Is cervical cancer a part of

the Lynch Spectrum?

HNPCC annual meeting October 12, 2017

Karina Rønlund

MD, Ph.D. Department of Clinical Genetics

OUH and Vejle Hospital

Lynch Syndrom

• Inherited in an autosomal dominant manner.

• Germline pathogenic variant in one of four mismatch repair (MMR) genes (MLH1, PMS2, MSH2 and MSH6)

• Associated with tumor microsatellite instability (MSI)

• Life time risks for cancer

- Colon cancer 52-82%

- Endometrie cancer 25-60%

- Ventrikel cancer 6-13%

- Ovarie cancer 4-12%

- Tyndtarm 3-6%

- Galdeveje 1,4-4%

- Øvre urinveje 1-4%

- Hjerne/CNS 1-3% 2

Lynch Syndrom

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HNPCC - register

Data from the Danish HNPCC-register:

• 575 patients with gynaecological cancers

• 262 patients with gynaecological cancers and mutation

• 29 patients with cervix uteri cancer / in situ carcinoma in cervical tissue (CIN III)

Tissue samples from 19 patients.

22.08.08

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Tissue samples

Immunohistochemical staining of mismatch repair (MMR) proteins (MLH1, MSH2, PMS2 and MSH6).

MLH1 clon ES05, Novo Casta

MSH2 clon 25D12 Novocastra

MSH6 clon 44 Transduction Lab

PMS2 clon A16-4 BD PharMingen

Visualised with Envison Flex+, analyzed on Autostaíner, DAKO.

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HPV Genotyping

LINEAR ARRAY® HPV Genotyping Test HPV genotypningtest includes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP6108 (high-risk in bold).

INNO-LiPA® HPV Genotyping PCR-based methods that detects 32 different HPV DNA-genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 70, 73, 81, 82, 83 og 89).

Koilocyt, HPV-infektion

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Tissue samples

Immunohistochemical staining of p16 protein (p16).

Diffuse p16 ekspression is a surrogat marker for high-risk human papillomavirus (HPV). E7 oncoprotein abrogates host cell retinoblastoma protein (pRB). Transcription factor E2F then stimulates S phase and p16 over-expression.

P16 clon JC8 Santa Cruz

Visualised with Envison Flex+

Analyzed on Autostaíner, DAKO.

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Results Nr. Familiy Gene status Age Year Histology MLH1 PMS2 MSH2 MSH6

17 Mutation detected MLH1 47 1977 Squamous cell carcinoma n n n n

12 Amst II + mutation detected MLH1 43 1999 Squamous cell carcinoma loss loss n n

11 Amst II + mutation detected MLH1 56 2005 Udifferentiated carcinoma (cervix uteri) loss loss n n

28 Mutation detected MSH2 35 1997 Adenosquamous carcinoma (polyp in cervix) n n loss loss

5 Amst I + mutation detected MLH1 82 1982 Adenocarcinoma (cervix uteri) n n n n

14 Mutation detected MSH2 carrier 41 1983 Adenocarcinoma (cervix uteri) n n loss loss

23 Mutation detected MSH2 obligate carrier 46 1990 Adenocarcinoma (portiobiopsi) n n loss loss

1 Amst I + mutation detected MLH1 30 1988 Squamous cell carcinoma in situ n n n n

4 Amst I + mutation detected MLH1 obligate carrier 48 1982 Squamous cell carcinoma in situ n n n n

6 Amst II + mutation detected MSH2 carrier 27 1990 Squamous cell carcinoma in situ n n n n

8 Amst I + mutation detected MSH2 carrier 28 1992 Squamous cell carcinoma in situ n n n n

13 Amst II + mutation detected MLH1 carrier 27 2004 Squamous cell carcinoma in situ n n n n

19 Amst II + mutation detected MSH2 40 1977 Squamous cell carcinoma in situ n n n n

22 Mutation detected MSH2 obligate carrier 64 1992 Squamous cell carcinoma in situ n n loss loss

25 Mutation detected MLH1 40 1992 Squamous cell carcinoma in situ n n n n

2 Amst I + mutation detected MLH1 38 1976 Squamous cell carcinoma in situ n n n n

24 Mutation detected MLH1 carrier 32 1988 Severe dysplasia n n n n

21 Amst I + mutation detected MSH2 carrier 32 1997 Serere dysplasia n n n n

26 Amst II + mutation detected MSH2 carrier 29 2006/09 Moderate dysplasia n n n n

Results Nr. Familiy Gene status Age Year Histology MLH1 PMS2 MSH2 MSH6

17 Mutation detected MLH1 47 1977 Squamous cell carcinoma n n n n

12 Amst II + mutation detected MLH1 43 1999 Squamous cell carcinoma loss loss n n

11 Amst II + mutation detected MLH1 56 2005 Udifferentiated carcinoma (cervix uteri) loss loss n n

28 Mutation detected MSH2 35 1997 Adenosquamous carcinoma (polyp in cervix) n n loss loss

5 Amst I + mutation detected MLH1 82 1982 Adenocarcinoma (cervix uteri) n n n n

14 Mutation detected MSH2 carrier 41 1983 Adenocarcinoma (cervix uteri) n n loss loss

23 Mutation detected MSH2 obligate carrier 46 1990 Adenocarcinoma (portiobiopsi) n n loss loss

1 Amst I + mutation detected MLH1 30 1988 Squamous cell carcinoma in situ n n n n

4 Amst I + mutation detected MLH1 obligate carrier 48 1982 Squamous cell carcinoma in situ n n n n

6 Amst II + mutation detected MSH2 carrier 27 1990 Squamous cell carcinoma in situ n n n n

8 Amst I + mutation detected MSH2 carrier 28 1992 Squamous cell carcinoma in situ n n n n

13 Amst II + mutation detected MLH1 carrier 27 2004 Squamous cell carcinoma in situ n n n n

19 Amst II + mutation detected MSH2 40 1977 Squamous cell carcinoma in situ n n n n

22 Mutation detected MSH2 obligate carrier 64 1992 Squamous cell carcinoma in situ n n loss loss

25 Mutation detected MLH1 40 1992 Squamous cell carcinoma in situ n n n n

2 Amst I + mutation detected MLH1 38 1976 Squamous cell carcinoma in situ n n n n

24 Mutation detected MLH1 carrier 32 1988 Severe dysplasia n n n n

21 Amst I + mutation detected MSH2 carrier 32 1997 Serere dysplasia n n n n

26 Amst II + mutation detected MSH2 carrier 29 2006/09 Moderate dysplasia n n n n

Results

Tumor tissue IHC staining from Patient 14 shows a loss of expression of MSH2 and loss of MSH6. Expression of MLH1 and PMS2 were normal. In MSH2 and MSH6 the intern control (stroma cells and lymfocyttes) are still positive, but the islands containing tumor cells has lost expression.

MLH1 MLH1 PMS2 PMS2

MSH2 MSH2 MSH6 MSH6

Tumor tissue IHC staining from Patient 12 shows a loss of expression of MLH1 and loss of PMS2. Expression of MSH2 and MSH6 were normal. In the figure it is also illustrated how the epithelium with dysplasia/ carcinoma in situ has normal expression of MLH1 and PMS2, while the invasive tumor has lost the expression.

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Results

Nr. Histology Linear array Inno Lipa p16 IHC

17 Squamous cell carcinoma negative HPV 16 strong positive

12 Squamous cell carcinoma negative HPV 16 strong positive

11 Udifferentiated carcinoma (cervix uteri) negative negative focal

28 Adenosquamous carcinoma (polyp in cervix) negative negative focal

5 Adenocarcinoma (cervix uteri) invalid negative strong positive

14 Adenocarcinoma (cervix uteri) negative negative focal

23 Adenocarcinoma (portiobiopsy) negative negative focal

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Strong positive Immunohistochemical staining for p16 indicates HPV infection. Focal staining does not

Results

Nr. Histology Linear array Inno Lipa p16 IHC

17 Squamous cell carcinoma negative HPV 16 strong positive

12 Squamous cell carcinoma negative HPV 16 strong positive

11 Udifferentiated carcinoma (cervix uteri) negative negative focal

28 Adenosquamous carcinoma (polyp in cervix) negative negative focal

5 Adenocarcinoma (cervix uteri) invalid negative strong positive

14 Adenocarcinoma (cervix uteri) negative negative focal

23 Adenocarcinoma (portiobiopsy) negative negative focal

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Loss of MMR expression

Strong positive Immunohistochemical staining for p16 indicates HPV infection. Focal staining does not

Conclusion

• The results indicate a relation between the MMR mutation found in

families and loss of MMR gene ekspression in cervical tumours.

• Loss of MMR gene expression seems to be a late event in the carcinogenesis of cervical cancer.

• Our data suggest that women with Lynch syndrome are at higher risk of developing adenocarcinomas than the background population.

• Normally HPV can be detected in at least 95% of adenocarcinomas of the cervix and in close to all squamous cell carcinomas. However, we could not detect HPV in any of the cervical adenocarcinomas in our material.

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Acknowledgement

Facilities, staff and know-how were provided by the department of Clinical Genetics and Clinical Pathology at Vejle Hospital. Special thanks to Ms. Kaja Skovgård Jensen, Ms. Tinna Herløv Jensen and Ms. Rita Andersen for their excellent assistance, and the staff at the Danish HNPCC register. Supported by the Danish Association of Medical Women, Svejgårdsvej 23, 2900 Hellerup

Marianne Waldstrøm MD Department of Clinical Pathology Vejle Hospital