Hereditary Non-Polyposis Colon Cancer and Microsatellite instability

Hereditary Non-Polyposis Colon Cancer and Microsatellite instability Byoung Kwon Kim M.D., M.S., IFCAP Department of Pathology and Medical Genetics Institute, Green Cross Reference Laboratory

Agenda Long history of HNPCC and mismatch repair system Microsatellite and it s instability Clinical implication of MSI Q&A

Agenda Long history of HNPCC and mismatch repair system Microsatellite and it s instability Clinical implication of MSI Q&A

Colorectal cancer Sporadic colorectal cancer (CRC) Hereditary (familial) CRC Lynch et al., 2004

Colorectal cancer carcinogenesis Normal gland Adenoma Carcinoma Vogelstein et al., 2004; Vilar et al., 2010

Colorectal cancer carcinogenesis Important concept of CRC carcinogenesis - Multistep genetic alteration in accordance with pathologic alteration - Oncogene / Tumor suppressor gene - Chromosomal (structural) instability Vogelstein et al., 2004; Vilar et al., 2010

Earlier records on Hereditary Non- Polyposis Colon Cancer (HNPCC) Cancer Family G was described by Aldred Warthin in 1895 and reported in 1913. Henry T. Lynch described 2 Midwestern kindred's as having a cancer family syndrome in 1966.

HNPCC vs. sporadic colorectal cancer Autosomal dominant trait (familial) Mean age of diagnosis; 41 years Proximal location of splenic flexure; 68% More than one; 7% Less metastatic tendency (good prognosis) Extra-colonic cancer risk; stomach, endometrium, biliary system, urinary system Lynch et al., 1966, Myrho et al., 1997

Questions Polygenic or monogenic? AD trait à probable monogenic nature What causes HNPCC? à mapping and cloning of the possible gene

Genetic mapping Linkage analysis using 345 microsatellite markers à narrow down to D2S*** (Peltomaki et al., 1993)

Genetic mapping Z max at D2S123 with no recombination (Peltomaki et al., 1993)

Genetic mapping Microsatellite syntany around D2S123 11cM 14cM 5cM D2S119 D2S5 D2S123 D2S136 D2S5; Previously known as located at 2p15~16 (Peltomaki et al., 1993)

Genetic mapping Aaltonen et al., (1993) studied different families from those studied by Peltomaki et al., (1993). Linkage analysis using microsatellite markers and formalin-fixed paraffin-embedded (FFPE) normal and tumor tissues.

Genetic mapping Aaltonen s mapping result Not consistent LOD score!!! à genetic heterogenity of HNPCC families involved in his study? (Aaltonen et al., 1993)

Genetic mapping Aaltonen s unexpected results Wide spread alteration in short repeat DNA sequences, (CA) n dinucleotide repeat fragments (Aaltonen et al., 1993)

Genetic mapping Aaltonen s unexpected results Normal chromosomal structure (No chromosomal instability) 13% of non-familial (sporadic) CRC showed identical alteration in microsatellite sequences with preference to proximal (right-sided) location (later reports; 10~15% of sporadic CRC) ~existence of a gene on chromosome 2 which is neither an oncogene nor a tumor suppressor gene, but rather a gene leading to genomic instability ~ (cf. chromosomal instability in sporadic CRC) (Aaltonen et al., 1993)

? Perspective from microbial geneticist Proof reading ~10-10 REAL WORLD Proof reading? Proof reading Base selection Replicative polymerases complex ; family A (e.g. T7 Pol), family B (e.g. T4 Pol, Pol δ, Pol ϵ) and family C (e.g. E. coli Pol III). Proofreading-defective derivatives (designated A, B, C, and RT, for viral reverse transcriptase) Kunkel TA, 2004

Earlier records on mismatch repair system Cloning of the hexa mismatch-repair gene of Streptococcus pneumoniae and identification of the product. (Martin et al., 1986) Nucleotide sequence of the hexa gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexa to muts of Escherichia coli and Salmonella typhimurium. (Priebe, S.D. et al. 1988)

Perspective from microbial geneticist Prokaryote mismatch repair (MMR) system E. coli Mut HLS pathway (Mut H, MutL, MutS + UveD(helicase II)) acting on recombination intermediate with mis-paired bases (Wagner et al., 1976) E. coli also has a long patch (~2Kb) excision repair system (Modrich et al., 1989) Eukaryote mismatch repair (MMR) system S. cerevisiae has MMR system (Holliday et al., 1964) MutS homology, MSH was identified and characterized (MSH2 by Reeman et al., 1992, MSH3 by New et al., 1993, MSH4 by Ross-McDonald et al., 1993) MutL homology, MLH1 & PMS1 by Kramer et al., 1981 Mutant msh2, pms1 or mlh1 increased rates of expansion and contraction of dinucleotide repeats (Strand et al., 1993)

Bacterial MMR system During DNA replication Horvat M & Stabuc B, 2011

Bacterial MMR system During DNA replication Horvat M & Stabuc B, 2011

Bacterial MMR system During DNA replication Horvat M & Stabuc B, 2011

Perspective from microbial geneticist Certain types of human hereditary and sporadic cancer show similar alteration in short repeat DNA sequences like in microbes (Aaltonen et al., 1993, Ionove et al., 1993, Thibodeu et al., 1993) possibility of existence of human homologous protein of MMR system

Perspective from microbial geneticist bacterial muts vs. yeast msh2 protein

Perspective from microbial geneticist Degenerate PCR strategy using total RNA RT template ---TGPNM--------------------F(ATV)TH(FY)----- TGPNM à ß FATH(FY) ß FTTH(FY) ß FVTH(FY) (Fishel et al., 1993)

Cloning of HUMAN MSH2 2727 bp ORF 909 amino acids; 41% homology with S. cerevisiae msh2 (Fishel et al., 1993)

Cloning of human MSH2 MutS MMR protein superfamily (Fishel et al., 1993)

Chromosome mapping Hybrid chromosome analysis using Chinese hamster cell line Chromosome 2!!!

Chromosome mapping Mouse chr 17 ßà Human chr 2 syntany Linkage analysis using (B6xM. spretus)f1 back cross Mapping!!! Chr2p22-21 (cf. 2p16~15 by Peltomaki et al., 1993)

Gene to disease causality Mutation analysis of hmsh2 with small HNPCC family (pph blood, screening with SSCP à direct sequencing) normal Top strand (forward) Bottom strand (backward) normal HNPCC HNPCC (Fishel et al., 1993)

Summary of genes and mapping of HNPCC subtypes phenotype location Gene/locus authors Type 1 2p21 MSH2 Fishel et al., 1993; Leach et al., 1994 Type 2 3p22.2 MLH1 Lindblom et al., 1993 Type 3 2q32.2 PMS1 Nicolaides et al., 1994 Type 4 7p22.1 PMS2 Nicolaides et al., 1994 Type 5 2p16.3 MSH6 Wijnen et al., 1999 Type 6 3p24.1 TGFBR2 Lu et al., 1998 Type 7 14q24.3 MLH3 Liu et al., 2003 Type 8 2p21 EPCAM Chan et al., 2006

Summary of genes and mapping of HNPCC subtypes phenotype location Gene/locus authors Type 1 2p21 MSH2 Fishel et al., 1993; Leach et al., 1994 Type 2 3p22.2 MLH1 Lindblom et al., 1993 Type 3 2q32.2 PMS1 Nicolaides et al., 1994 Type 4 7p22.1 PMS2 Nicolaides et al., 1994 Type 5 2p16.3 MSH6 Wijnen et al., 1999 Type 6 3p24.1 TGFBR2 Lu et al., 1998 Type 7 14q24.3 MLH3 Liu et al., 2003 Type 8 2p21 EPCAM Chan et al., 2006

Kariola R, 2004

Mismatch repair system E. coli system Yeast system Vilar E & Gruber SB, 2010 Kariola R, 2004

MMR defect and carcinogenesis -HNPCC- Germline mutation 1 st hit MMR gene Somatic mutation 2 nd hit Malfunction Dysfunction Alteration in expression level MMR defect Global genomic instability Increased mutation rate in satellite DNA (genes with microsatellite in coding region) Increased mutation rate in non-satellite DNA

MMR defect and carcinogenesis -sporadic colon cancer- Promoter hypermethylation MMR gene Alteration in expression level MMR defect Promoter hypermethylation Global genomic instability Increased mutation rate in satellite DNA (genes with microsatellite in coding region) Increased mutation rate in non-satellite DNA

Colorectal cancer carcinogenesis -dual pathway- ~85% Chromosomal instability (CIN) ~15% Microsatellite instability (MSI)

Agenda Long history of HNPCC and mismatch repair system Microsatellite and it s instability Clinical implication of MSI Q&A

Satellite DNA Equilibrium density gradient centrifugation concentration gradient using cesium chloride analysis of buoyant density of sheared DNA fragments Satellite peak was identified in Yeast-Moustacchi E & Williamson DH, 1966 Bacteria-Douthit HA & Halvorson HO, 1966 Mouse-Flamm et al., 1966 Human-Corneo et al., 1967

Repeated sequences in genome Category Tandem repeat Interspersed repeat Genomic island Repeat sequences Satellite DNA (VNTR-minisatellite, STR-microsatellite) transposon retrotransposon SINEs Alu, MIR DNA transposon Genomic island LINEs LTR LINE1, LINE2 MER4, HERV, retroposon MER1, MER2, mariners

Microsatellite Synonym ; Short tandem repeat (STR) Definition varies among researchers (arbitrary) Repeat unit; 1~6bp, 2~4bp, 2~6bp, 1~10bp Length; five times or more, eight times or more, five times to 5000 times

Microsatellite Schlotterer C & Harr B, 2001

Microsatellite General characteristics Comprise 3% of the human genome with more than one million loci Frequency; di->mono->tetra->tri- Single copy with length polymorphism Slightly higher mutation rate than other genomic area during replication Mostly located in intergenic and intronic area, a few cases in coding region (TGFbRII, IGFRII, Bax, E2F4, hmsh3, hmsh6 ) Application Forensic science/paternity test/linkage analysis (mapping)/population genetics (HNPCC screening)

Replication slippage 4 5 6 7 8 9 10 11 If intact MMR restoration restoration Mismatch error is very rare but happens even in intact MMR system! Eg.) Meiosis (sperm); rare but happens! à polymorphism, disease

Replication slippage 4 5 6 7 8 9 10 11 If defective MMR Higher rate of error during synthesis by Taq pol.(pcr reaction) à stutter bands of repeated DNA sequence Higher rate of error during cell division(mitosis) of cancer à MSI and global genomic instability

Definition of Microsatellite instability Definition of instability; aberrant peak in tumor, aberrant morphology in tumor, 5bp shift MSI is defined as a change of any length due to either insertion or deletion of repeating units, in a microsatellite within a tumor when compared to normal tissue. (Boland et al., in NCI workshop, 1998) stable unstable Oslo University Hospital, http://www.ous-research.no/home/lothe/methods/2766

Which microsatellites are used?

Microsatellite instability

Agenda Long history of HNPCC and mismatch repair system Microsatellite and it s instability Clinical implication of MSI Q&A

-Colorectal cancer (n=509) -MSI analysis in all cases -Genetic analysis of hmlh1 & hmsh2 in all cases (thus, considering genetic features first clinical features later)

Aaltonen et al., 1998

Aaltonen et al., 1998

Immunohistochemistry and/or MSI analysis 42 patients with colorectal carcinoma 11 met the Amsterdam criteria 31 were suspected HNPCC families. IHC + MSI analysis+ sequencing of MLH1, MSH2 using normal and tumor tissues Christensen et al., 2002

Immunohistochemistry and/or MSI analysis Christensen et al., 2002

Germline mutation in MLH1 or MSH2 N=13 Wild type in MLH1 or MSH2 N=18 Christensen et al., 2002

Germline mutation (MLH1 or MSH2) (n=13) 중에서 IHC 시행한환자 11 명, 이중에서 IHC 상에 MLH1 and/or MSH2 loss 있는경우 (n=8) Sensitivity ; 8/11 =72.7% Christensen et al., 2002

MLH1 or MSH2 유전자 WT 인환자 (n=18) 중에서 IHC 한환자 18 명, IHC 상 MLH1 and/or MSH2 loss 있는경우 3 명 (MLH1 만주로 loss) Specificity ; 15/18=83.3% Christensen et al., 2002

Germline mutation (MLH1 or MSH2) (n=13) 중에서 MSI 시행한환자 12 명, 이중 MSI-H 인경우 11 명 Sensitivity ; 11/12 =91.7% Christensen et al., 2002

MLH1 or MSH2 유전자 WT 인환자 (n=18) 중에서 MSI 시행한환자 13 명, MSI-H 인경우 4 Specificity ; 9/13=69.2% Christensen et al., 2002

Germline mutation (MLH1 or MSH2) (n=10) 중에서 MSI-H + IHC loss; 10 Sensitivity ; 10/10 =100% MLH1 or MSH2 유전자 WT 인환자 (n=13) 중에서 MSI-H + IHC loss; 5 Specificity ; 8/13=61.5% Christensen et al., 2002

MSI and prognosis In 1966, HNPCC show better survival than stage-matched sporadic CRC patients. Clinically HNPCC 10~15% of sporadic CRC show MSI and have similar pathologic features like HNPCC. Control ; sporadic CRC Any relationship between MSI and prognosis? à sporadic CRC with MSI vs. sporadic CRC without MSI

Population based series of 607 sporadic CRC Gryfe et al., 2000

Gryfe et al., 2000

Gryfe et al., 2000

Gryfe et al., 2000

Large series of sporadic CRC (n~1300) ; MSI vs. MSS Samowitz et al., 2001

MSI and prognosis 4 meta-analysis results Horvat M & Stabuc B, 2011

MSI and chemo-response Several cell-based studies, xenograft studies à discordant results, slightly favor 5-FU 570 patients with CRC previously enrolled in 5 phase III trials of adjuvant chemotherapy (5-FU) à MSI and response? Ribic et al., 2003

Vilar E & Gruber SB, 2010

Ribic et al., 2003

Ribic et al., 2003

Ribic et al., 2003

(MSI+chemo and MSS+chemo) No chemo HR = 1 (MSS/MSL+chemo) (MSI-H+chemo) Ribicet al., 2003

Clinical implication of MSI Screening method of HNPCC combined with IHC of MMR proteins Good prognostic marker in sporadic CRC No benefit for 5-FU based chemotherapy

녹십자의료재단 MSI 검사의차별점 종양부분병리전문의직접리뷰 à analytical sensitivity Multiplex PCR 결과 equivocal 시에는다시 simplex 로 PCR 시행 à failure rate

녹십자의료재단에서가능한 HNPCC 관련검사항목 IHC hmlh1 MSH2 MSH6 Genetic testing MSI testing; FFPE tissue B-RAF mutation testing; FFPE tissue K-RAS mutation testing; FFPE tissue hmlh1 germline genetic testing; blood hmsh2 germline genetic testing; blood

Agenda History of HNPCC and mismatch repair system Microsatellite and it s instability Clinical implication of MSI Q&A