Loss Of Mismatch Repair Proteins Research Articles

18 - Loss of DNA mismatch repair proteins characterises Grade group 3-5 prostate carcinoma and did not correlate with serum PSA level

18 - Loss of DNA mismatch repair proteins characterises Grade group 3-5 prostate carcinoma and did not correlate with serum PSA level

Genetic, Epigenetic, and Immunologic Profiling of MMR-Deficient Relapsed Glioblastoma.

In-depth characterization of recurrent glioblastoma (rGBM) might contribute to a better understanding of the mechanisms behind tumor progression and enable rGBM treatment with targeted drugs.Experimental Design: In this study, GBM samples were collected at diagnosis and recurrence from adult patients treated with Stupp protocol. Expression of mismatch repair (MMR) proteins was evaluated by IHC, followed by whole exome sequencing (WES) of tumor samples showing loss of MSH6 reactivity. Established genetic, epigenetic, and immunologic markers were assessed by standard methods and correlated with loss of MMR proteins and patient survival. Expression of MMR proteins was partially or completely lost in 25.9% rGBM samples. Specifically, 12 samples showed partial or total MSH6 expression reduction. Conversely, 96.4% of GBM samples at diagnosis expressed MMR markers. WES disclosed lack of variants in MMR genes in primary samples, whereas two MSH6-negative rGBM samples shared a c.3438+1G>A* splicing MSH6 variant with a potential loss of function effect. MSH6-negative rGBM specimens had high tumor mutational burden (TMB), but no microsatellite instability. In contrast, GBM samples with partial loss of MMR proteins disclosed low TMB. MMR-deficient rGBM showed significant telomere shortening and MGMT methylation and are characterized by highly heterogeneous MHC class I expression. Multilevel profiling of MMR-deficient rGBM uncovered hypermutated genotype uncoupled from enriched expression of immune-related markers. Assessment of MHC class I expression and TMB should be included in protocols aiming to identify rGBM patients potentially eligible for treatment with drugs targeting immune-checkpoint inhibitors.

Abstract P2-08-21: Mismatch repair protein loss is a prognostic and predictive biomarker in breast cancers regardless of microsatellite instability

Abstract Despite the approval of pembrolizumab in all tumors showing mismatch-repair (MMR) deficiency and/or microsatellite instability (MSI), there are currently no companion diagnostics for MMR status assessment in breast cancer. Here, we sought to define the diagnostic and prognostic role of MMR and MSI testing in breast cancer patients. We subjected 444 breast cancers to MMR immunohistochemistry (IHC) and MSI analysis. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and MMR-heterogeneous (hMMR) based on the loss of immunoreactivity; MSI was defined by the instability in the five indicators recommended by the National Cancer Institute for endometrial and colorectal cancers. Correlation of MMR status with patients' survival was assessed using the Kaplan-Meier estimator. In 75 patients (17%) the loss of MMR proteins was homogeneous, classified as dMMR, while 55 cases (12%) were hMMR. The prevalence of cancers with loss of the MMR proteins was homogeneous across ER+ breast cancers (15-19% for dMMR and 10-18% for hMMR tumors). The level of overlap between IHC and MSI analysis was 9% (p<0.0001). Among ER+/HER2- carcinomas, pMMR and hMMR patients displayed better survival rates (p=0.008). In chemo-treated ER-/HER2- breast cancers, the dMMR status was a marker of good prognosis (p<0.001). Our study documents the clinical impact of MMR testing in a large series of breast cancers, using the most commonly adopted diagnostic tools and criteria. We show that MMR protein loss is a rather common event in breast cancer and has a remarkable degree of intra-tumor heterogeneity, therefore making the analysis of a small area of the tumor, or a small biopsy, of little clinical value. Our investigation supports the concept that MSI occurs rarely in breast cancer and demonstrate that this condition is restricted to a minority of tumors with MMR protein loss. These data suggest that MMR IHC and MSI analysis should not be considered as interchangeable tests in the diagnostic workup of breast carcinomas. Finally, our observations indicate that the complete loss of at least one of the MMR proteins assessed by IHC is able to identify high-risk ER+/HER2- breast cancers that can potentially benefit from pembrolizumab therapy, whereas first-line chemotherapy shows comparatively good results in dMMR ER-/HER2- breast cancers. Citation Format: Fusco N, Lopez G, Corti C, Pesenti C, Colapietro P, Ercoli G, Gaudioso G, Faversani A, Gambini D, Despini L, Blundo C, Vaira V, Miozzo M, Ferrero S, Bosari S. Mismatch repair protein loss is a prognostic and predictive biomarker in breast cancers regardless of microsatellite instability [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-21.

Abstract P1-06-02: Mismatch repair protein loss in breast cancer: Clinicopathological associations in a large British Columbia cohort

Abstract Background: Alterations to mismatched repair (MMR) pathways are a known cause of cancer (particularly colorectal and endometrial). Recently, the FDA approved pembrolizumab for use in MMR-deficient (MMRD) cancers of any type, and the diagnosis can be made by immunohistochemistry (IHC) or genomic methods. In breast cancer, mutational process analyses indicate MMRD occurs in about 2% of breast cancer (Cancer Res; 77; 4755-62, 2017) and recent functional studies have shown associations with resistance to endocrine therapy and sensitivity to CDK4/6 inhibitors (Cancer Discov; 7; 1168-83, 2017). To date, insufficient cases have been assembled to power meaningful associative or survival studies. Herein, the strong correlation between IHC-determined loss of MLH1, PMS2, MSH2 or MSH6 and genomic evidence allowed the assessment of MMRD on a large tissue microarray (TMA) series linked to detailed biomarkers and long-term outcome data. Methods: IHC markers MLH1, PMS2, MSH2 and MSH6 were optimized on the Ventana automated stainer for application to breast cancer TMAs. The patient cohort consists of females from British Columbia diagnosed with primary invasive breast carcinoma in 1986-1992, referred to the British Columbia Cancer Agency for treatment and follow-up. TMA blocks were sectioned and stained. Slides were scored by a pathologist and only nuclear positivity was evaluated positive. Loss of nuclear positivity for any one of the four tested marker defined MMRD. Clinicopathological associations were tested by Chi-square, and survival by Kaplan-Meier plot with log rank test. Result: 1635 cases were interpretable for all MMR markers. 31 cases (1.9%) met criteria for MMRD. 6 cases had paired losses (4 MLH1-PMS2 loss, 2 MSH2-MSH6 loss) and the remaining 25 cases had singular MMR loss (11 PMS2 loss, 10 MLH1 loss, 3 MSH6 loss, 1 MSH2 loss). Deficiency of the the MutL complex (MLH1/PMS2) predominated over the MutS complex (MSH2/MSH6). Among the demographic and pathological variables assessed – age, grade, tumour size, lymphovascular invasion, nodal and menstrual status – high grade is associated with MMRD (p=0.014). In terms of biomarker, MMRD is significantly associated with PR negativity (p=0.003) and PD-L1 expression (p=0.049), but not with ER, Her2, Ki67, or basal breast cancer IHC markers, nor does MMRD significantly correlate with any of the established major intrinsic subtypes of breast cancer. Tumor infiltrating lymphocyte (TIL) counts are higher in MMRD cases (p=0.009). Although statistically not significant (small numbers), Kaplan-Meier plots of survival analysis demonstrated a trend for MMR loss to be associated with decreased breast cancer disease-specific and overall survival. Conclusion: This large series assessed by IHC corroborates findings from smaller genomic series that MMRD is present in about 2% of breast cancers. MMRD tumors are more likely to be high grade, low PR and immunologically active (higher PD-L1 expression and TIL counts). MMR deficiency is present across all major molecular subtypes (luminal, HER2, basal). Given the efficacy of PD1/PDL1 targeting agents in MMR deficient tumors of other types, evidence for the activity of these agents in MMR deficient breast cancers should be actively sought. Citation Format: Cheng AS, Leung SC, Gao D, Anurag M, Nielsen T, Ellis MJ. Mismatch repair protein loss in breast cancer: Clinicopathological associations in a large British Columbia cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-02.

Alterations in ERBB2 and BRCA and microsatellite instability as new personalized treatment options in small bowel carcinoma

BackgroundCarcinomas of the small bowel are rare tumors usually with dismal prognosis. Most recently, some potentially treatable molecular alterations were described. We emphasize the growing evidence of individualized treatment options in small bowel carcinoma.MethodsWe performed a DNA- based multi-gene panel using ultra-deep sequencing analysis (including 14 genes with up to 452 amplicons in total; KRAS, NRAS, HRAS, BRAF, DDR2, ERBB2, KEAP1, NFE2L2, PIK3CA, PTEN, RHOA, BRCA1, BRCA2 and TP53) as well as an RNA-based gene fusion panel including ALK, BRAF, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET and ROS1 on eleven formalin fixed and paraffin embedded small bowel carcinomas. Additionally, mismatch-repair-deficiency was analyzed by checking the microsatellite status using the five different mononucleotide markers BAT25, BAT26, NR-21, NR-22 and NR-27 and loss of mismatch repair proteins using four different markers (MLH1, MSH6, MSH2, PMS2).ResultsIn five out of eleven small bowel carcinomas we found potentially treatable genetic alterations. Three patients demonstrated pathogenic (class 5) BRCA1 or BRCA2 mutations – one germline-related in a mixed neuroendocrine-non neuroendocrine neoplasm (MiNEN). Two additional patients revealed an activating ERBB2 mutation or PIK3CA mutation.Furthermore two tumors were highly microsatellite-instable (MSI-high), in one case associated to Lynch-syndrome. We did not find any gene fusions.ConclusionOur results underscore, in particular, the relevance of potentially treatable molecular alterations (like ERBB2, BRCA and MSI) in small bowel carcinomas. Further studies are needed to proof the efficacy of these targeted therapies in small bowel carcinomas.

Targeted next-generation sequencing in the detection of mismatch repair deficiency in endometrial cancers

Mismatch repair deficiency represents a biomarker of immunotherapy response and a phenotypic feature of Lynch syndrome-associated endometrial cancers. Using a targeted next-generation sequencing assay, we identified molecular features of mismatch repair deficiency, specifically insertion and deletion mutations in mononucleotide repeats, and established thresholds for the number of such mutations to classify endometrial cancers as mismatch repair deficient, proficient, or indeterminate. Sequencing classification was compared to the loss of MLH1, MSH2, MSH6, or PMS2 expression by immunohistochemistry. A total of 259 endometrial cancers were classified by sequencing as mismatch repair deficient (n = 48, 19%), proficient (n = 199, 77%), or indeterminate (n = 12, 5%). Sequencing findings were concordant with loss of expression of at least one mismatch repair protein in 47 of 48 (98%) cases classified as deficient and retained expression of all four proteins in 190 of 199 (95%) cases classified as proficient. Of the 12 cases classified as indeterminate, 7 (58%) demonstrated mismatch repair protein loss. Overall, targeted next-generation sequencing exhibited a high rate of concordance with immunohistochemistry for mismatch repair deficiency; however, sequencing was indeterminate in a few cases and demonstrated a false negative rate of 5%. Although we recommend implementation of a mismatch repair deficiency algorithm for laboratories performing next-generation sequencing cancer panels, immunohistochemistry remains a cost-effective screening method for mismatch repair deficiency in endometrial cancer.

PD-L1 and CD8 are associated with deficient mismatch repair status in triple-negative and HER2-positive breast cancers

Triple-negative and HER2-positive breast cancers (BCs) are more aggressive than hormone receptor-positive/HER2-negative BCs and show higher levels of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression. Recently, US Food and Drug Administration approved anti-PD-L1 immunotherapy for solid tumors with deficient mismatch repair (MMR). In this study, we aimed to examine the prevalence of deficient MMR and its association with checkpoint immune markers in BCs. Immunohistochemistries (IHCs) with anti-MMR proteins (MLH1, PMS2, MSH2 and MSH6) and multiplex IHCs with anti-PD-L1, anti-CD8 or anti-CD163 were performed on tissue microarrays (TMAs) with 101 triple-negative BCs (TNBC) and 197 HER2-positive BCs. Additional IHCs for MMR proteins were also performed on whole-tissue sections from selected cases. Thirteen cases (4.4%) showed complete loss of MMR protein on TMAs, including 7 TNBCs (6.9%) and 6 HER2-positive BCs. On whole-tissue sections, only one of 13 cases showed complete loss of MMR proteins, while the other 12 cases showed partial loss. PD-L1 expression was identified in 37% of cases and was significantly higher in TNBCs than in HER2-positive BCs (71% versus 19%). Furthermore, BCs with complete/partial loss of MMR demonstrated significantly more PD-L1 and CD8 expressions than BCs with preserved MMR proteins. Although complete loss of MMR proteins exists in an extremely low frequency, partial loss is not uncommon in BCs. The association of partial loss of MMR proteins with increased PD-L1 and CD8 expression suggests a potential use of MMR testing as a screening method for anti-PD-L1 immunotherapy in BCs.

Discordant loss of mismatch repair proteins in advanced endometrial endometrioid carcinoma compared to paired primary uterine tumors

ObjectiveA subset of endometrial cancer is characterized by deficiencies in the mismatch repair (MMR) pathway. MMR testing is a well-established tool to screen for Lynch syndrome, but has also become a companion diagnostic test for immunotherapy. We compared the MMR status of primary and paired metastatic endometrial cancer to determine whether MMR deficiency can occur specifically in advanced endometrial cancer compared to primary tumor. MethodsMatched primary uterine and metastatic endometrioid adenocarcinoma from 2009 to 2018 at our institution were identified. PMS2 and MSH6 protein expression in metastatic and matched primary tumor was assessed using clinically validated immunohistochemistry methods for Lynch syndrome screening. MLH1 promoter hypermethylation and microsatellite instability (MSI) were performed in discordant cases. Results29 patients were identified with paired primary endometrial endometrioid adenocarcinoma and metastasis or recurrence after the original hysterectomy. Fourteen of 29 cases (48.2%, 14/29) were found to be MMR deficient at the metastatic or recurrent site. Two patients (6.9%, 2/29) showed discordant MMR status with PMS2 protein loss at the metastatic sites and intact expression in the primary uterine tumors. Both discordant cases exhibited abnormal subclonal loss at primary site and MLH1 promoter hypermethylation. High levels of microsatellite instability (MSI-H) was confirmed in one discordant metastatic site. ConclusionAdvanced endometrial cancer can rarely (~7%) show somatic loss of MMR protein expression in recurrent or metastatic sites compared to matched paired primary tumor. MMR testing of recurrent or metastasis should be considered for guiding immunotherapy if primary uterine tumor exhibits abnormal subclonal MMR loss.

Mismatch Repair Protein Loss as a Prognostic and Predictive Biomarker in Breast Cancers Regardless of Microsatellite Instability.

BackgroundBreast cancers that harbor mismatch-repair (MMR) deficiency and/or microsatellite instability (MSI) might be sensitive to immune checkpoint blockade, but there are currently no specific guidelines for assessing MMR status in breast cancer. Here, we sought to define the clinical value of MMR immunohistochemistry (IHC) and MSI analysis in breast cancers.MethodsWe subjected 444 breast cancers to MMR IHC and MSI analysis. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and MMR-heterogeneous (hMMR) based on the loss of immunoreactivity; MSI was defined by instability in the five indicators recommended by the National Cancer Institute for endometrial and colorectal cancers. Correlation of MMR status with patients’ survival was assessed using the Kaplan-Meier estimator. Statistical tests were two-sided.ResultsLoss of MMR proteins was homogeneous (dMMR) in 75 patients (17%) and heterogeneous (hMMR) in 55 (12%). Among luminal breast cancers, there were similar frequencies of dMMR and hMMR tumors. Overall, the rate of discrepancy between IHC and MSI analysis was high (91%). Women with Luminal B-like dMMR carcinomas (n = 44) showed shorter overall survival (median = 77 months, range = 0–115 months) than those with pMMR (n = 205) or hMMR (n = 35) tumors (median = 84 months, range = 0–127 months) (P = .008). On the contrary, patients with estrogen receptor-negative breast cancers treated with chemotherapy lived longer in cases of dMMR (n = 9) than pMMR (n = 33) or hMMR (n = 7) tumors, with 87 months of median survival (range = 73–123 months) for the former compared with 79 months (range = 8–113 months) for the latter two categories (P < .001).ConclusionsImmunohistochemistry and MSI are not interchangeable tests in breast carcinomas. MMR protein loss is a more common event than MSI and shows intra-tumor heterogeneity. MMR IHC allows the identification of clinically relevant subclasses of breast cancer patients, provided that multiple areas of the tumor are analyzed.

Routine Molecular Analysis for Lynch Syndrome Among Adenomas or Colorectal Cancer Within a National Screening Program

It is important to identify individuals with Lynch syndrome because surveillance programs can reduce their morbidity and mortality from colorectal cancer (CRC). We assessed the diagnostic yield of immunohistochemistry to detect Lynch syndrome in patients with advanced and multiple adenomas within our national CRC screening program. We performed a prospective study of all participants (n= 1101; 55% male; median age, 66 years; interquartile range, 61-70 years) referred to the Erasmus MC in The Netherlands after a positive result from a fecal immunohistochemical test, from December 2013 to December 2016. Colon tissues were collected from patients with advanced adenomas, ≥4 nonadvanced adenomas, or CRC, and analyzed by immunohistochemistry to identify patients with loss of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, or PMS2): a marker of Lynch syndrome. Specimens from patients with loss of MLH1 were analyzed for MLH1 promoter hypermethylation. Patients with an MMR-deficient tumor or adenoma without MLH1 promoter hypermethylation were referred for genetic analysis. At colonoscopy, 456 patients (41%) (65% male; mean age, 67 years; interquartile range, 63-71 years) were found to have CRC and/or an adenoma eligible for analysis by immunohistochemistry. Of 56 CRCs, 7 (13%) had lost an MMR protein and 5 had hypermethylation of the MLH1 promoter. Analyses of tumor DNA revealed that 2 patients without MLH1 promoter hypermethylation had developed sporadic tumors. In total, 400 patients with adenomas were analyzed. Of the examined adenomas, 208 (52%) had a villous component and/or high-grade dysplasia: 186 (47%) had a villous component and 41 (10%) had high-grade dysplasia. Only 1 adenoma had lost an MMR protein. This adenoma was found to have 2 somatic mutations in MSH6. In a CRC screening program in The Netherlands for individuals aged 55 to 75 years, routine screening for Lynch syndrome by immunohistochemistry analysis of colon tissues from patients with advanced and multiple adenomas identified no individuals with this genetic disorder.

PO-338 Recurrent glioblastoma: a complex scenario dominated by loss of MMR proteins

IntroductionGlioblastoma (GBM) is the most common primary brain tumour in adults and the Stupp protocol represents the standard of care. However, the tumour invariably relapses suggesting marked intra-tumour genetic heterogeneity enabling rapid adaptation to therapy. In-depth characterisation of recurrent GBM (rGBM) might contribute to better understand mechanisms behind tumour progression and enable rGBM treatment with targeted drugs.Material and methodsMatched GBM samples have been collected at diagnosis and recurrence from adult patients (n=57) treated with the Stupp protocol. Expression of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, MSH6) was evaluated by IHC, followed by exome sequencing of 3 pairs showing loss of MSH6 reactivity as well as of 3 MSH6 positive pairs. In addition, established genetic and epigenetic markers of GBM were investigated along with their correlation with loss of MMR proteins and patients’ survival.Results and discussionsAccording to IHC results, 13 out of 52 rGBM samples (25%) lacked expression of MMR proteins. In particular, 11 among the 13 samples (85%) showed partial or total reduction of MSH6 expression. Conversely, almost all GBM samples at diagnosis (96.4%) stained positive for the 4 MMR markers. Consistent with IHC data, exome sequencing disclosed lack of variants in MMR genes in primary samples whereas rGBM samples lacking MSH6 expression were mutated in the abovementioned genes and shared a c.3438+1G>A* splicing variant in MSH6 with a potential loss of function effect. Moreover, MSH6 negative relapsed specimens were characterised by 30 to 100-fold more variants compared to the matched primary ones and lacked microsatellite instability. Notably, MMR deficiency was associated with significant telomere shortening. Conversely, the tumour pairs expressing MMR proteins showed an almost comparable number of mutations in primary versus relapsed samples and absence of variants in MMR genes both in the initial tumours and in their recurrent counterpart.ConclusionOur study shows that IHC staining is a valuable tool to identify a subset of rGBM patients with alterations in MMR genes linked to high mutational burden and, hence, potentially eligible for drugs targeting immune checkpoint inhibitors.

Abstract 3361: Are mismatch repair (MMR) defects and microsatellite instability (MSI) relevant in renal cell and prostate carcinomas? Their significance for future therapies

Abstract Background Few studies have investigated mismatch repair (MMR) defects in renal and prostatic cancers. Normally, MMR accurately detects and corrects mistakes made during replication of a DNA strand. But, a defective MMR system is very likely to miss copy-number errors, particularly in microsatellites (short tandem base repeats). Alterations in function of MMR genes can result in microsatellite instability (MSI), known to facilitate tumor progression and poor prognosis. MMR deficiency is observed frequently in colonic and endometrial carcinoma as part of Lynch's syndrome, as well as occasionally in other neoplasms. In our study, we assayed MMR expression in a series of kidney tumors representing the spectrum of renal malignancies and in a wide-ranging group of prostate cancers (for which clinical information and Gleason grade was obtained in all cases). Materials and methods Immunohistochemical (IHC) staining of FFPE sections from 45 kidney cancers and 30 prostatic tumors was performed. The main MMR proteins of interest are MSH2 and MLH1, plus their heterodimer partners MSH6 and PMS2; staining was directed at these 4 targets. Antibody sources were Abcam, Cambridge, MA 02139 (MLH1, MSH6, PMS2) and Agilent Technologies, Wilmington, DE 19808 (MSH2). Also, PD-L1 expression was evaluated using antibody from Cell Signaling, Danvers, MA 01923. Results Forty-five cases of renal cell carcinoma (RCC) comprised 12 cases of clear cell VHL (von Hippel Lindau) related cancer, 11 papillary type 1, 11 chromophobe, and 11 HLRCC (hereditary leiomyomatosis and renal cell carcinoma). Only one case of chromophobe RCC with sarcomatoid differentiation showed complete loss of MLH1 and PMS2 (2% of 45 renal tumors). Of prostate cancers, only one of 30 tumors (3%) was completely negative for both MLH1 and PMS2; that tumor was Gleason grade 8. The remaining 29 cases were positive for all MMR proteins. Discussion Our study suggests that neither kidney nor prostate cancers are likely to possess significant MMR defects. In our series, clear cell, papillary and HLRCC tumors did not lack MMR expression; one chromophobe case negative for MMR (and presumed liable to MSI) may have developed genomic changes as disease progressed to a more aggressive, sarcomatoid pattern. Prostate cancer with MMR defects also appears to be rare, and we found no correlation with grade and stage. MMR defects lead to highly unstable microsatellite sequences and altered gene expression, so while only a small proportion of these tumors exhibited loss of MMR proteins, it is possible that in these few cases the loss may be significant and that such patients may benefit from new modalities of treatment. IHC detection of MMR defects (as predictors of probable MSI) may prove a useful prognostic tool in identification of rare renal and prostatic cancers likely to exhibit greater genetic instability and neoplastic advance. Citation Format: Xu Naizhen, Marston Linehan, Peter A. Pinto, Maria J. Merino. Are mismatch repair (MMR) defects and microsatellite instability (MSI) relevant in renal cell and prostate carcinomas? Their significance for future therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3361.

Molecular and Pathological Features of Gastric Cancer in Lynch Syndrome and Familial Adenomatous Polyposis.

Lynch syndrome (LS) and familial adenomatous polyposis (FAP) are autosomal dominant hereditary diseases caused by germline mutations leading to the development of colorectal cancer. Moreover, these mutations result in the development of a spectrum of different tumors, including gastric cancers (GCs). Since the clinical characteristics of GCs associated with LS and FAP are not well known, we investigated clinical and molecular features of GCs occurring in patients with LS and FAP attending our Institution. The Hereditary Tumor Registry was established in 1994 at the Department of Oncologic Gastroenterology, CRO Aviano National Cancer Institute, Italy. It includes 139 patients with LS and 86 patients with FAP. Patients were recruited locally for prospective surveillance. Out of 139 LS patients, 4 developed GC—3 in the presence of helicobacter pylori infection and 1 on the background of autoimmune diseases. All GCs displayed a high microsatellite instability (MSI-H) and loss of related mismatch repair (MMR) protein. One of the FAP patients developed a flat adenoma, displaying low-grade dysplasia at the gastric body, and another poorly differentiated adenocarcinoma with signet ring cells like Krukenberg without HP infection. LS carriers displayed a risk of GC. The recognition of HP infection and autoimmune diseases would indicate those at higher risk for an endoscopic surveillance. Regarding FAP, the data suggested the need of suitable endoscopic surveillance in long survivals with diffuse fundic gland polyps.

PO-467 Unclarified cases of microsatellite instability analyses for lynch syndrome diagnostics

IntroductionLynch syndrome (LS) is an autosomal dominantly inherited form of colorectal cancer (CRC) and is implicated in 2%–4% of CRC cases. LS develops from a mutation in one allele of one of the DNA mismatch repair (MMR) genes, most commonly MLH1 and MSH2, or less frequently MSH6 and PMS2. Loss of functional MMR proteins leads to defects in DNA repair and, subsequently, high DNA microsatellite instability (MSI-High). LS diagnostics currently consists of an analysis of MMR protein expression by means of Immunohistochemistry (IHC) and a molecular analysis to detect MSI. During our 10 year experience in performing LS diagnostics we have encountered few cases where IHC and MSI analyses do not match. This study is to clarify these inconsistent molecular alterations which will contribute to our understanding of LS diagnostics.Material and methodsOf 2335 LS cases 27 (1.1%) showed discrepant IHC and MSI results. To clarify this, IHC and MSI analyses were repeated if possible with different antibodies and different MSI markers (mononucleotide instead of dinucleotide markers). Using MS-MLPA MLH1 hypermethylation, BRAF V600E mutation status and LOH of the MMR genes was analysed. Finally, germline and somatic mutations in MMR genes were analysed by using NGS. Protein modelling was also performed to visualise structural protein changes.Results and discussionsAmong these 27 cases, due to the decline of patients for further testing, 4 cases were excluded from further analyses. Of the remaining cases in 2 cases using a different antibody explained the unexpected results and in 2 cases switching from dinucleotide to mononucleotide MSI markers. In respectively 8 and 4 cases somatic and germline mutations explained the IHC and MSI results. Finally, in 2 cases protein modelling of identified mutations explained the presence of protein staining in MSI-high cases. The rest cases cannot be further studied due to the lack of tissues.ConclusionBased on these results, we have concluded: 1. Mononucleotide markers are more sensitive to detect microsatellite instability. 2. Protein modelling can explain presence of protein staining for mutations that do not cause a major conformational change. 3. Somatic sequencing is a valuable addition to Lynch syndrome diagnostics.

Holocrine Poroma: A Distinctive Adnexal Tumor.

There are 36 cases of complex poroid tumors with folliculosebaceous and apocrine differentiation reported in the literature. The authors evaluated 111 poroid tumors including 63 typical eccrine poromas and 48 poroid tumors with folliculosebaceous elements. Folliculosebaceous poroid tumors (FSPT) had basaloid and squamous cells (100%), ducts with steatocystoma-like cuticles and holocrine secretions (89.6%), infundibular follicular structures (66.7%), and entrapped sebocytes (56.3%). No definite apocrine decapitation secretions in FSPT were found. Immunohistochemistry was strongly positive for CK903 and focally positive for CAM5.2, epithelial membrane antigen, and carcinoembryonic antigen. No loss of MLH-1, MSH-2, or MSH-6 mismatch repair proteins was found. FSPT had distinctive features that differentiate them from eccrine poromas including the frequent head and neck locations (62.5% vs. 20.6%, P < 0.01), squamous cytology (100% vs. 1.6%, P < 0.01), more prominent cytoplasmic vacuolization (score 1.4/4.0 vs. 0.3/4.0, P < 0.01), presence of infundibular follicular structures (score 1.2/4.0 vs. 0.03/4.0, P < 0.01), presence of ducts with steatocystoma-like cuticles (89.6% vs. 0.0%, P < 0.01), and less stromal hyalinization (score 1.0/4.0 vs. 2.5/4.0, P < 0.01). The authors propose that FSPT are distinctive benign tumors originating from the sebaceous gland duct and are therefore best categorized as holocrine poroma.

A phase II study of frontline paclitaxel/carboplatin/bevacizumab, paclitaxel/carboplatin/temsirolimus, or ixabepilone/carboplatin/bevacizumab in advanced/recurrent endometrial cancer

ObjectivePaclitaxel and carboplatin (PC) is a standard initial therapy for advanced endometrial cancer. We evaluated the efficacy and tolerability of incorporating three novel agents into initial therapy. MethodsIn this randomized phase II trial, patients with chemotherapy-naïve stage III/IVA (with measurable disease) and stage IVB or recurrent (with or without measurable disease) endometrial cancer were randomly assigned to treatment with PC plus bevacizumab (Arm 1), PC plus temsirolimus (Arm 2) or ixabepilone and carboplatin (IC) plus bevacizumab (Arm 3). The primary endpoint was progression-free survival (PFS). Comparable patients on the PC Arm of trial GOG209 were used as historical controls. Secondary endpoints were response rate, overall survival (OS), and safety. ResultsOverall, 349 patients were randomized. PFS duration was not significantly increased in any experimental arm compared with historical controls (p > 0.039). Treatment HRs (92% CI) for Arms 1, 2, and 3 relative to controls were 0.81 (0.63–1.02), 1.22 (0.96–1.55) and 0.87 (0.68–1.11), respectively. Response rates were similar across arms (60%, 55% and 53%, respectively). Relative to controls, OS duration (with censoring at 36 months), was significantly increased in Arm 1 (p < 0.039) but not in Arms 2 and 3; the HRs (92% CIs) were 0.71 (0.55–0.91), 0.99 (0.78–1.26), and 0.97 (0.77–1.23), respectively. No new safety signals were identified. Common mutations and rates of mismatch repair protein loss are described by histotype. Potential predictive biomarkers for temsirolimus and bevacizumab were identified. ConclusionPFS was not significantly increased in any experimental arm compared to historical controls. NRG Oncology/Gynecologic Oncology Group Study GOG-86P.

Prevalence and molecular characteristics of DNA mismatch repair protein-deficient sebaceous neoplasms and keratoacanthomas in a Japanese hospital-based population.

Muir-Torre syndrome (MTS) is currently considered as a clinical variant of Lynch syndrome (LS). The clinical significance of the screening of patients with MTS-associated cutaneous tumors for the identification of LS has not yet been established. In addition, the prevalence and molecular characteristics of mismatch repair (MMR) protein deficiency in such tumors has scarcely been investigated in the Japanese population. Immunohistochemistry (IHC) for MMR proteins (MLH1, MSH2, MSH6 and PMS2) was performed in formalin-fixed paraffin-embedded sections prepared from 16 sebaceous neoplasms (SNs) resected from 13 patients and 32 keratoacanthomas (KAs) resected from 31 patients at our institution between January 2005 and March 2014. Tumors showing MMR protein loss were further subjected to genetic analysis for detecting the presence of germline and/or somatic alterations of the MMR genes to identify the precise molecular mechanisms underlying the protein loss. Among the 16 SNs resected from 13 patients, eight SNs resected from five patients (38.5%) showed loss of expression of MMR proteins (MLH1/PMS2 loss, one patient; MSH2/MSH6 loss, four patients). Genetic analyses showed a pathogenic germline MSH2 mutation in one patient, somatic hypermethylation of the MLH1 promoter region in one patient, and somatic alterations of MSH2 without detectable germline mutations of MSH2 in three patients. None of the KAs examined in the study showed any loss of MMR protein expression. The efficacy of routine screening of cutaneous neoplasms known to be associated with MTS by IHC for MMR proteins to identify LS may be fairly limited. MMR protein loss as determined by IHC in SNs is not always diagnostic of LS, and appears, in most cases, to be a result of somatic inactivation of the MMR genes.

Extent of field change in colorectal cancers with BRAF mutation.

Sporadic colorectal cancers with BRAF mutations constitute two distinct subgroups of colorectal cancers. Recent studies have linked the presence of the BRAF mutation to a familial inheritance pattern. This was a proof-of-concept study that aimed to examine: (a) the extent of field change in sporadic colorectal cancers with BRAF mutation; and (b) the extent of resection margins required and the pattern of DNA mismatch repair protein loss in these tumours. Eight microsatellite instability-high tumours with positive BRAF mutation from an existing histopathological database were selected for BRAF mutation and mismatch repair protein analysis. All the resection margins were negative for BRAF mutation. Three tumours had loss of MLH1 and PMS2 expressions, and five tumours had no protein loss. Six peritumoral tissues were negative and one was positive for BRAF mutation. The results suggest that any early field change effect is restricted to the immediate vicinity of the tumour and is not a pan-colonic phenomenon. Current guidelines on resection margins are adequate for BRAF mutation-positive colorectal cancers. Any suggestion of a hereditary link to these tumours is likely not related to germline BRAF gene mutations. The pattern of protein loss reinforces previous findings for the two subgroups of BRAF mutation-positive colorectal cancers.

Clinicopathologic characteristics of double primary endometrial and colorectal cancers in a single institution.

To investigate the clinicopathologic and genetic correlations between double primary endometrial and colorectal cancer related to Lynch syndrome and to analyze germline mutations in mismatch repair genes in endometrial cancer patients in Korea. Thirteen patients diagnosed with pathologically endometrial and colorectal cancer between January 2005 and November 2016 in a single institution were enrolled in the study. The medical records were retrospectively analyzed. The genetic mutational information of endometrial cancer in Korea was retrieved from the literature review. Endometrial cancer was diagnosed first in eight (62%) patients, and one patient was diagnosed with colorectal cancer first. Endometrioid adenocarcinoma was reported in 10 of 13 (77%) endometrial cancer patients. Endometrial cancer was found at the low uterine segment in three patients. Three of four patients had high microsatellite instability. The loss of mismatch repair proteins was confirmed in 7 of 11 cases using immunohistochemistry. Four patients fulfilled clinical criteria based on a family history of cancer. Overall, the incidence of suspected Lynch syndrome was 77% (10/13). Four of them underwent genetic testing and three were found to have a pathogenic germline mutation. A possible founder mutation, c.1757_1758insC in MLH1, was observed in 21 germline mutation information from literature review. The present study describes the clinicopathologic data of double primary endometrial and colorectal cancer patients and supports that these patients should undergo closed approach for Lynch syndrome. Moreover, a possible founder mutation in Korean endometrial cancer patients was identified.

The Relationship Between Mismatch Repair Deficiency and PD-L1 Expression in Breast Carcinoma.

Mismatch repair (MMR) deficiency in solid tumors has recently been linked to susceptibility to immunotherapies targeting the programmed cell death-1 (PD-1)/programmed cell death-1 ligand (PD-L1) axis. Loss of MMR proteins has been shown to correlate with tumoral PD-L1 expression in colorectal and endometrial carcinomas, but the association between expression of MMR proteins and PD-L1 has not previously been studied in breast carcinoma, where MMR deficiency is less common. We assessed the relationship between PD-L1 and MMR protein expression by immunohistochemistry in 245 primary and 40 metastatic breast carcinomas. Tumoral staining for PD-L1 was positive in 12% of all cases, including 32% of triple-negative cancers. MMR deficiency was observed in 0.04% of breast cancers; the single MMR-deficient case was a high-grade, triple-negative ductal carcinoma which showed dual loss of MLH1 and PMS2 proteins and expressed PD-L1. Two ER carcinomas initially were scored with MMR protein loss in tissue microarray format but were subsequently shown to be MMR-intact on whole sections. Analysis of MMR gene mutation in The Cancer Genome Atlas corroborates low frequency of MMR deficiency for invasive breast cancer. MMR protein expression is therefore unlikely to show utility as a screen for immunotherapeutic vulnerability in this tumor type, and may provoke unwarranted genetic testing in patients unlikely to have a heritable cancer syndrome. PD-L1 may be a more clinically relevant biomarker for anti-PD-1/PD-L1 therapies in this setting.