Abstract 631: Trans-species analysis of central nervous system developmental-specific replication repair deficiency reveals differential patterns of gliomagenesis and response to immunotherapy.

Is <i>hEXO1</i> a Cancer Predisposing Gene?

Familial Mutations in PMS2 Can Cause Autosomal Dominant Hereditary Nonpolyposis Colorectal Cancer

Monoallelic germline mutations in one of the DNA mismatch repair (MMR) genes cause Lynch syndrome, with a high lifetime risks of colorectal and endometrial cancer at adult age. Less well known, is the constitutional mismatch repair deficiency (CMMRD) syndrome caused by biallelic germline mutations in MMR genes. This syndrome is characterized by the development of childhood cancer. Patients with CMMRD are at extremely high risk of developing multiple cancers including hematological, brain and intestinal tumors. Mutations in MMR genes impair DNA repair and therefore most tumors of patients with CMMRD are hypermutated. These mutations lead to changes in the translational reading frame, which consequently result in neoantigen formation. Neoantigens are recognized as foreign by the immune system and can induce specific immune responses. The growing evidence on the clinical efficacy of immunotherapies, such as immune checkpoint inhibitors, offers the prospect for treatment of patients with CMMRD. Combining neoantigen-based vaccination strategies and immune checkpoint inhibitors could be an effective way to conquer CMMRD-related tumors. Neoantigen-based vaccines might also be a preventive treatment option in healthy biallelic MMR mutation carriers. Future studies need to reveal the safety and efficacy of immunotherapies for patients with CMMRD.

A Prospective, Multicenter, Population-Based Study of BRAF Mutational Analysis for Lynch Syndrome Screening

N-acetyltransferase (NAT) 2 acetylator status and age of onset in patients with hereditary nonpolyposis colorectal cancer (HNPCC)

Mono-allelic germline pathogenic variants (PV) in one of the mismatch repair (MMR) system genes cause Lynch syndrome, associated mainly with colon and endometrial cancer in adults. Germline PVs in DNA polymerase epsilon (POLE) are associated with a dominantly inherited syndrome which confers risk for polyposis and colon cancer. Brain tumors have been described as part of Lynch syndrome and POLE associated syndrome, mostly in adults. Constitutional mismatch repair deficiency (CMMRd) is caused by bi-allelic mutations in the MMR genes, associated with multiple café au lait macules (CAMs) and high incidence of pediatric cancer, including brain tumors. Both MMRD and POLE associated tumors have high tumor mutation burden (TMB), however, microsatellite status is usually unstable in MMR tumors, and stable in POLE. Germline POLE and CMMRd tumors have different mutational signatures, as is signature of MMR tumors with secondary somatic POLE. We describe a 4.5 y/o male who presented with a grossly metastatic SHH-activated, TP53-wildtype desmoplastic medulloblastoma. Physical examination was noted for CAMs. Family history was positive for a heterozygous POLE variant with variable clinical manifestations. Immunohistochemistry of the tumor showed loss of nuclear expression of the MMR gene PMS2, specifically in tumor cells. Analysis showed exceptionally high TMB (up to 276 Mut/Mb) and both the MMR and the POLE signatures. Germline analysis detected the familial POLE variant as well as a de novo heterozygous PMS2 PV. The phenotype of the patient together with the tumor’s features, led us to classify this case as a CMMRd-like. The patient had a partial response to intensive chemotherapy and is currently on immunotherapy without radiation. Collectively, our data suggest that heterozygous simultaneous germline mutations in MMR and polymerase genes can lead to novel “POLYNCH syndrome” that manifests with an ultra-hypermutant aggressive tumor and requires appropriate treatment and surveillance.

&amp;lt;i&amp;gt;Introduction&amp;lt;/i&amp;gt;: Constitutional Mismatch Repair Deficiency (CMMR-D) syndrome is a rare tumour predisposition and polyposis syndrome that presents in childhood. It is caused by mutations in mismatch repair (MMR) genes that result in a tumour spectrum including colorectal cancers, high-grade gliomas, non-Hodgkin T-cell lymphomas and leukaemias. It is characterized by biallelic germline mutation of one of four MMR genes that can be identified by immunohistochemistry. Immunohistochemistry is used in screening for Lynch syndrome (LS); however, the pattern of loss-of-staining in the background, non-tumour tissue is unique to CMMR-D syndrome. CMMR-D syndrome is seen in LS families and occurs as a result of consanguinity or founder effect. In South Africa, LS families in the Western and Northern Cape Provinces show a unique &amp;lt;I&amp;gt;MLH1&amp;lt;/I&amp;gt; c1528C&amp;gt;T mutation. The diagnosis of CMMR-D syndrome includes clinical findings outlined in the European Consortium’s Care of CMMRD document and confirmation of biallelic mutation in one MMR gene. Immunohistochemistry can be used in the diagnosis of CMMR-D syndrome by identifying cases for targeted molecular genetic tests. Loss of staining of the affected gene in the background, non-tumour tissue, is a key feature of CMMR-D syndrome. &amp;lt;i&amp;gt;Methods&amp;lt;/i&amp;gt;: A retrospective analysis of archival, formalin fixed paraffin embedded tissue was performed on specimens of children attending Red Cross Children’s Hospital with tumours that form part of the CMMR-D spectrum, outlined by the Care for CMMRD criteria. We used the criteria of high-grade gliomas (WHO Grade III or IV) occurring before 25 years of age, cutaneous lesions suggestive of CMMR-D syndrome and patients with a first or second degree relative diagnosed with LS. Immunohistochemistry was performed and the staining pattern was documented using a modified Allred Scoring system. Specific attention was given to the characterization of the staining pattern of the background normal tissue. &amp;lt;i&amp;gt;Results&amp;lt;/i&amp;gt;: 21 samples evaluated from 18 patients. 16 samples represented brain tumours. Three inadequate samples were excluded. 12 samples showed intact staining. Two samples showed staining of unknown significance. Four samples from 3 different patients showed staining patterns compatible with MMR deficiency. Of these four samples, three samples showed loss of staining in background non-tumour tissue with positive external control. &amp;lt;i&amp;gt;Conclusion&amp;lt;/i&amp;gt;: MMR immunohistochemistry can be used in the evaluation of CMMR-D syndrome. The pattern and scoring of both the tumour and the background non-tumour tissue is critical. The diagnosis of CMMR-D syndrome depends on clinical application of Care for CMMRD criteria, MMR immunohistochemistry in conjunction with molecular genetic testing.

Functional Significance and Clinical Phenotype of Nontruncating Mismatch Repair Variants of

Colon and Endometrial Cancers With Mismatch Repair Deficiency Can Arise From Somatic, Rather Than Germline, Mutations

Dear Editor: We read with interest the recent letter to the editor written by Victoria Fretwell and Paul Rooney, regarding their 15year-old patient with reported biallelic mutations [1]. Unfortunately, there are significant errors in this report, and we are writing to highlight our findings and concerns. The authors describe a 15-year-old girl with rectal cancer who was found to have mutations in both the MLH1 and MSH6 genes. The authors incorrectly refer to this as Bbiallelic inheritance.^ Although unusual to have two mutations in two different mismatch repair genes, this is not consistent with the definition of biallelic inheritance. The definition of biallelic mutations is Bpathogenic sequence alterations, albeit not necessarily identical, present in both copies of the same gene^ [2]. The case described by Fretwell and Rooney instead has double heterozygous mutations. Biallelic mutations in mismatch repair genes results in constitutional mismatch repair deficiency (CMMRD) syndrome. CMMRD syndrome is characterized by features similar to neurofibromatosis type 1 (NF1), in addition to an increased risk for childhood onset hematologic malignancies, brain tumors, and Lynch syndrome cancers [3]. Individuals with double heterozygous mutations in mismatch repair genes are not expected to have features similar to those with CMMRD syndrome and likely have similar cancer risks as those with Lynch syndrome. A similar scenario is seen for double heterozygous mutations in BRCA1 and BRCA2, which have a similar phenotype to individuals with monoallelic BRCA1 or BRCA2mutations [4]. There is another case report of a woman with mutations in bothMLH1 andMSH6 who was diagnosed with endometrial cancer at the age of 41 years [5]. This is a typical age of diagnosis for a patient with a single MMR gene mutation. Additionally, despite the authors’ declaration, theirs is not the first case in the literature of an individual with both MLH1 and MSH6 mutations. We would also would like to highlight a few other concerns we have. One concern is that the variants found inMLH1 and MSH6were not included in this report. There is no way for the reader to determine if the MLH1 and MSH6 variants were indeed pathogenic. The authors also provide no information on whether the patient has signs of CMMRD syndrome or if microsatellite instability and/or mismatch repair immunohistochemistry tumor analyses were performed, all of which might provide insight into the etiology of this young girl’s rectal cancer. Another major disquiet is that the authors allude to making treatment decisions based on the biallelic inheritance and further allude to the girl’s imminent early death based on mistaken information. In summary, the basis of this report, biallelic inheritance, is factually incorrect. Given the erroneous and missing information presented, this case has little scientific merit as presented and leaves us with more questions and concerns, than answers.

Hereditary and Familial Colon Cancer

e15791 Background: Tumors with mismatch repair-deficiency (MMRD) have a high mutational burden and have good responses to immunotherapy (Le, NEJM, 2015). We describe the natural course, clinicopathological, and genomic status of MMRD PDAC patients (pts) at Memorial Sloan Kettering Cancer Center (MSKCC). Methods: MSKCC institutional registry and ICD billing database queried from 2006-2016 for PDAC pts with genetically confirmed mutations in mismatch repair (MMR) genes. Mutation # determined via MSK-IMPACT, a targeted tumor next generation sequencing (NGS) test (Cheng, J Mol Diagn, 2015). Results: 5/607 (0.8%) PDAC pts had Lynch syndrome (LS) (confirmed germline mutations) (Table 1). Of the 5 LS pts, all had &gt; 10 mutations in NGS, with 4 of 5 having &gt; 50 mutations. 4 of 5 (80%) are alive at last follow-up (survival 30-314 months). N=4 had extensive personal/family history of cancer. Of N=3 who had resected disease, all 3 had recurrence at 11, 49 and 311 months, and all are alive (survival: 69-314 months). Of N= 2 pts that had unresectable tumors, one passed away at 30 months while the other is on checkpoint inhibitor trial and is alive at 30 months. In contrast, 7/607 (1.1%) PDAC pts had somatic mutations in MMR genes with an average of 5.7 mutations in NGS, with 4/7 having &lt;5 mutations. 4/7 (57%) are deceased at last follow-up (survival: 10-42 months). Conclusions: All cases with germline mutations in the MMR genes, with one exception, had high mutation #. All cases with somatic mutations in the MMR genes had low mutation #. Germline mutations in MMR genes and high mutational burden may predict for a prognostically favorable subgroup of PDAC pts with high susceptibility to immune oncology agents. [Table: see text]

21067 Background: Hereditary non-polyposis colorectal cancer (HNPCC) is a genetic disorder that results in an increased risk of early onset colorectal cancer (CRC). HNPCC is caused by germline mutations in DNA mismatch repair (MMR) genes, including MLH1 and MSH2, and is transmitted in an autosomal dominant manner. De novo germline mutations in MMR genes are exceedingly rare. We now describe a case of a de novo germline mutation in MLH1 associated with early onset CRC in a young woman with a negative family history. Methods: We present a case of a 31-year-old Caucasian female who presented with abdominal pain. Colonoscopy revealed a moderately differentiated adenocarcinoma with focal mucin production involving the sigmoid colon, with a final staging of T4N0M0. Genetic testing was offered because of her young age at diagnosis, having fulfilled the Bethesda guidelines. Microsatellite instability (MSI) testing using a panel of six microsatellite loci was performed on the tumor sample from the affected patient. Immunohistochemistry (IHC) testing for MLH1 and MSH2 was performed. Following counseling and with informed consent, DNA was isolated and sequenced using bi-directional PCR of the MLH1 gene. All exons of MLH1 and MSH2 were analyzed by standard Southern blot methods. Paternity was established using eight genetic loci. Results: The patient's tumor revealed high MSI and complete absence of MLH1 immunoreactivity. MSH2 IHC staining was normal. A large deletional mutation involving exons 5–12 of MLH1 was identified by Southern blot analysis. The patient's parents and siblings were tested and found to have wild type MLH1. Paternity was confirmed with greater than 99.9% certainty. Conclusions: De novo mutations in MMR genes are a rare cause of HNPCC. We report the first case of a large de novo deletion in the MLH1 gene accounting for early onset CRC. Such de novo mutations, albeit rare, must be considered in patients who present with early onset CRC and a negative family history. These results support the use of the Bethesda guidelines to identify individuals who may carry mutations in the MMR genes. No significant financial relationships to disclose.

Background: Paired tumor-normal targeted next-generation sequencing (NGS) is primarily used to identify actionable somatic mutations, but can also detect germline variants including pathogenic germline mutations in DNA mismatch repair (MMR) genes that underlie Lynch syndrome. In the present study we examined paired NGS data from lung cancer patients to identify germline mutations in MMR genes. As lung cancer is not one of the recognized Lynch syndrome-associated neoplasms, we also investigated whether these lung cancer cases are due to Lynch syndrome or are instead sporadic cancers occurring in Lynch syndrome patients.Methods: A retrospective study of 1,179 lung cancer patients with available paired NGS data was performed to identify germline mutations in the MMR genes MLH1, MSH2, MSH6, and PMS2, and evaluate tumor mutation burden (TMB). Microsatellite instability (MSI) testing was done on select cases with MMR gene mutations by either NGS or PCR/capillary electrophoresis approach. Immunohistochemistry (IHC) for MMR proteins was performed in select patients.Results: Pathogenic or likely-pathogenic germline mutations in PMS2, MSH2, or MSH6 were detected in 0.5% (6/1,179) of lung cancer patients; three of the patients had a family history of colon or gastric cancer. The median age at diagnosis of these cases was 68.5 years old. None of these six patients exhibited MSI or loss of MMR protein expression. Among them, no second hit somatic mutations in MMR genes (including single-nucleotide variants, small insertions or deletions and copy number alterations) were detected, and the median TMB was 4.5 muts/MB. Subsequent genetic testing of family members identified new Lynch syndrome cases in two first-degree relatives.Conclusion: These data imply that lung cancers in Lynch syndrome patients are unrelated to the underlying Lynch syndrome diagnosis and occur spontaneously. Nonetheless, paired tumor-normal NGS can identify germline mutations to help reveal Lynch syndrome in cancer patients. This has important implications for cancer screening and risk reduction in these patients and their families.

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLHI, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLHI exon 6, and two missense mutations in MLHI exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes.