples can also be employed alone or in combination as needed. In a preferred embodiment, the glucocerebrosidase gene and its pseudogene will be sufficient as it affords specific deletional regions of 19 bases and 55 bases in exons 1 and 9, respectively.
The competitive template PCR (CT-PCR) reaction for the glucocerebrosidase gene/pseudogene pairing (exons 1 and 9) provides a novel and sensitive means to quantitate the degree of DNA degradation. The results from this assay, together with the data from the OD and qPCR steps, provide discriminating information on DNA degradation. With the current techniques used in the art, the conditions of Table 1, for example, are only poorly discriminated in fine needle biopsy cytology specimens using microscopic evaluation. This information in turn can be used to distinguish the conditions listed in Table 1, or any breast conditions discussed herein.
Another aspect contemplates the use of competitive template PCR (CT-PCR). This technique enhances quality determination of DNA and analytic validation. Replicate aliquots are used as substrates to amplify a segment of DNA present in the for example glucocerebrosidase gene (GCS) that is identical in base sequence to that in its pseudogene, except for a 55 base pair deletion present in the latter (glucocerebrosidase gene (GenBank D13286) and its pseudogene (GenBank D13287). A second deletion of 19 bases present in the GCS gene, but is absent in the pseudogene is also available for similar application. The result is two amplicons that theoretically should be generated in equal amounts. Preferential amplification of a shorter allele from among a mixture of templates is a common observation when amplifiable DNA is of low quality or otherwise rate limiting. The relative fluorescent intensities of the true GCS/pseudo GCS is thus a measure of effective ability to amplify the starting DNA, in turn a measure of DNA integrity. This determination is coupled with absolute determination of fluorescent content serving as a measure of total amplicon production. The closer to a value of one (1.0) for ratio of true GCS gene/pseudo GCS gene, the higher the absolute fluorescence, the more reproducible the replicate analysis, and the greater confidence exists that minute starting quantities of template DNA are being accurately represented in the final analysis. Analysis in triplicate is performed to determine consistency in PCR amplification for the templates present in a particular sample. This is vital for the effective molecular pathology analysis of paucicellular fluid samples. These techniques can be combined with analysis of microdissected tissue samples and cytology samples. Another gene/pseudogene combination that be substituted here is the human carboxyl ester lipase (CEL) gene (GenBank Accession No. M94579) and its pseudogene (GenBank Accession No. M94580). Similar examples that could be substituted with equal effectiveness.
If the microdissected cellular sample is found to have excellent quality starting DNA, as described by the criteria in the previous paragraph, then mutational analysis may proceed using thresholds for significant allelic imbalance that are at the 95% confidence level for the range of variation in normal tissue specimens for each unique pairing of polymorphic markers. However, if there is evidence of limiting DNA, as reflected by CT-PCR, greater allowance must be given to imbalances that favor the preferential amplification of the shorter microsatellite allele. When CT-PCR indicates a relatively mild impairment, then mild imbalances favoring the shorter allele may be ignored. When greater impairment is indicated by CT-PCR, the specimen can be regarded as possessing low quantities of relatively poor quality DNA. As a further check, specific imbalances for genomic loci can be repeated in duplicate, and the same criteria applied as that for true GCS/pseudo GCS pairing and/or other gene/pseudogene pairings.
The CT-PCR reaction is performed in replicate using increasing amounts of starting DNA. When performed in this manner the results will inform the user concerning the total amount of amplifiable DNA in the test sample, and the extent of degradation of the DNA into smaller sized fragments.
The presence of adequate amounts of good quality DNA is reflected by ratios of allelic height peaks in the about 0.8 to about 1.0 range. This value indicates that the longer length allele is essentially amplified at an equivalent rate to that of the shorter allele. When the DNA content is suboptimal for balanced PCR amplification, there will be a tendency to delete the longer allele, and thus the ratio will tend to fall. As a higher concentration of starting DNA is used, one will see an improvement in the ratio into the range of about 0.8 to about 1.0 that is associated with adequate amounts of good quality DNA. With very low quantities of DNA, “allelic dropout” and PCR failures will be seen with wide fluctuations in the amplifiability of individual alleles, as exemplified in Table 2.
When DNA is degraded, there will be a range of DNA fragments, from short to long, in the specimen. Very short fragments may not be adequate for PCR amplification, while slightly longer fragments will tend to favor amplification of the short over the longer sized allele. Thus, a stable lower peak height ratio value will be seen for all concentrations (Table 2). The combination of low amounts of poor quality DNA will show the expected effects as exemplified in Table 2. As shown, the presence of very low amounts and/or very poor quality DNA will result in failures and allelic imbalance that will not be possible to specifically trace to a particular cause.
3.5 Mutation analysis and other molecular analysis of DNA in aspirate for Allelic Imbalance (loss of heterozygosity [LOH])
Chromosomal allelic loss, commonly referred to as loss of heterozygosity (LOH), is a major cause of tumor suppressor gene inactivation. Thus, detection of LOH from microsatellite markers closely linked to key tumor suppressor genes serves as an excellent surrogate marker for gene inactivation. The materials and methods herein use a panel of LOH markers on specimens, together with analysis of DNA quality and quantity to predict the presence of cancer or neoplasia, and to diagnose and treat these conditions.
PCR amplification may be used to generate amplicons of less than 200 nucleotides using synthetic oligonucleotide primers flanking each microsatellite (amplicons can range in size from about 40 to less than 200 and every integer value in between). Allele peak heights and lengths may be used to define the presence or absence of an allelic imbalance (i.e., LOH) for a given sample. Allelic imbalance is reported when the ratio of polymorphic allelic bands for a particular marker is beyond about 95% confidence limits for the variation in peak heights for individual allele pairings derived from an analysis using non-neoplastic specimen samples. In general, the value of the ratio is below 0.5 or above 2.0. Preferably, the allele ratio is two standard deviations beyond the average for the ratio of the specific pairing of polymorphic alleles. This will provide the lowest threshold for detection of significant allelic imbalance (LOH). However, other algorithms for defining LOH can be used, so long as they are applied uniformly across different specimens such as using the allele ratio of the non-neoplastic sample as a denominator that is divided into the allele ratio for the lesional samples. It is understood that minor amounts of LOH will not be detected. However, these minor LOH mutations may not be causally related to clonal expansion or provide significant malignant growth properties.
Allelic imbalance mutations are treated as genomic deletions associated with tumor suppressor genes. The ratio of allele peak heights is a measure of an admixture of mutated and non-mutated cells (or mutated and non-mutated DNA), and varies according to the individual pairing of specific, microsatellite marker alleles. Allele ratios of 2.0 or 0.5 is said to be present when 50% of the total DNA is derived from cells possessing the loss. The deviation from the ideal normal ratio of about 1.0 indicates which specific allele is affected. Allele ratios below about 0.5 or above about 2.0 are mathematically correlated with the proportion of cells affected by genomic loss. The order of mutation acquisition may be arranged in a temporal sequence reflecting the proportion of cells affected by specific microsatellite marker loss. Markers displaying more extreme ratios are considered to have been acquired earlier in the disease process. This conclusion is based on the premise of clonal expansion. A “clonal expansion” occurs when tumor cell populations progressively replace each other by accruing mutations, which are causally associated with increasing malignant phenotype.
3.5.1 Nucleic Acid Amplification
Nucleic acid amplification can be carried out, for example, as follows using the DNA preparation as described above or in the examples. One microliter aliquots are removed for PCR amplification of individual polymorphic microsatellite markers. Other sized aliquots of DNA can also serve equally well. Nucleic acid amplification can be carried out according to manufacturer's instructions (e.g., using GeneAmp kit, Applied Biosystems, Foster City, Calif.). Other variations on the PCR reaction can apply equally. Fluorescent labeled oligonucleotide primers are employed for quantitative determination of allelic imbalance based on the peak height ratio of polymorphic microsatellite alleles. Other quantitative system for PCR and/or qualitative approach can be substituted.
3.5.2 Allelic Imbalance Determination
Allelic imbalance determination can be carried out as follows. Post-amplification products are electrophoresed, and relative fluorescence determined for individual allele peak height (e.g., using GeneScan ABI3100, Applied Biosystems, Foster City, Calif.). The ratio of peaks are calculated by dividing the value for the shorter-sized allele by that of the longer-sized allele. Thresholds for significant allelic imbalance have been determined beforehand in extensive studies using normal (i.e., non-neoplastic) specimens representing each unique pairing of individual alleles for every marker used in the panel. Peak height ratios falling outside of two standard deviations beyond the mean for each polymorphic allele pairing were assessed as showing significant allelic imbalance. In each case, the non-neoplastic tissue targets are used to establish informativeness status and then to determine the individual pattern of polymorphic marker alleles. Having established significant allelic imbalance, it is then possible to calculate the proportion of cellular DNA that was subject to hemizygous loss. For example, a polymorphic marker pairing whose peak height ratio was ideally 1.00 in normal tissue with a standard deviation in non-neoplastic tissue of 0.23, could be inferred to have 50% of its cellular content affected by hemizygous loss if the peak height ratio was 0.5 or 2.0. This requires that a minimum of 50% of the DNA in a given sample be derived from cells possessing deletion of the specific microsatellite marker. The deviation from ideal normal ratio of 1.0 indicated which specific allele was affected. In a similar fashion, allele ratios below 0.5 or above 2.0 could be mathematically correlated with the proportion of cells affected by genomic loss. Other algorithms for quantitative determination of allelic imbalance can be used with equal effectiveness.
At this point the proportion of cells or DNA accounting for the imbalance will be determined for each marker. This information is used with the information derived from microscopic analysis of the tissue section or cytology cells from where the samples originated. Particular attention here is paid to the extent of non-neoplastic cell inclusion because these cells with provide normal DNA to the analysis. The inclusion of non-neoplastic cellular elements is not limited and is only mentioned in order to provide greater understanding for the quantitation of allelic imbalance. The inclusion of some degree of non-neoplastic cellular elements will not interfere with the final characterization of genomic deletion expansion. The proportion of cells or DNA affected by imbalance is determined for markers pertaining to a specific genomic deletion. A gradient of reduced mutation involvement indicates that a proportion of tumor cells with an expanded deletion are present in the area from which the sample was derived. This analysis is sensitive for detected genomic deletion up to the threshold representing two standard deviations from the mean for normal allele peak ratios.
3.5.3 Cytology Microdissection
Cytology microdissection is carried out in the same manner as topographic genotyping with attention paid to the microscopic cytology appearance of the cells for selection to perform genotyping. For topographic genotyping, see U.S. Pat. No. 6,340,563, which is incorporated by reference in its entirety for all purposes. Target clusters of cytology cells are marked with xylene resistant ink on the reverse face of the glass slide. The cover slips are removed, and designated cellular targets are microdissected off the glass slides using a manual approach or other technique for specimen microdissection, such as laser capture microdissection, etc. The microdissected cells are then processed for DNA quality determination and mutation detection as described herein.
The analysis of microdissected cellular material is carried out independently to that of the free DNA in the collected fluid sample. The results of each analysis may be compared to each other. While the two types of specimens would appear to be equivalent, they are in fact not equivalent. The microdissected cell genotyping informs the user of the alterations present in the cells themselves. The free-fluid genotyping informs the users of the changes affecting a wider distribution of cells encompassing all the cells with the potential to contribute DNA into the fluid collection. For example, changes upstream of the fluid collection can be detected by analyzing the DNA that is collected in a downstream location. Other possible scenarios are shown in Table 3.
3.5.4 Genomic Deletion Expansion
A series of polymorphic markers are selected which may be in the form of microsatellites or single nucleotide polymorphisms. These are specifically designed to cover the location of the deletion and adjacent DNA both on the centromeric and telomeric side. A representative example is shown in Table 4 for the APC gene. The same approach can be applied to any gene or interest across all chromosomes. Also, the specific markers may vary according to the genomic roadmap. The greater the number of markers used which cover the DNA on either side of the genomic deletion, the more precise the existence and extent of genomic deletion expansion will be able to be characterized. Thus, any number of markers may be used. In Table 4, four markers are shown for simplicity, two on either side of the deleted gene. However, any number of markers can be used. Allelic imbalance analysis is then performed for each of the polymorphic markers. This involves PCR followed by electrophoresis to determine the balance status for polymorphic alleles. Any system of allelic imbalance analysis can be substituted and will yield the same results. At the conclusion of this step, the extent of the genomic deletion will be defined according to the status of the location of the various polymorphic markers. This will be described in more detail below with several clinical case examples provided.
This analysis is then carried out in a quantitative manner to determine the proportion of cells and/or DNA affected by imbalance. The concepts described here can be applied to any source of DNA, as well as to biological fluid specimens.
As shown in Table 5, the APC gene region is subject to deletion damage. Polymorphic microsatellites are varying distances away from the deleted region, which demonstrates progressively small proportions of affected cells. Thus, the deletion is shown to be expanding. The rate of expansion is quantitatively expressed by the diminishing proportion of cells manifesting this change in the topographic sites from where the source of DNA has been obtained. The use of additional markers for allelic balance representing genomic sites at different distance from the gene-of-interest will provide the user with a clearer understanding on how rapidly the genomic deletion is expanding.
3.5.5 Point Mutational Analysis
The fluid DNA and/or the microdissected cells can be evaluated for the presence of point mutational change as one of several different forms of cancer related damage.
BETA-CATENIN, EXON 3: POINT MUTATIONS ARE FOUND BETWEEN CODONS 32 TO 41 (Italicized, underlined and bolded)
The domains used for primers in amplifying the region of the beta-catenin gene that is mutated are underlined with a single underline. The section in underlined, italicized and in bold is the region of the beta-catenin third exon that is affected by mutational change.
The beta-catenin gene undergoes localized point mutational change in certain forms of human cancer. Oligonucleotide primers, designed according to the genomic sequence available from many different sources, is used to amplify the region potential subject to point mutational change. Another example is the defined acquisition of point mutational change in the b-raf gene, which occurs frequently in melanocytic tumors, papillary carcinoma of thyroid, and other neoplasms. The protein exhibits a mutation wherein a valine is introduced (underlined “V”). The responsible nucleotide change producing the valine is shown in the lower panel of Table 7 in enlarged, underlined letters. The sequences to which the forward and reverse primers were prepared in the b-RAF sequence are indicated by the dotted line. Thus, the forward primer was 5′-TTTCTTCATGAAGACCTCACAG-3′ and the reverse primer, which is antisense to the b-RAF nucleic acid depicted, is 5′-ACAAAATGGATCCAGACAACTG-3′.
The coding exon in the lower panel of Table 7 is highlighted in bold, representing sites of mutational change that alters protein sequence.
Another example of point mutational change is detection of an EGFR point mutation in lung, non-small cell carcinoma and many other forms of neoplasia (Table 8).
The coding exon is in bold, representing site of mutational change that alters protein sequence. The upstream and downstream primers utilized respectively for Exon 19 of EGFR are 5′-CGTCTTCCTTCTCTCTCTGTC-3′ and 5′-ACCCCCACACAGCAAAGC-3′. The upstream and downstream primers utilized respectively for Exon 21 of EGFR are 5′-AGCAGGGTCTTCTCTGTTTC-3′ and 5′-GGCTGACCTAAAGCCACC-3′. Other primers can also be utilized.
3.5.6 Copy Number Analysis
Copy number analysis is used to search for and quantitatively characterize homozygous deletion and/or specific gene amplification. The simplest approach to accomplishing this objective is to perform a duplex PCR reaction for a gene-of-interest coupled with a comparator gene that is assumed not to vary with respect to cancer progression. This approach is not effective, because the two individual nucleic acid amplification reactions operate independently. More importantly, the independent nature of the reactions reside mainly in the operating characteristics of the amplification primers with respect to hybridization and denaturation. The Human Genome Database (Internet: <URL:www.gbd.org>) and Ensembl Genome Browser (Internet: <URL:www.ensembl.org>) was searched for primer sequences that were highly similar, especially with respect to the 3′ end of the primers for the gene-of-interest and the comparator gene. This led to defining primer sequences for duplex PCR amplification in which the 3′ end of the primers were highly similar. For example, the amplification products of both reactions would be close in size, such that the amplification reactions would operate in a nearly equivalent fashion. This is exemplified with the CDKN2A gene, which can undergo homozygous deletion in many types of human cancer. The comparator gene containing a sequence of near identity to CDKN2A was the APP gene situated on chromosome 22 (Table 9). The underlined areas indicate the precisely homologous sequences of base pairs in the upstream (solid underline) and downstream (dashed underline) primers used for duplex amplification.
The bolding and underlined domains the domains in which forward and reverse primers are designed to amplify the noted gene. Each sequence in Table 9 is oriented in a 5′ to 3′ orientation.
Note that the 10 bases at the 3′ end of the upstream primers (sequences in bold and underlined) for each gene are identical in sequence. Similarly, there is identity of the seven bases at the 3′ end of the downstream primer (sequences in bold only). This homology together with the design of each primer having similar melting temperatures ensures that the duplex reaction will proceed in parallel relationship to each other. The ratio of peaks for each product using labeled primers on capillary electrophoresis provides a quantitative relationship with respect to the relative content. Using normal tissue, cytology cells, and biological fluid samples as a source of non-neoplastic DNA and sample variation, it is possible to define thresholds for significant gene loss or gain in pathologic samples. This approach is ideally suited for use with minute specimen samples, but can be used on larger samples. It opens the way for copy number analysis of individual genes in clinical samples of the type obtained in routine patient management.
3.5.7 Microsatellite Instability
Free DNA collected from fluid samples and microdissected cell clusters can be evaluated for shifts in microsatellite repeats to support the finding of DNA repair gene mutational damage. A simple yet thorough approach, ideally suited to microdissection based analysis, is to carefully remove representative areas of cancer or precancerous lesions together with non-neoplastic tissue. The microdissected samples are then evaluated for shifts in microsatellite band positions.
The marker panel that will be used to develop this microdissection genotyping assay will consist of two groups of 5 microsatellite markers recommended and published by authorities in this field. The criteria for determination of instability will follow closely that currently suggested in the literature. Patients will be classified as having either microsatellite stable, low level instability (1 out of 5 unstable markers), or high level instability (2-5 out of 5 unstable markers). The testing will be configured to use the second marker group in subjects initially found to have low level instability. Alternative microsatellites can be used with similar effectiveness, as may be required with different cancers.
3.5.8 Altered DNA Methylation Status
Certain common approaches for characterization of changes in DNA methylation status, such as methylation specific PCR, will not function properly when using minute amounts of fixative-treated DNA from clinical specimens. Disclosed is a more robust system in which bisulfite modification is performed on tissue section and cytology specimens, while still adherent to glass slides. Non-methylated cytosines are first converted to thymidines, and then the specimens are microdissected.
The PCR primers are carefully formulated to avoid overlapping potential methylated cytosines at C-G dinucleotide pairs. Methylation status is then assessed using capillary electrophoresis within the amplicons. The amplicons are formulated to include as many potential C-G dinucleotide pair overlaps, thereby improving allele discrimination. This approach can be used for simple and effective methylation status detection with microdissected tissue samples. An example for the CDKN2A gene is shown in Table 10.
In Table 10, the promoter sequence of the gene has been shown after bisulfite modification. Another example is shown for the MGMT gene in Table 11:
In Table 11, the promoter sequence of the gene has been shown after bisulfite modification.
The protocol for in situ bisulfite modification is as follows:
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- 1. Deparaffinize four-micron thick tissue sections.
- 2. Treat tissue sections with Proteinase K (10 mg/ml, for 30 minutes) or other proteinase or proteinase cocktail.
- 3. Using Koplin jars (or equivalent container), incubate tissue sections in 0.2 N NaOH for 20 minutes.
- 4. Incubate overnight in 3 M sodium bisulfite containing hydroquinone.
- 5. Rinse tissue sections in 0.3 N NaOH; three rinses for 10 minutes each.
- 6. Pass through graded alcohol and air dry. Tissue is ready for microdissection.
It is to be expected that in situ bisulfite conversion will not proceed to completion, and thus a proportion of DNA collected in the microdissection will not have undergone conversion. Primers are designed to take advantage of the presence of non-methylated cytosines at the 3′ end of the oligonucleotide. The presence of unmodified DNA is not expected to interfere, given that PCR primers recognize only modified DNA. Moreover, the assay can be expected to operate in a stoichiometric manner in relationship to quantity the content of modified DNA. This assumption is reasonable and supported by preliminary data.
Traditional DNA methylation assays based on bisulfite modification require the use of highly purified extracted DNA to allow full conversion. The use of consensus primers to amplify both methylated and unmethylated forms of modified DNA, makes the novel reaction as described here insensitive to a large degree by the extent of bisulfite modification. So long as partial modification does not discriminate between methylated and unmethylated forms of modified DNA, the efficiency of modification will be not critical. Efficiency of modification will be assessed by running of specific primers and probes in parallel on both lesional and non-neoplastic microdissected samples.
Shown in Table 12 are exons 6-9 of the PAX8 gene and the first exon of the PPARgamma gene. Single nucleotide polymorphisms (SNPs) are indicated in bold and underlined. Exon sequences are in bold. The downstream primer in the PPARgamma gene is dotted underlined. Upstream primers can be created in each exon of the PAX8 gene for singleplex PCR reactions to test for the presence of gene rearrangement.
3.5.9 DNA Gene Rearrangement
Gene rearrangements can be detected using genomic DNA in fixative-treated specimens and fluid provided that the amplicon length is kept to a minimum. The longer the amplicon, the less likely the amplification will be successful. Positive detection of the rearrangement by positive nucleic acid amplification provides support for the existence of specific mutational alterations. An example is shown for the PAX/PPARgamma gene rearrangement present in certain forms of thyroid neoplasia (Table 12).
The most common approach to detect this rearrangement in aspirated thyroid fluid or microdissected thyroid cells is to convert the RNA to cDNA and then attempt to detect the rearrangement using primers designed to amplify a long segment of the rearrangement. This is less favored when dealing with fixative-treated, stained cytology, or free DNA from biological fluid samples, because the amplification may fail to work due to the poor quality of the DNA. Seeking to minimize the length of the amplified segment, separate primers can be fashioned for each of the PAX exons that may be subject to translocation (Table 12). A similar approach can be used on other genes to detect other gene rearrangements.
3.6 Analysis of Mutation Accumulation Sequence
Another aspect of the materials, methods, and kits diclosed relates to the essential need to make greater use of the detected mutations. This aspect involves delineating a time course of detectable mutation accumulation in addition to simply noting the presence or absence of one or more mutations.
Mutation accumulation is also important to the diagnosis, prognosis, and treatment plan of a patient with an anomaly. The time course of mutation acquisition is currently not part of any system of pre-cancer or cancer diagnosis, classification, or characterization. Currently, the emphasis is entirely upon cataloging the presence of specific mutational alterations without regard for their temporal occurrence in relationship to each other. For example, DNA expression arrays and proteomic analysis of tumors makes no attempt to align the detected alterations in terms of a time line of sequential acquisition. Time course considerations are overlooked, because there currently are no methods available for use with clinical tissue specimens that would permit such a delineation to be obtained.
Temporal sequence of mutation acquisition can, however, exert a dramatic effect upon cancer diagnosis and prognostication. This is exemplified by mutational acquisition in gliomas. Gliomas that acquire deletion of chromosome 1p and/or 19q manifest a greater sensitivity to chemotherapy with longer survival. The mechanism to account for this phenomenon is unclear, but may be related to mutational damage to DNA repair genes situated at 1p/19q that are involved in glioma tumorigenesis but are damaged with respect to nucleic acid repair. As a consequence of the damage, perhaps the chemotherapy is more effective. Thus, if the 1p/19q mutation is the first mutational change acquired, all subsequent glioma cells clonally derived from the affected cells will bear this mutational alteration. Although other mutational events may occur during the clonal expansion of tumor cells, the 1p/19q mutation will be essentially locked into all glioma cells. Treatment targeting the 1p/19q mutation may be expected to dramatically affect the entire neoplasms. On the other hand, if the 1p/19q deletion is acquired later in the time course of mutation acquisition, a significant component of the glioma will lack this alteration and may be able to resist chemotherapy. This may be the basis for recent observation that not all gliomas bearing a 1p/19q deletion show favorable treatment responsiveness.
Methods and materials for enhancing DNA amplification can be used in combination with materials and methods to determine the time course of mutation acquisition. The approach is based on the established concept of clonal expansion of phenotypically more aggressive tumor cells. Clonal expansion is a unidirectional process replacing precursor neoplastic cells with a dominant tumor cell population of cells containing progressively more mutations.
The first approach is carried out in tissue section using a microdissection genotyping technique. Thus, this aspect can be performed on a patient who has provided both a biological fluid sample as well as a tissue sample from which both types of data can be provided or based only a tissue sample. This approach may not be feasible for all fluid samples or in specimens composed of microdissected cells. Mutations present over a wider area of tumor are representative of having been acquired earlier in development of, for example, a breast cancer. Mutations present more focally are acquired generally more recently acquired by the anomaly.
Quantitative determination of allelic imbalance mutation or other forms of mutation are arranged according to the degree of relative DNA involvement. Mutations with greater DNA involvement can be assigned earlier temporal acquisition, while those with relatively less involvement can be defined as occurring later in time. By greater “DNA involvement” is meant a greater proportion of the DNA is subject to mutational change for a particular marker. Other marker mutations involving a greater proportion of the same DNA occur earlier in time, while those occurring later in time involve a lesser proportion of the target DNA.
Clinical validation for these concepts was demonstrated in gliomas classified according to the timing of 1p/19q deletion (Table 13). The latter has been associated with better treatment responsiveness. However, this is not true for all patients. The subset of patients having acquired 1p/19q early with telomeric involvement of 1p by deletion showed significantly better treatment responsiveness. These experiments were performed on tissue specimens, but the same analysis can be applied to fluid or cytology specimens alone or in combination with tissue samples.
Determination of the temporal sequence of mutation acquisition has not previously been effectively performed on fixative-treated clinical specimens or in a clinical context. Delineation of the time course of mutation acquisition is provided herein and is important to the diagnosis, prognosis, and treatment of a patient's disease. The identical profile of mutations between different individual patients with the same microscopic form of cancer may not be expected to behave in exactly the same manner. The order by which the mutations are accumulated may greatly influence the final biological behavior of the cancer. This is especially true for response to treatment where earlier acquisition of treatment responsive mutations will be associated with greater therapeutic responsiveness in the treated patient due to the presence of those mutations. For example, it is shown that gliomas which acquire the 1p/19q deletion earlier in tumorigenesis are more responsive to treatment than gliomas that do not have deletions early in tumorigenesis (see Table 16).
Assuming a model of allelic loss with minimal non-neoplastic cell DNA inclusion, the percentage of mutated DNA may be determined for each marker. When two or more mutations, (e.g., K-ras-2 and/or other allelic imbalance mutations), are detected, their time course of accumulation is inferred by the proportion of total DNA manifesting the alteration. In the case of K-ras-2 oncogene point mutation, involvement of 100% of cells is considered to be present when the intensity of the mutated base on sequencing is equal to or greater than the normal sequence base pair. This can be determined qualitatively by visual comparison and/or more precisely by quantitation. Alternatively, any method that provides relative amounts of mutated cell population can be used.
During the final evaluation of all the data from the methods, the presence of mutational change must first be analyzed with respect to the quantity and quality of DNA. It is important to do this analysis of mutational change, because the presence of low amounts of poor quality DNA can produce a false-positive detection of deletion-type mutations due to a phenomenon in the PCR reaction called allelic drop-out.
These analytical methods may be used to diagnose cancer, pre-cancerous states or non-neoplastic conditions. The analytical methods may also be used to determine the prognosis for a patient suffering from cancer, pre-cancerous state, or non-neoplastic condition. The analytical methods may be further used to determine a course of therapy or combination of therapeutic modalities for a patient suffering from cancer, a pre-cancerous condition, or an alternative neoplastic condition, as discussed herein. Because the methods allow for sensitive prediction of cancer and pre-cancerous conditions, a physician or other of skill in the art can use the methods to more accurately determine the prognosis of the condition. Furthermore, if a physician has advanced warning that cancer is present, treatment may be begun earlier and more carefully tailored to the condition (including surgery, radiation therapy, chemotherapy, and biologic therapy). To this end, the physician skilled in the art will be familiar with diagnosis, prognosis and treatment of breast anomalies. See, for example, D
The insight gathered through temporal sequence of mutation acquisition analysis of acquired mutational damage was used to create a molecular classification of gliomas (
3.6.1. Quality Control Measures
Quality control measures to monitor the deletion expansion analysis are provided which consist of recommendations for replicate analysis at both the specimen level (sample replication) as well as replication of the individual marker results (marker replication) so that it may be most effectively applied to clinical specimens.
As in the case of all clinical analysis, it is important to have quality control measures in place to ensure that recorded results are accurate and reproducible. There are multiple layers of quality control to monitor analytic precision. First, the taking of multiple topographic samples provides a means for separate sample validation (
A preferred method for analytical validation can be achieved by comparing the molecular analysis from biopsy sampling of a particular tissue or organ to that obtained from analysis of microdissected samples from the resected specimen. A high degree of concordance should be evident.
4. Data Acquisition, Compilation, and Weighting
As data is obtained from the methods described herein, it can be further assessed via computer algorithms that weight the data obtained by the methods discussed with other risk factors. Fields of data addressed by such an algorithms include, but are not limited to: case number, accession date, collection date, name of physician, name of patient, social security number of patient or other identifier, information about the patient such as age, date-of-birth, any other person to be copied on the data report, patient history, including information from pathology report, ICD-9 code, pre-operative diagnosis, post-operative diagnosis, information on any procedures performed, and the final diagnosis. Other fields considered by the algorithm include materials received relevant to the patient, such as stained and blank slides and the pathology report itself, as well as comments regarding tissue sections or fluid provided for analysis. Such comments may include information as the microdissection and comments from the pathologist, as well as to allelic imbalance in each target area and mutation time course.
Using breast cancer as an example, the risk factors can be weighed and placed within a relational database. The data by the computer algorithms can include, but are not limited to patient age, reproductive history of the patient (e.g., age at menarche, parity and age at first birth, age at menopause, and use of exogenous estrogens), family history (e.g., age at diagnosis, laterality of breast cancers to be confirmed with pathology reports when possible, presence of other cancers especially ovarian cancer, and the number of unaffected relatives), prior breast biopsies (e.g., number and histologic diagnoses of those biopsies), alcohol intake, smoking, diet, prior cancer diagnosis, cancer recurrence, and exposure to ionizing radiation.
Additionally, as data is acquired and a patient is staged according to a commonly accepted staging system, that data again can be added to the data obtained by the methods herein, and the compiled data weighted to provide the treating physician with a prognosis and treatment plan for the patient. Most staging for breast cancer involves analysis of the primary tumor (ranking of Tx, T0, Tis, and T1-T4), regional lymph node involvement (which can involve clinical analysis (N) and/or pathologic analysis (pN)), distant metastases (M), and stage grouping (i.e., TMN) of the prior factors.
EXAMPLESAlthough the present materials, methods and kits have been described in detail with reference to examples below, it is understood that various modifications can be made without departing from their spirit, and would be readily known to the skilled artisan.
Example 1A 57 year old woman was found to have a right pleural effusion and a possible right lower lobe mass on chest X-ray. A portion of the aspirated pleural fluid was prepared for cytology examination. Scattered clusters of epithelial cells were judged to have atypical cytologic features and were designated as atypical. A definitive diagnosis of cancer could not be made with certainty. Mutational analysis was performed in search of the presence and cumulative amount of mutational change.
Materials and Methods. The cover slip from the cytology slide was removed and two microdissections were performed termed here “standard” and “new.” One microdissection was performed using standard microdissection and standard PCR conditions, while the other was performed with new modifications described in detail as follows (referred to as “new microdissection”).
“Standard microdissection” was performed under stereomicroscopic observation (Olympus SZ-PT) and using a # 11 scalpel (Feather Scalpels). Clusters of cytology cells were removed and placed in 50 μL 10 mM Tris-HCl, pH 7.
The “new microdissection procedure” was performed under stereomicroscopic observation (Olympus SZ-PT) and using a #11 scalpel (Feather Scalpels). Clusters of cytology cells were removed and placed into 50 μL of 0.5% NP-40, as well as 100 mM NaCl; 25 mM EDTA, pH 8.6; and 10 mM Tris, pH 8.3 (final composition pH can be anywhere within about 7.0 to about 8.6 and every 0.1 value in between that range). The buffer was prepared using deionized, filtered water. Microdissected cells were added to a point of ideal turbidity. Ideal turbidity was reached when particulate material was clearly present, however it was just possible to see through the turbid fluid to the other side. To this ideally turbid collection of cells, proteinase K was added to reach a final concentration in the collection tube of 2 mg/mL proteinase K. The tube was then incubated at 37° C. for two hours. After this, the collection tube was placed in boiling water for 5 minutes to deactivate the proteinase. The tube was then centrifuged at 5,000 rpm for 10 minutes in a table-top centrifuge.
Standard Nucleic Acid Amplification Procedure. One microliter aliquots of the standard microdissected and collected cells were transferred into a 200 microliter tube containing 12.5 μL PCR buffer (GeneAmp kit, Perkin-Elmer-Cetus, Norwalk, Conn.), deoxyribonucleic acid and specific oligonucleotide primers (0.1-1.0 μM concentration) for the following panel of microsatellite markers:
Discrimination between reactive versus neoplastic proliferation: D1S1193, D1S407, D3S2303, D3S1539, D5S592, D5S615, D9S254, D9S251, D10S1173, D10S520, D17S974, D17S1289, D17S1161, D21S1244, D22S532 Specific primer sequences for these microsatellites are set forth in Table 14.
New Nucleic Acid Amplification Procedure. A 12.5 μL PCR reaction was prepared as for the standard PCR procedure. One microliter aliquots of the new microdissected and collected cells were transferred into a 200 microliter tube containing PCR buffer (GeneAmp kit, Perkin-Elmer-Cetus, Norwalk, Conn.), deoxyribonucleic acid, and specific oligonucleotide primers for the microsatellite markers as described above. The aliquots were pipetted from just above the centrifuged pellet. Magnesium chloride was added to reach a level of 8 mM. Then sucrose was added to reach a concentration of 12 gram percent.
At this point all subsequent steps were identical between the standard and new amplification methods for broad panel marker analysis. PCR amplification was performed under the same cycling condition of 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 2 minutes. Forty cycles of amplification were performed. The products of amplification were mixed with deionized formamide and electrophoresed using capillary electrophoresis (ABI GeneScan 3100, Applied Biosystems, Mountainview, Calif.).
Results. All reactions run under standard recommended conditions without MgCl2, sucrose, nonionic detergent, and proteinase K. Use of the standard conditions described above either failed to yield a detectable amplicon or produced significant allelic imbalance due to allelic dropout, because of inadequate amounts of DNA for effective amplification (Tables 15 and 16). All reactions run using the “new procedure” with the use of MgCl2, sucrose, a nonionic detergent and proteinase K produced large amounts of amplicon and reproducible results (Tables 15 and 16).
Amplification reactions run under standard PCR conditions failed to show evidence of polymorphic alleles (Table 15). Clearly, the amplifiability of the DNA was such that there was insufficient amounts of template for effective amplicon production. These reactions were also extended in cycle number, and yet these failed to generate detectable allele band product.
As seen in Table 15, there is no detectable amplicon. It is the failure to detect the amplicon that demonstrates the conditions used for this example were inadequate for this specimen. In contrast, the sample treated with the “new” protocol showed excellent amplification of product (Table 15).
In a few instances, such as with the tetranucleotide marker D5S592, amplification product was present. However, there was evidence of allele dropout with replicate analysis varying widely in degree of imbalance as depicted in Table 15. Thus, the results using the standard procedures could not be trusted from such operating conditions. In contrast with the new protocol, replicate analysis shows excellent concordance of the results (Table 15).
When the buffer comprising proteinase K and a nonionic detergent (NP-40) was used in combination with amplification in the presence of MgCl2 and sucrose, abundant product for polymorphic markers were produced. At the same time, replicate analysis showed very little variation in the ratio of peak heights indicating good reliability (Table 15).
At this point, it was decided to evaluate each of the components used to enhance amplifiability to establish their individual contribution. First, three different concentrations of magnesium chloride were used in the PCR reaction consisting of 2 mM, 8 mM, and 12 mM. From Table 16 it is clear that accurate and robust amplicon production was achieved with 8 mM MgCl2 (Table 16). No product was detected with either the 2 mM or 12 mM MgCl2 concentrations (top and bottom panels respectively).
1The values present in the boxes represents relative fluorescence measured by capillary electrophoresis.
1The values present in the boxes represents relative fluorescence measured by capillary electrophoresis.
A 60 year old male with severe heartburn underwent an upper GI biopsy of the esophagus and was found to have invasive esophageal adenocarcinoma (
The question presenting to the diagnosing pathologist was whether the lung nodule represented a metastasis of the prior esophageal cancer or a de novo lung cancer. If the lung cancer was de novo, the patient would be treated for the lung cancer and not for metastatic disease. The patient still would be considered as disease-free for the esophageal cancer. Immunohistochemical stains of the aspirated lung material were not helpful in clarifying this issue.
Materials and Methods. The identical methods used in the “new” procedure in case 1 was used here with the following changes. The following markers were used: D18S814 and k-ras-2 point mutation. The primer sequences used were as follows:
The primers are provided in a 5′ to 3′ direction.
The mutational fingerprint of the each tumor, generated by broad panel PCR and capillary electrophoresis, which was performed as discussed in Example 1, showed a totally discordant profile of mutations between the two tumors including the specific alleles affected (red versus blue for the different copies). The panel also showed a different accumulation of mutations temporally. Therefore, the tumor was diagnosed as a de novo lung tumor. The patient was successfully treated for de novo lung cancer and remains disease free.
Because the biopsy and the fine needle aspiration of the lung are both minute in size, the novel advances described herein was essential to successful resolution of this issue in this patient.
Example 3 A 55 year old woman underwent fine needle aspiration (FNA) of a pancreatic head region mass after work-up for weight loss (
Materials and Methods. The identical methods used in the “new” procedure discussed in Example 1 supra was used for this example with the following differences. Tissue sections were microdissected from unstained four-micron thick paraffin sections. Individual clusters of pancreatic cytology cells were microdissected for mutational analysis to discriminate between reactive, benign, and malignant causes of the pancreatic mass lesions (
A 48 year old male with longstanding alcohol overuse was found in a workup for recent onset diabetes mellitus to have a pancreatic cyst (
Materials and Methods. The identical methods used in the “new” procedure in Example 1 above were used here with the following additional steps. The pancreatic cyst fluid underwent DNA extraction by running 200 μL of the cyst fluid through a Qiagen column. The isolated DNA was quantified by optical density (OD) measurement (Nanodrop technique).
Notwithstanding the very small amount of DNA present in each PCR reaction (i.e., about 5 ng/μL), PCR was performed on 1 μL aliquots of the extracted DNA from the pancreatic cyst fluid. A 12.5 μL PCR reaction was prepared as for the standard PCR procedure. One microliter aliquots of the new microdissected and collected cells were transferred into a 200-μL tube containing PCR buffer (GeneAmp kit, Perkin-Elmer-Cetus, Norwalk, Conn.), deoxyribonucleic acid, and specific oligonucleotide primers for the following panel of microsatellite markers. The oligonucleotide primers and panel of markers utilized are as described in the examples above. Magnesium chloride was added to reach a level of 8 mM. Then sucrose was added to reach a concentration of 12 gram percent. PCR amplification was performed under the same cycling condition of 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes. Forty cycles of amplification were performed. The products of amplification were mixed with deionized formamide and electrophoresed using capillary electrophoresis (ABI GeneScan 3100, Applied Biosystems, Mountainview, Calif.) as described above. A detailed mutational profile was obtained by capillary electrophoresis and indicated the presence of 5 allelic imbalance mutations and point mutation of the k-ras-2 oncogene (
Thus, six mutations were sufficient for a molecular pathology diagnosis of pancreatic cancer even in the face of inadequate cytology. The pancreas was resected and a small focus of early pancreatic cancer was discovered in one part of the cyst wall (
A 65 year old woman was found to have a solitary pancreatic cyst in the tail of her pancreas. Fine needle aspiration was performed at three time points separated by six month intervals (
Materials and Methods. The methods described referred to as “new” procedure in Examples 1 and 4 were used here with the following additions. The quality of DNA was assessed by quantitative PCR for k-ras-2 exon 1 production (Icycler, Biorad). Sample optical density amount of DNA concentration (8.63 ng/μL) was adjusted to a value of about 5 ng/μL with water. The number of qPCR cycles may be used as a marker of DNA quality. A lower the number of cycles required to reach a desired threshold is indicative of higher quality DNA. In general, if over 30 qPCR cycles are required, then the DNA quality is considered suboptimal due to, for example, allelic imbalance resulting from inadequate amounts of template DNA. Specifically, Ct values (i.e., threshold values for quantitative PCR product detection) over 30 cycles is considered evidence of poor quality of DNA, especially if the DNA quantity present is above about 2.0 ng/μL. Ct values of about 29-30 are considered borderline and representative of poor quality DNA. Cycle values of 29 or less are indicative of good quality DNA and are generally characteristic of the breast anomaly being malignant. Additionally, values of 29 or less may indicate the presence of neoplasia. The lower the Ct value, the more likely neoplastic cell proliferation is malignant. These values may vary based on the conditions of the assay and amounts of DNA employed. Cancers that proliferate slowly, are well differentiated in growth pattern, and are relatively less cellular may be expected to show borderline Ct values that are significantly higher than 29-30 cycles. The PCR reaction was performed as described above with the addition of 1 microliter of sybr green for signal detection.
Results. Cytology examination of each sampling was interpreted as insufficient for diagnosis. Broad panel mutational analysis revealed valuable discriminating information over the course of the three aspirations (
Two patients with advanced cirrhosis were awaiting autologous liver transplantation. Both patients developed two nodules. For both patients, the nodules were in the right and in the left lobes of the liver. The nodules were highly suspicious for hepatocellular carcinoma (HCC). Each patient underwent liver biopsy of both their tumor deposits as a basis for stratification for receipt of a liver transplant.
Materials and Methods. The methods were identical to those described in Example 1 with the new lysis and amplification procedure. The same primers and panel of markers were utilized for obtaining a molecular analysis. The “new” procedure and materials were also utilized to enhance DNA amplifiability.
Results. The data on the patient depicted in the upper portion of
The patient's data depicted in the lower panel of
A 75 year old man was found to have both a squamous cell carcinoma of the anterior floor of mouth and the base of tongue (
Materials and Methods. The methods were identical to those described in Example 1 with the new lysis and amplification procedure. The same primers and panel of markers were utilized for obtaining a molecular analysis. The “new” procedure and materials were also utilized to enhance DNA amplifiability.
Results. The mutational profiles of all three deposits of cancer were determined. It was clearly shown that the cervical lymph node metastasis was from the base of tongue tumor and not the anterior oral cancer. Moreover, the two oral cancers could be shown to be independent, primary cancers. The full molecular pathogenesis of multifocal oral cancers was defined based upon the novel insights defined in this application.
Example 8A 27 year old man had blood in stool and found to have a large cecal intestinal mass, the presence of which is highly suspicious for colon cancer. Biopsies of the mass were obtained, with some pieces showing adenocarcinoma. At issue was whether this patient might have an inherited DNA repair gene mutation causing cancer susceptibility. Family history was not possible, because the patient was adopted and had no knowledge of his natural parents.
Materials and Methods. The methods and materials were identical to those described in Example 1 with the new lysis and amplification procedure.
Results. By searching for microsatellite instability on microdissected targets of the endoscopic cecal biopsies, it was possible, using PCR/capillary electrophoresis as described above, to determine the presence of instability. This in turn enabled the surgeon to resect not only the area of cancer, but also the entire colon save the rectum given the high risk for development of a second colon cancer. The patient was greatly helped with significant cost savings to the healthcare system by the ability to detect microsatellite instability in tiny biopsy specimens.
Example 9A 56 year old woman was found to have approximately 50 adenomatous polyps of the colon during a surveillance colonoscopy. Ten of the polyps were removed for microscopic examination and confirmed to be small, early adenomatous polyps. At issue was whether the patient had an inherited cancer susceptibility syndrome, such as familial polyposis, to account for these numerous polyps. Family history was unproductive and the patient lacked health insurance and the financial resources to have the extensive gene sequencing performed to make a determination of the presence of an inheritable syndrome.
Materials and Methods. The materials and methods were identical to those described in the prior examples.
Results. Using PCR/DNA sequencing, three intragenic single nucleotide polymorphisms (SNPs) of the APC gene were analyzed for allelic imbalance. In addition, several microsatellites in proximity to the APC gene were also evaluated using PCR/capillary electrophoresis (as described in Example 1 above) or fragment length determination. Two of the three SNPs were found to be informative. Only the SNP for exon 15 was found to be informative. SNPs have the advantage that they are more numerous across the genome than microsatellites but the major disadvantage is that they are not as polymorphic. The single base wobbles between two alteratives, say T or G, meaning that the possibilities in the population are TG, TT, or GG. Thus, one cannot obtain more than 50% of the population being informative (TG) for any one SNP.
Specifically, the SNPS showed loss of heterozygosity with the same direction of imbalance between different alleles in all the polyps. This provided a very high degree of certainty that the APC gene carried a constitutional, germ-line mutation, which was causing the reproducible loss in all neoplastic lesions. Thus, without DNA sequencing, the mutation was detected and the presence of an APC gene germ-line mutation was established. At the same time, other potential mutated genes need not be viewed as the fundamental cause of cancer susceptibility. As for all examples here, this critically depended upon the unique insights provided in this application enabling minute fixative treated biopsy specimen to more effectively amplify for multigene/multimarker analysis.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
The application claims priority of U.S. Provisional Applications Nos. 60/620,926 filed Oct. 22, 2004; 60/631,240 filed Nov. 29, 2004; 60/644,568 filed Jan. 19, 2005; 60/679,969 filed May 12, 2005; and 60/679,968 filed May 12, 2005, all of which are herein incorporated in their entirety for all purposes. Also incorporated by reference in their entirety for all purposes are U.S. Patent Applications filed Oct. 24, 2005 entitled “Molecular Analysis of Cellular Fluid and Liquid Cytology Specimens for Clinical Diagnosis, Characterization, and Integration with Microscopic Pathology Evaluation” and “Dynamic Genomic Deletion Expansion and Formulation of Molecular Marker Panels for Integrated Molecular Pathology Diagnosis and Characterization of Tissue, Cellular Fluid, and Pure Fluid Specimens”.
All references cited above are incorporated herein in their entirety for all purposes.
Claims
1. A kit for amplification of a nucleic acid from a sample with low nucleic acid concentration comprising: (a) a buffer comprising a nonionic detergent; (b) a proteinase; (c) XCl2, wherein X is magnesium or manganese; and (d) sucrose; and wherein XCl2 and sucrose when admixed with the sample for amplification of DNA have a final concentration of about 5 mM to about 10 mM XCl2 and about 5 gram percent to about 15 gram percent sucrose.
2. The kit of claim 1, wherein the sample is fixative-treated, fixative-treated and stained, a liquid cytology, or a liquid biological sample.
3. The kit of claim 1, wherein the nucleic acid is DNA.
4. The kit of claim 1, wherein the nonionic detergent is Nonidet P40 (NP-40), Tween, Triton X, or Nikkol.
5. The kit of claim 4, wherein the nonionic detergent is present in the amount of about 0.1% to about 3.0%.
6. The kit of claim 5, wherein the nonionic detergent is Nonidet P40.
7. The kit of claim 4, wherein the nonionic detergent is Nonidet P40 and is present in an amount of about 1.0%.
8. The kit of claim 1, wherein the proteinase is proteinase K, pronase, subtilisin, thermolysin, papain, or a combination thereof.
9. The kit of claim 8, wherein the proteinase or proteinase combination is present in the amount of about 0.1% to about 3.0%.
10. The kit of claim 9, wherein the proteinase is proteinase K.
11. The kit of claim 8, wherein the proteinase is proteinase K and is present in an amount of about 1 mg/mL to about 5 mg/mL.
12. The kit of claim 11, wherein the proteinase K is present in the amount of about 2 mg/mL.
13. The kit of claim 1, wherein XCl2 is MgCl2.
14. The kit of claim 1, wherein XCl2 is MgCl2 and is present in the amount of about 6.0 mM to about 10.0 mM.
15. The kit of claim 14, wherein the MgCl2 is present in the amount of about 8.0 mM.
16. The kit of claim 1, wherein the sucrose is present in the amount of about 12 gram percent.
17. A method for amplification of DNA of a sample with low DNA concentration comprising:
- (a) combining a sample of cellular containing material and an aqueous buffer comprising a nonionic detergent;
- (b) adding a proteinase to the buffer and the sample and incubating the buffer, the sample, and proteinase at a temperature suitable for proteinase digestion of protein;
- (c) deactivating the proteinase;
- (d) adding sucrose to the sample being amplified to a final concentration of about five gram percent to about 15 gram percent; and
- (e) adding XCl2 to the sample being amplified to a final concentration of about 6.0 mM to about 10.0 mM.
18. The method of claim 17, wherein about a 1 μL to about a 5 μL sample is amplified.
19. The method of claim 17, wherein the buffer further comprises sodium chloride, EDTA, and Tris.
20. The method of claim 17, wherein the sample is fixative-treated, stained, fixative-treated and stained, a liquid cytology, or a biological liquid specimen.
21. The method of claim 17, wherein the nonionic detergent is Nonidet P40 (NP-40), Tween, Triton X, or Nikkol.
22. The method of claim 21, wherein the nonionic detergent is NP-40 and is present in the amount of about 1.0%.
23. The method of claim 17, wherein the proteinase is proteinase K, pronase, subtilisin, thermolysin, papain, or a combination thereof.
24. The method of claim 23, wherein the proteinase is proteinase K and is present in an amount of about 1.0 mg/mL to about 5.0 mg/mL.
25. The method of claim 24, wherein the proteinase K is present in the amount of about 2.0 mg/mL.
26. The method of claim 17, wherein said XCl2 is MgCl2 or MnCl2.
27. The method of claim 26, wherein the XCl2 is MgCl2 and is present in the amount of about 5.0 mM to about 10.0 mM.
28. The method of claim 17, wherein sucrose is present in the amount of about 12.0 gram percent.
29. The method of claim 17, wherein the proteinase is proteinase K and is present in the amount of about 2 mg/mL; the sucrose is present in the amount of about 12.0 gram percent; the nonionic detergent is nonidet P-40, which is present in the amount of about 1%; and XCl2 is MgCl2 and is present in the amount of about 8.0 mM.
Type: Application
Filed: Oct 24, 2005
Publication Date: Jun 1, 2006
Inventors: Sydney Finkelstein (Pittsburgh, PA), Patricia Swalsky (Pittsburgh, PA)
Application Number: 11/255,980
International Classification: C12Q 1/68 (20060101); C12P 19/34 (20060101); C12N 1/08 (20060101);
