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Department of SGE NIMS Hyderabad 23-03-2009

Department of SGE NIMS Hyderabad 23-03-2009. A GENETIC AND EPIDEMIOLOGICAL STUDY OF HEREDITARY NON-POLYPOSIS COLORECTAL CANCER IN COLORECTAL CARCINOMA PATIENTS PRESENTING TO A TERTIARY CARE HOSPITAL.

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Department of SGE NIMS Hyderabad 23-03-2009

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  1. Department of SGE NIMS Hyderabad 23-03-2009 A GENETIC AND EPIDEMIOLOGICAL STUDY OF HEREDITARY NON-POLYPOSIS COLORECTAL CANCER IN COLORECTAL CARCINOMA PATIENTS PRESENTING TO A TERTIARY CARE HOSPITAL

  2. This study is being done in collaboration with center for dna fingerprinting and diagnostics(cdfd)

  3. Abbreviations • CRC colorectal carcinoma • HNPCC Hereditary Non-polyposis Colorectal Cancer • APC adenomatouspolyposis coli • MSI micro satellite instability • MMR mismatch repair • PCR polymerase chain reaction

  4. Outline • Background • Aim • Materials • Methods • DNA isolation • Genomic • Tumor • Microsatellite instability • Immunohistochemistry for MMR genes • PCR • Sanger’s sequencing

  5. What is HNPCC(Lynch Syndrome) • Definition is based on demonstration of mutation in MMR genes • No clinical definition • Amsterdam’s criteria helps in clinically defining HNPCC

  6. Amsterdam II criteria:

  7. What is Amsterdam's criteria

  8. What are Bethesda guidelines

  9. Tests for HNPCC • Screening tests • Confirmatory tests

  10. Background • Colorectal carcinoma in India is biologically a different disease compared to western population. • Occurs in young age • Rectal carcinoma constitutes half of the cases • Incidence is less • Majority of young CRC patients are sporadic • Late presentation • No studies on CRC genetics from India • No reported cases of HNPCC from India

  11. Background • 90% of CRC in west occur after 50years • India 50-60% <50years • WHY?

  12. Background • In west 10% occur in young • 30% of these are HNPCC • This stimulated us to study about HNPCC and colorectal carcinoma in our own population

  13. Vogelstein’s pathway

  14. Background • HNPCC is caused by MSI, the hallmark of HNPCC • MSI is caused by MMR genes mutation • MMR genes are also called caretaker genes • MMR gene mutation causes HNPCC

  15. Background • Bethesda guidelines are for MSI screening • It is likely that Bethesda guidelines are not useful in Indian population. • This hypothesis will be either proved or disproved by our study.

  16. Life time risk of cancer in HNPCC

  17. Primary objective • To study the incidence of HNPCC in CRC patients presenting to NIMS. • To study the mutations causing HNPCC • To identify any new mutation • Study the usefulness of MSI & IHC in diagnosing HNPCC • To screen first degree relatives of HNPCC patients for MMR mutation

  18. Secondary objective • To assess age distribution , sex distribution , site distribution of CRC • To assess pathologic characteristics of CRC • To assess stage of presentation

  19. INCLUSION CRITERIA • CRC, diagnosed in patients < 50 years • Synchronous or metachronous CRC or other tumors associated with HNPCC, regardless of age • CRC with of MSI-H or specific histology diagnosed < 60 years. • HNPCC related cancer <50yrs in 1 first degree relative • HNPCC associated tumor at any age in two first- or second-degree relatives

  20. Exclusion criteria • All patients with neoadjuvant CT/RT are excluded from study. • All patients who are HIV/HBsAg +ve. • Patients with FAP

  21. Methods • DNA isolation • Genomic • Tumor • Microsatellite instability • Immunohistochemistry for MMR genes • PCR • Sanger’s sequencing

  22. Case 1 • 24yr female with bleeding PR • Sigmoid carcinomas at two synchronous locations-T2N0M0 • Left hemi-colectomy T2N0M0 x2 • 3 yrs later - multiple carcinomas • Caecum –T3N0M0 • Duodenum-T2N0M0 • Jejunum –T2N0M0

  23. DNA isolation • Genomic DNA • Blood – easy to isolate • Normal tissue • Tumor DNA • H&E stained 5micron slide • Identify tumor cells • Scrape the cells • Isolate DNA

  24. DNA isolation from blood • 3ml blood • Potassium EDTA bottle • Store at 40⁰ c • Should not be frozen • If frozen - should not be thawed • Blood should not be stored for more than 7 days because yield will come down

  25. DNA isolation from blood • Centrifuge blood after adding erythrocyte lysis buffer –30min • Centrifuge @10000 rpm for 10min • Discard supernatant fluid • WBC pellet • Add proteinkinase , DDS • Incubate at 37⁰ C for 12hours • Purification of DNA

  26. DNA isolation from blood • Purification is a lengthy procedure • WBC pellet is digested with proteinkinase • Intra cellular contents are released • Repeated purification and centrifugation • Protein heaviest at bottom • DNA next to protein • Need careful extraction

  27. Once DNA is isolated it can be stored at -20⁰ c for a long time for many reactions • Genomic DNA is highly concentrated • Should be very careful otherwise it will contaminate other reactions

  28. PCR • First described in 1985, Nobel Prize for Kary Mullis in 1993 • The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermusaquaticus, discovered in hot springs.

  29. PCR • The primary materials, or reagents, used in PCR are: • DNA nucleotides, the building blocks for the new DNA • Template DNA, the DNA sequence that you want to amplify • Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA • DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA

  30. PCR

  31. Sample tray and micropipettor. Each tray holds 96 samples

  32. PCR • The result is a dramatic amplification of a the DNA that exists between the primers. • The amount of amplification is 2 raised to the n power; • n represents the number of cycles that are performed. • After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012

  33. STEP-1 • Denaturation at around 94°C : • During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle).

  34. STEP-2 • Annealing at around 54°C : • Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached

  35. STEP-3 • Extension at around 72°C : • The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

  36. THERMOCYCLER

  37. Sitamahalakshmi

  38. AGAROSE GEL ELECTROPHORESIS A method used to separate macromolecules like proteins and nucleic acids (ie DNA/RNA) based on their size and electric charge

  39. Sequencing • “Sequencing” means finding the order of nucleotides on a piece of DNA . • Nucleotide order determines Amino acid order, and by extension, protein structure and function (proteomics) • An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect

  40. Sequencing • Historically there are two main methods of DNA sequencing: • Maxam & Gilbert, using chemical sequencing • Sanger, using dideoxynucleotides. • Modern sequencing equipment uses the principles of the Sanger technique.

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