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REVIEW ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 2  |  Page : 209-216
Molecular pathology for the general pathologist: Second oration of the Indian college of pathologists


Department of Pathology, St. Georges University School of Medicine, St. Georges, Grenada, West Indies

Click here for correspondence address and email

Date of Web Publication12-Jun-2010
 

   Abstract 

Molecular diagnostic tools form a necessitity in the modern practice of pathology. This review aims to introduce some basic principles of molecular diagnostics to a pathologist involved in the practice of histology. Some common clinical scenarios and the application of molecular techniques in those settings to obtain an accurate diagnosis is discussed.

Keywords: Diagnosis, histopathology, molecular pathology

How to cite this article:
Bhusnurmath SR. Molecular pathology for the general pathologist: Second oration of the Indian college of pathologists. Indian J Pathol Microbiol 2010;53:209-16

How to cite this URL:
Bhusnurmath SR. Molecular pathology for the general pathologist: Second oration of the Indian college of pathologists. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Nov 21];53:209-16. Available from: http://www.ijpmonline.org/text.asp?2010/53/2/209/64322



   Introduction Top


Most of us, as general pathologists, are apprehensive of the area of molecular pathology. It conjures images of 'high tech labs', in depth knowledge of chemistry, a high degree of research experience and a tough task of discovering new genes. Most of the middle level and senior pathologists are trained in pathology when molecular pathology was either nonexistent or in its infancy. Hence they are reluctant to talk about it, use it in practice or teach it to postgraduates and medical students. Molecular Pathology, however, plays a pivotal role in the current practice of medicine in diagnostic, screening and therapeutic applications to the extent that it would be unsafe for any medical doctor, let alone a pathologist to be ignorant of the common applications. One need not be an actual molecular pathologist to understand and apply its practical utilities in the day to day practice of medicine. This article has used a couple of common clinical scenarios to illustrate the basic applications and, in the process, explain a little bit about the techniques themselves without making it too technical. It is hoped that this would encourage a friendlier attitude towards the topic among those who are uninitiated so far.

Brief Outline

There are different levels at which a diagnosis can be made by a pathologist; like gross anatomic diagnosis, histopathology, cytology etc. We can take this one step further when needed to study the abnormalities in deoxyribose nucleic acid (DNA), Ribose nucleic acid (RNA) or proteins in a cell. [1] This is what is molecular pathology in simple terms. One does not have to be a research scientist or possess a highly sophisticated research laboratory to do this. There are so many simple and well established techniques that can be performed at a low cost that are essential for the current practice of medicine. It is mandatory for every practicing pathologist, if not every graduating medical doctor to be aware of them.

The material for the study of DNA, RNA or proteins can be obtained from a variety of sources like intact tissues or cells from fresh tissue, frozen tissue, paraffin embedded tissue, blood cells, saliva, semen etc. At the outset, it must be emphasized that no test result should be interpreted in isolation. All the data, including clinical features, and all the laboratory data must be considered together to make the final diagnosis. Chromosomal analysis has been in use for a long time but many of the damages to the DNA that result in disease are far too subtle to be detected by this technique. The DNA molecules are very stable and can survive for a long time without much change. A disease may result from just one molecule in the DNA being changed. Studying the DNA for such changes is some times called as "gene biopsy". The applications of molecular pathology encompass several useful situations like diagnosis of inherited disease, prediction and diagnosis of neoplastic disease, prognosis of a given neoplasm, transplantation, infectious disease, identity and paternity testing etc.

Illustrative Cases

The following cases illustrate some of the techniques in common usage.

Patient 1

A middle aged female who was a nonalcoholic complained of constant tiredness. She had a family history of liver disease of unknown nature. Physical examination revealed a slightly enlarged liver and mildly elevated transaminases. The serum transferin saturation and the serum ferritin were elevated. A hereditary overload of iron, namely hemochromatosis was suspected. The liver biopsy confirmed excess iron deposition of iron in the hepatocytes [Figure 1]. There are several situations in which iron can accumulate in the liver. There was a need to look for any genetic basis for the disease.

It is well established that the majority of the patients with hemochromatosis have a genetic mutation (HFE gene) in the DNA fragment near the human leucocyte antigen (HLA) zone. This chain has a length of 387 base pairs. The test to investigate the mutation uses some specific enzymes secreted by bacteria, called restriction enzymes. These can read the DNA sequence and break the chain at specific points. The enzymes are commercially available. If one exposes the particular normal 387 bp chain of HFE gene to a restriction enzyme called Rsal, the enzyme splits the normal fragment in to two fragments of 247 and 140 base pairs each. However, if there is a mutation (C282Y mutation), the same enzyme splits the chain in to 3 fragments of 247, 111 and 29 base pairs each. There is another much rarer mutation in hemochromatosis (H-63D mutant) in which another fragment of DNA, which is 208 bp in length, is mutated [Figure 2]. In this normal gene when you expose it to another restriction enzyme called Mbol, it splits it in to two fragments of 138 and 70bp. If there is a mutation, the Mbol fails to split the chain and the result is still a chain of 208bp. Since the length of the fragments obtained varies, the term restriction fragment length polymorphism-RFLP has been applied to this phenomenon. All these features have been well established. One does not have to reinvent the wheel. Thus to look for the gene, one has to extract DNA from the patients cells, expose the selected fragment of the DNA to these two restriction enzymes and then look for the lengths of the resultant fragments. The dilemma is that the quantity of the DNA fragments thus produced is very small and is difficult to detect. We need to amplify this product to make it easy to detect. This is achieved by a process called polymerase chain reaction (PCR).

The principle of PCR is very simple. The selected DNA fragment is used as a template to build multiple copies. The other ingredients added are the primers (short strands of DNA synthetically made, commercially available and inexpensive), dTNPs (which are the building blocks), taq polymerase enzyme and a buffer. All these are put in a test tube and the test tube goes in to a small machine. The machine alternatively heats and cools the test tube. Each time it is heated the DNA fragment splits into the two duplicate strands. When it is cooled, the rest of the building blocks under the action of the polymerase enable the complementary copy of DNA strand to be made along each existing strand. Thus, with each heating and cooling cycle, multiple copies of DNA are made. The amplification is exponential. Theoretically, by 35 cycles there should be 34 billion copies although in reality the number would be less. In a matter of few hours there would be a large quantity of the DNA fragments which would make it easy to detect. How do we know if this fragment is normal or abnormal?

The product has to be matched with DNA of known molecular weight or base pairs- positive and negative controls. The contents of the test tube are poured over a slot on glass slide with agarose gel spread over it. A buffer is also poured to make the movements of the molecules easy. An electric current is applied. The DNA molecules move from the positive to the negative pole. The distance moved is proportional to the molecular size. Smaller molecules move faster and farther. A little dye can then be added to visualize the bands of the proteins. The size of the molecules is compared to the positive and negative controls to get the correct reading example. if it matches with the 247 bp positive control, the molecule is also 247 bp in size. A variant of this technique using polyacrylamide gel instead of agarose gel can be performed. The process is called gel electrophoresis. The dye used most commonly is ethidium bromide (EtBr). It binds to the DNA and is easily visible under ultraviolet light. One has to ensure that appropriate positive and negative controls are used to get accurate results.

The PCR technique has been further automated into what is called real time PCR in which multiple fluorescent dyes are labeled to the probes and through laser recordings on a computer, the results are ready as a print out in 15 to 20 minutes. This yields better quantifications and has wider applications.

The patient under discussion was detected to have a c-282Y mutation by PCR after using the restriction enzyme Rsal [Figure 3]. There was negative result for the H-63 mutation. The liver biopsy revealed extensive deposits of iron with histological evidence of cirrhosis. The defective genes had led to excessive iron absorption and deposition in various organs like liver, heart and joints. The frequency of hemochromatosis is 1 in 200 in the general population in the US but the incidence in India is not clear. The detection of the mutant gene in the patient made it mandatory for the family members to be screened as it runs in families. It would then make it possible for measures to be taken to prevent actual tissue damage.

This patient illustrated an example of the use of molecular pathology in the diagnosis of inherited disease. Some other inherited diseases that can be screened like this include cystic fibrosis, sickle cell disease, hemophilia, Huntington's disease, Fragile X syndrome, neurofibromatosis etc. The process of PCR is generic and applied in many situations for the detection of DNA mutations.

Patient 2

A 34-year-old female had come for a routine check up. She had a strong family history of cancer. Her mother had breast cancer at the age of 36 years, the paternal grand mother was diagnosed with breast cancer at the age of 57 years, and the paternal grand parents also had died of prostrate cancer and melanoma respectively. She was worried about getting cancer. [3]

Most cases of breast cancer are sporadic while some are a result of an inherited susceptibility gene. This is suggested by the occurrence of cancer spanning several generations, younger age at onset, bilateral tumors and the development of a second cancer in an individual. In our patient, one needs to look for the genetic mutations of BRCA 1 and BRCA 2. [2] If present, it suggests a 56% risk for breast cancer and 16% risk for ovarian cancer.

The BRCA1 is located on chromosome 17 at band 17q21 and BRCA 2 on chromosome 13 at band 13q12.3. There is a strong difference between these mutations and the one in hemochromatosis. In BRCA it is not one mutation but a collection of over 200 mutations, any one of which can lead to the cancer susceptibility. These are tumor suppressor genes. If one had to screen for each of the 200 mutations, one will have to perform 200 separate PCRs with 200 different restriction enzymes and probes. This would not be practical. Hence the DNA fragment is isolated and the selected fragment is sequenced in an automated fashion. [4] The reaction mix contains PCR fragments of different lengths each terminating in a dideoxynucleotide bound to a fluorescent dye. The automatically sequenced DNA is run on a polyacrylamide gel and detected by laser. A computer generated print out is obtained that shows each one in a different color. It can then be matched with a known data base. This technique is called automated sequencing.

Our patient was detected to have mutation in the BRCA1. This finding enabled the physician to explain the relative risks of developing breast and ovarian cancer to the patient. [5] One could explore the possibilities of prophylactic mastectomy, frequent screening for preneoplastic lesions/early malignancy and life style changes. The family members also needed to be screened for these genetic mutations.

The other mutations of tumor suppressor genes that can be screened to detect tumor susceptibility include RET, p53, APC etc. If RET is detected it indicates a susceptibility for Multiple endocrine neoplasia-MEN-2 syndrome. This indicated a high risk for future medullary carcinoma of thyroid and one may contemplate a preemptive thyroidectomy at a young age even before any evidence of actual tumor is detected.

Patient 3

A 38-year-old female presented with weight loss and blood in the stools. Investigations revealed an adenocarcinoma in the ascending colon. She was too young to have a carcinoma of the colon and the right side of the colon is an uncommon site for this tumor. Both these features led to a suspicion of an inherited genetic mutation like HNPCC (hereditary non-polyposis colon cancer). Her father was also diagnosed to have colonic cancer at the age of 42 years. The HNPCC gene related colonic cancer is characterized by younger age at diagnosis, right sided tumors, and additional tumors involving endometrium, ovary, stomach, small bowel, ureter, kidney etc. The first degree relatives also exhibit a higher frequency of these tumors.

The HNPCC mutation leads to defective DNA repair enzymes. Thus DNA damages are not repaired and mutant cells multiply leading to cancer. The abnormality is located in the segments of DNA called microsatellites. These are short repetitive segments of DNA scattered through out the genome predominantly in the noncoding regions. A mutation leads to a change in the length of the microsatellite compared to normal. This could be due to deletion or insertion. If the DNA repair enzymes are normal this would be easily corrected. If the mis-match repair (MMR) system fails to correct this during DNA replication it leads to new DNA with variable lengths of microsatellites. This is termed microsatellite instability. [6],[7] This leads to frame shift mutation in the coding region of DNA e.g. for TGF β in HNPCC.

The detection of microsatellite instability is done by amplifying the DNA for a panel of microsatellite loci by PCR from dissected tissue or on automated analyzers. It can also be done by gel electrophoresis after PCR which will show an extra band in the tumor DNA.

Our patient was positive for HNPCC. What are the implications? [8] She has an 80% life time risk of developing endometrial cancer in addition to the colonic cancer she already has. She may be considered for prophylactic hysterectomy and salphingo-oopherectomy at the time of colectomy. In her relatives who turn out to be positive for HNPCC one needs to do regular colonoscopy, colposcopy, transvaginal ultrasound and endometrial aspirations from a very young age.

The preceding cases illustrated the application of molecular pathology to screen for tumor susceptibility genes. The next ones will look at d iagnostic applications.

Patient 4

A 67-year-old man presented with chronic fatigue and anemia. Physical examination revealed generalized lymph-adenopathy and splenomegaly. The peripheral blood revealed leucocytosis. A lymph node biopsy was performed. It revealed a diffuse effacement of the architecture by monotonous looking lymphocytes. A diagnosis of non-Hodgkin's lymphoma was suspected. A molecular pathology test was needed to look for monoclonality of the cells to confirm the diagnosis. In B cell lymphomas only one clone of B cells proliferate. The IgH chain will be rearranged in one molecular size only where as in reactive lymph node it would be polyclonal. If the DNA from these lymphocytes is extracted and a PCR performed, one will be able detect all the IgH molecules of only one size because of the heavy chain rearrangement. Similarly in a T cell lymphoma the T cell receptors on the cell membrane are rearranged in a monoclonal fashion.

The DNA hybridization was described by Dr Southern and goes by the name of Southern blot technique. The principle is very simple. The extracted DNA is subjected to electrophoresis on agarose gel. The nucleic acids are separated according to their size. The gel is fragile and one needs to transfer the separated fragments to a more stable platform. A buffer is poured on the gel. A sponge is placed on top of it. A nitrocellulose paper is placed on top of the sponge. A couple of paper towels are stacked on top of the nitrocellulose paper. The papers are gently tapped. By capillary action the nucleic acid fragments are sucked from the gel and get attached to the nitrocellulose paper which is then peeled out. It is then hybridized with labeled probes. The bands are then easily detected.

If RNA is extracted instead of DNA it is called Northern blotting. Western blotting refers to protein blotting.

In this patient the paraffin tissue was subjected to DNA extraction which was tested for IgH and TCR rearrangement using appropriate positive and negative controls on acrylamide gel electrophoresis. There was a positive result for monoclonal IgH rearrangement and negative one for TCR rearrangement [Figure 4]. This confirmed the diagnosis of B cell lymphoma. [9],[10] This test can also be sued for detecting recurrence of the tumor in a very small sample of cells.

Molecular pathology has several applications in hematological malignancies including documentation of clonality, assignment of lineage, involvement of oncogenes, mutation analysis, detection of minimal residual disease or recurrence, targeted therapy etc.

Patient 5

A 52-year-old female complained of constant tiredness, fever, loss of appetite and night sweats. Physical examination revealed pallor and splenomegaly. Blood counts revealed leucocytosis with a shift to the left. Bone marrow examination revealed increased myelocytes suggesting chronic myeloid leukemia. The diagnosis was not a problem but a decision had to be taken whether Gleevic could be used for treatment. Majority of the CML patients have a 9:22 translocation (Philadelphia chromosome) that leads to the production of a chimeric fusion gene Bcr.Abl the product of which is a tyrosine kinase that is resistant to inactivation. Persistent action of the mutant tyrosine kinase leads to uncontrolled cell proliferation and leukemia. Gleevic is an inhibitor of Bcr.Abl kinase. [11],[12] It will selectively knock off leukemic cells without harming normal hematopoietic cells and hence very useful if there is this mutation. Chemotherapy with usual cytotoxic drugs would knock off the normal cells along with the leukemic cells putting the patient in danger. Thus it is important to confirm the presence of this mutation before therapy is decided.

The translocation can be confirmed by cytogenetic studies which are time consuming. Another useful procedure to look for it is called fluorescent in situ hybridization (FISH). Fluorescent dye tagged probes are used e.g. red for chr 9 and green for chr 22. They are hybridized on the nuclei of intact leukemic cells. If red and green signals are detected separately as dots then there is no translocation. If one sees a yellow dot it indicates a combination of red and green and hence the translocation [Figure 5]. One could also do a PCR with two primers and detect a single final product. Gleevic would be the treatment of choice if the translocation is detected. A similar application is used in gastrointestinal stromal tumor (GIST) with C-kit mutation where it is treated with Imatinib, which has an action just like Gleevic.

The preceding examples showed the applications using DNA. The next case illustrates the use of RNA.

Patient 6

A 32-year-old male presented with a mass in the upper right thigh close to the hip joint. The resected specimen revealed a soft tissue tumor composed of malignant spindle cells. The immunocytochemistry suggested a sarcoma but could not specify the cell of origin. The age of the patient and the location suggested a synovial sarcoma but the usual biphasic pattern was missing. It is well documented that more than 90% of synovial sarcomas have t (x:18) translocation. SSX1 is involved in the biphasic pattern and SSX2 in the monophasic pattern. [13] This translocation produces a chimeric mRNA which in turn leads to a chimeric protein. RNA is unstable unlike DNA and can not be amplified. In this patient, RNA was extracted from the tumor tissue. Reversed transcriptase enzyme was applied to generate DNA from RNA. The DNA was then amplified using PCR using primers x and 18. If there was no translocation one would see two separate end products while if there is translocation there would be a single final product. This procedure of using RNA and creating DNA is called Reverse transcriptase polymerase chain reaction- RTPCR. The patients sample demonstrated a positive result for a single final product and the diagnosis of monophasic synovial sarcoma was confirmed [Figure 6].

A similar RTPCR testing could be done in patients with chronic myeloid leukemia (patient 5) starting with mRNA of Bcr.Abl.

The next two cases will illustrate the use of molecular pathology for prognosis and treatment in tumors already diagnosed.

Patient 7

A 52-year-old female presented with a lump in the right breast. It was firm and adherent to the skin and deeper tissues. Fine needle aspiration cytology (FNAC) and later lumpectomy confirmed it to be an invasive duct carcinoma. This tumor needs to be further tested for the degree of expression of epidermal growth factor (HER 2 neu). [14],[15] Twenty five per cent of the patients of breast cancer are positive for this. The higher the degree of expression, the greater is the aggressiveness of the tumor. The protein product of the mutant gene can be measured by immunocytochemistry using labeled specific antibodies (grading 0 to 3+). The immunocytochemistry does not give any idea of the gene amplification. The gene can be looked at by hybridizing the nuclear DNA with a specific probe. The tissue is heated which will melt the DNA and then a probe is put on it. The probe binds to the amplified multiple copies of the gene. The probe is tagged to a fluorescent dye to make it easily identifiable. This is called fluorescent in situ hybridization- FISH. One could use different colored probes for the gene and the protein product and measure the degree of gene amplification and protein expression simultaneously. HER2 over expression correlates with aggressive behavior and HER 2 gene amplification correlates with shortened disease free and overall survival of node positive patients. Further they can be treated with Herceptin which is a specific antibody to HER2 protein. Herceptin binds to the receptor and prevents the transmission of the signal for the multiplication of cells.

Patient 8

A three-year-old boy presented with flu like symptoms and abdominal pain. Physical examination revealed generalized lymph adenopathy and splenomegaly. The peripheral blood and bone marrow examination confirmed the diagnosis of acute lymphoblastic leukemia. He received chemotherapy which was tolerated very well. He had to be then placed on maintenance therapy with 6 mercaptopurine. There is a danger in placing all such patients on this therapy because some do not have the enzyme to metabolize it to 6 methyl mercaptopurine, which is inactive. The routine doses accumulate, produce toxicity and kill the patient. It is mandatory to test if the patient belongs to that small subset of people with a deficiency of that enzyme-Thiopurine methyl transferase (TMPT).

The basis for this variation of the enzyme in different people is called single nucleotide polymorphism (SNP). There are approximately 1.42 million SNPs in the human genome. Most of our genes have SNPs but this does not affect their function; a few do. SNPs are major factors that influence drug response. SNPs are not mutations because they are very common and seen in more than 1% of the population. It is a simple variation or polymorphism. The SNPs are called synonymous if there is no change in the amino acids and nonsynonymous if the aminoacids change.

In this child an SNP analysis was performed using the DNA of the tumor cells, PCR and gel electrophoresis using controls of normal (wild) type and mutant types as controls. The child was detected to have the mutant type of TPMT. [16] He was placed on a very low dose of 6 mercaptopurine as the normal dose would have killed him.

SNPs lead us to the idea that in future we may be looking at a situation of individualized medicine rather than one drug suits all. [17] The variation in response to common antihypertensive drugs could also be explained on a similar basis. Molecular pathology has several applications in infectious diseases like diagnosis, epidemiological studies, and identification of unknown organisms, monitoring viral loads etc. The DNAs of infectious agents are quite different from human DNA and hence quite easy to distinguish. The next case illustrates one such example.

Patient 9

A 45-year-old male had undergone an orthotopic liver transplantation for cirrhosis. A few months later he started feeling weak. The aspartate amino-transferase (AST) and alanine amino-transferase (ALT) levels were found to be elevated. The physician had to determine if the damage to the hepatocytes was a result of rejection or a viral infection like Epstein-Barr virus (EBV) which is common in such an immune suppressed situation. The treatment for each of these is quite different and the wrong choice can kill the patient. The liver biopsy revealed extensive lymphoplasmacytic infiltration in the portal tracts [Figure 7]. In-situ hybridization was done on the liver biopsy section using EBV probe with a tagged dye. There was no need to extract DNA from the tissue as the probe was hybridized on the tissue itself (in situ hybridization). The nuclei stained dark positive with the probe indicating EBV infection [Figure 8]. He was placed on the appropriate EBV therapy.

Several infections including viral (herpes, hepatitis B and C, CMV, papilloma, parvo, HIV, HTLV), bacterial (mycobacteria,  Neisseria More Details, E. Coli) etc can be tracked in a similar way or by PCR-based assays. Antiviral therapy and viral load can also be monitored for infections like HIV, CMV, HCV etc.[18],[19]

Another application of molecular pathology that is more popular with the lay public is the identity determination and paternity testing. This is based on the determination of the pattern of segments of DNA in the introns called variable number tandem repeats (VNTR). These are repeated sequences of base pairs whose function is not yet determined. The VNTRs contain from dozens to several thousand base pairs so that on gel electrophoresis they result in distinct bands. They are unique to each individual and are inherited from the parents and hence paternity testing is also easy. Any source of DNA like blood, tissue, hair, skin, semen etc can be extracted with Southern blot. Multiple specific probes with a known molecular (base pair) size are applied to determine the VNTR pattern. This is also loosely referred to as DNA finger printing. [20] [Figure 9] VNTRs are also useful in tissue antigen matching for transplants, tests for engraftment of transplanted bone marrow cells etc.[21]

Mitochondrial DNA also provides a unique source for determination of identity as it is inherited only from the mother.

A research application of molecular pathology is being extensively applied to tumors that could result in new routine tests for diagnosis and treatment of some of the tumors in the future. [22],[23] This is called gene chip or gene array. Thousands of known and commercially available DNA sequences are immobilized on the surface of a glass slide. They are hybridized with the DNA or RNA from a given tumor. The binding is determined using computer technology and fluorescent binding read with laser, e.g. a normal gene may show a green color, a tumor gene may show a red color and a red signal may indicate a combination of normal and mutant gene. The gene chip technology allows the identification of gene mutation, expression level of genes etc. Follow-up studies can decide if a particular gene mutation or gene expression can determine the behavior of tumor, response to chemotherapy or surgery etc.


   Conclusion Top


Some common clinical examples have been used to illustrate the applications of molecular pathology in the diagnosis of inherited disease, neoplastic disease, infectious disease, targeted therapy, transplantation, identity and paternity testing etc. These are powerful tools with extensive applications. One must be strongly sensitive to false positives and false negatives and the tests must be standardized using proper positive and negative controls. Ethical considerations are also important and should be seriously considered. No test is complete by itself and the total clinical features and all the lab data must be taken in to consideration for the maximum benefit to the patient. Some of these tests are so commonly being used in clinical practice these days that all the pathologists and all the future medical students should be sensitized to them. More tests will evolve as we move on and one should be ready to unlearn the extinct ones and learn the new ones continually through continuing medical education programs etc.


   Acknowledgment Top


I would like to express my profound gratitude to Prof Subrata Chakrabarti from the University of Western Ontario, London, Canada for his efforts to teach these segments to our medical students in Grenada and allowing me to use his material including cases and images freely in preparing this manuscript

 
   References Top

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2.Friedenson B. BRCA-1 and BRCA-2 pathways and the risk of cancers other than breast or ovarian. Med Gen Med 2005;7:60.  Back to cited text no. 2      
3.Calderon-Margalit R, Paltiel O. Prevention of breast cancer in women who carry BRCA1 or BRCA2 mutations: a critical review of literature. Int J Cancer 2004;112:357-64.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]  
4.Easton DF, Deffenbaugh AM, Pruss D, Frye C, Wenstrap RJ, Allen-Brady K, et al. A systematic genetic assessment of 1433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer - predisposition genes. Am J Hum Genet 2007;81:873-83.  Back to cited text no. 4      
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14.Lin F, Shen T, Prichard JW. Detection of Her-2/neu oncogene in breast cancer by chromogenic in situ hybridization in cytologic specimens. Diagn cytopathol 2005;33:376-80.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]  
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16.Stanulla M, Schaeffeler E, Flohr T, Cario G, Ehrauda A, Zimmerman M, et al. Thiopurine methyltransferase (tPMT) genotype and early treatment response to mercaptopurine in children with lymphoblastic leukemia. JAMA 2005;293:1485-9.  Back to cited text no. 16      
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21.del Rosario Uriarte M, Bonomi R, Azambuja C, Seuanez HN. Bone marrow reconstitution with normal autologous cells demonstrated by VNTR analysis after allogenic bone marrow transplantation for chronic myelogenous leukemia. Leukemia 1997;11:2213-4.  Back to cited text no. 21      
22.Check Heyden E. Gene chips unmask cryptic diseases. Nature 2008;455:274-5.  Back to cited text no. 22      
23.Tautravahi U, Wheeler P. Molecular genetic testing for prenatal diagnosis. Clin Lab Med 2003;23:481-502.  Back to cited text no. 23      

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Correspondence Address:
Shivayogi R Bhusnurmath
Department of Pathology, St. George's University School of Medicine, St. George's, Grenada
West Indies
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.64322

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