Loss Of Mismatch Repair Research Articles

Loss of mismatch repair signaling impairs the WNT-bone morphogenetic protein crosstalk and the colonic homeostasis.

The fine balance between proliferation, differentiation, and apoptosis in the colonic epithelium is tightly controlled by the interplay between WNT, Notch, and bone morphogenetic protein (BMP) signaling. How these complex networks coordinate the colonic homeostasis, especially if cancer predisposing mutations such as mutations in the DNA mismatch repair (MMR) are present, is unclear. Inactivation of the MMR system has long been linked to colorectal cancer; however, little is known about its role in the regulation of the colonic homeostasis. It has been shown that loss of MMR promotes the proliferation of colon epithelial cells that renders them highly susceptible to transformation. The mechanism through which MMR mediates this effect, yet, remains to be determined. Using an MMR-deficient mouse model, we show that increased methylation of Dickkopf1 impacts its expression, and consequently, the ability to negatively regulate WNT signaling. As a result, excessive levels of active β-catenin promote strong crypt progenitor-like phenotype and abnormal proliferation. Under these settings, the development and function of the goblet cells are affected. MMR-deficient mice have fewer goblet cells with enlarged mucin-loaded vesicles. We further show that MMR inactivation impacts the WNT–BMP signaling crosstalk.

Identification, incidence and clinical outcomes of patients (pts) with hypermutated prostate cancer (PC).

195 Background: A small proportion of metastatic PC exhibit outlier somatic mutation rates. The incidence, clinical course and treatment response of pts with hypermutation (HM) is poorly characterised. Methods: We performed targeted sequencing of 1047 plasma cell-free DNA samples and calculated somatic mutation burden. HM samples and available matched archival tissue were additionally subjected to whole exome sequencing. Trinucleotide mutational signatures and microsatellite instability (MSI) were determined via nonnegative matrix factorization and mSINGS, respectively. We evaluated PSA decline ≥50% from baseline (PSA50), time from androgen deprivation therapy (ADT) to castration-resistant prostate cancer (CRPC), median duration of 1st line CRPC therapy (1L CRPCT) and median OS (time from CRPC to death). The control cohort consisted of 199 CRPC pts treated with 1L abiraterone + prednisone (ABI+P) or enzalutamide (ENZ). Results: 671 samples from 434 pts had ctDNA% > 2 and were evaluable. The median mutation rate was 2.59/Mb (range, 0.9 – 155.6/Mb). 32 samples from 24 pts had > 11/Mb and fell above the 95th percentile for mutational burden. 10/24 pts had biallelic loss of mismatch repair (MMR) genes MSH2/6, and a further 5 pts without confirmed MMR defects had enrichment of trinucleotide signatures associated with MMR and/or were MSI high by mSINGS. The remaining 9 pts had either BRCA2 mutations or Kataegis (localized hypermutation). Clinical data was available for 10/15 MMR defective pts. Median age was 73.6 y. At diagnosis, 70% had Gleason score ≥8, 50% with M1 disease, median PSA was 22.8 (6.8 – 820). PSA50 with ADT (n = 8) or ADT + docetaxel (n = 2) was 100% in the castration sensitive setting. 5 pts had ENZ, 4 ABI + P, and 1 cabazitaxel in 1L CRPCT. Comparing the MMR defective with the control cohort, median time from ADT to CRPC was 9.1 m (95% CI 6.9 – 11.4) vs. 18.2 m (95% CI 15.1 – 21.3), p = 0.001; 1L CRPCT duration was 3.9 m (95% CI 1.3 – 6.5) vs. 8.4 m (95% CI 7.2 – 9.6), p = < 0.001; median OS was 13.1 m (95% CI 0.33 – 25.9) vs. 40.1 m (95% CI 32.4 – 47.8), p < 0.001. Conclusions: HM and MMR defects can be identified in a liquid biopsy. Although these pts can have poor outcomes with standard therapy, ctDNA may help selection for immunotherapy.

Incidence of Mismatch Repair Protein Deficiency and Associated Clinicopathologic Features in a Cohort of 104 Ovarian Endometrioid Carcinomas.

Patients with Lynch syndrome have up to a 24% risk of developing ovarian carcinoma, but universal mismatch repair (MMR) protein testing of ovarian carcinomas is not standard practice in most institutions. We reviewed 104 unselected ovarian endometrioid carcinomas (OEC) for various clinicopathologic features to determine if any are predictive of MMR loss. Immunohistochemistry for all 4 MMR proteins was performed followed by MLH1 promoter methylation analysis when indicated. Overall, patients had a mean age of 55 years and tumors averaged 12 cm. Most (72%) patients had stage I tumors, 63% were grade 1, and 30% had a synchronous stage IA endometrial endometrioid carcinoma. Peritumoral lymphocytes and intratumoral stromal inflammation were rare, but tumor-infiltrating lymphocytes averaged 47/10 high-power fields. Endometriosis was noted in 71%, adenofibromatous background in 14%, and both in 14% of tumors. Metaplastic changes were common and included squamous metaplasia (63%), clear cell change (32%), mucinous differentiation (24%), and sex cord-like elements (13%). When follow-up was available (n=99), 78% of patients were alive and well, 12% died from disease, 6% died from other causes, and 4% were alive with disease. Unmethylated, MMR-deficient OECs were identified in 7% of the cohort and included MSH2/MSH6 (n=4), MSH6 (n=2), and PMS2 (n=1). All these tumors were stage I, 71% grade 1, and 57% had a synchronous endometrial endometrioid carcinoma. Among patients in this group with follow-up (n=5), all were alive without evidence of disease (mean 150 mo). Given that no clinicopathologic features were associated with MMR deficiency on univariate analysis, this study highlights the importance of universal MMR screening in OECs.

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.

In search of effective therapies to overcome resistance to Temozolomide in brain tumours.

Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6-methylguanine (O6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease.

Heterogenous loss of mismatch repair (MMR) protein expression: a challenge for immunohistochemical interpretation and microsatellite instability (MSI) evaluation.

Immunohistochemistry (IHC) for mismatch repair (MMR) proteins is used to identify MMR status: being diffusely positive (intact/retained nuclear staining) or showing loss of nuclear tumour staining (MMR protein deficient). Four colonic adenocarcinomas and a gastric adenocarcinoma with associated dysplasia that displayed heterogenous IHC staining patterns in at least one of the four MMR proteins were characterised by next‐generation sequencing (NGS). In order to examine a potential molecular mechanism for these staining patterns, the respective areas were macrodissected, analysed for microsatellite instability (MSI) and investigated by NGS and multiplex ligation‐dependent probe amplification (MLPA) analysis of MLH1, MSH2, MSH6 and PMS2 genes, including MLH1 methylation analysis. One colonic adenocarcinoma showed heterogenous MSH6 IHC staining and molecular analysis demonstrated increasing allelic burden of two MSH6 frameshift variants (c.3261delC and c.3261dupC) in areas with MSH6 protein loss compared to areas where MSH6 was retained. Two colonic adenocarcinomas with heterogenous MLH1 staining showed no differences in sequence variants. In one of these cases, however, MLH1 was hypermethylated in the area of MLH1 loss. Another colon carcinoma with heterogenous PMS2 staining (but with retained MSH6) showed both MSH6 c.3261dupC and 3260_3261dupCC where PMS2 protein was lost and only c.3261dupC where PMS2 was retained. The gastric carcinoma showed complete loss of MSH6 in dysplastic foci, while the underlying invasive carcinoma showed retention of MSH6. Both these areas, however, were MSI‐high and showed the same MSH6 variant: c.3261delC. The gastric dysplasia additionally showed MSH6 c.3261dupC. In four of the five cases where MMR protein was lost, these areas were MSI‐high. Heterogenous MMR IHC (focal and/or zonal within the same tumour or between invasive and dysplastic preinvasive areas) is not always due to artefact and is invariably related to MSI‐high status in the areas of loss. An interesting aspect to this study is the presence of MSH6 somatic mutations irrespective of whether MSH6 IHC staining was intact or lost.

Patients with hMLH1 or/and hMSH2-deficient Metastatic Colorectal Cancer Are Associated with Reduced Levels of Vascular Endothelial Growth Factor-1 Expression and Higher Response Rate to Irinotecan-based Regimen.

The benefit of IFL (irinotecan, fluorouracil and leucovorin) regimen for metastatic colorectal cancer patients (mCRCs) with high levels of microsatellite instability (MSI-H) or loss of mismatch repair (dMMR) protein expression, is uncertain. This study investigated the association of tumour MMR-status and VEGF-1 expression with response to first-line IFL regimen in mCRCs. This prospective study analyzed patients diagnosed with mCRC between August 1st, 1998, and August 30th, 2003, at the Turku University Hospital, Finland. All patients received postoperative IFL regimen. Tumour expression of the MMR proteins, hMLH1 and hMSH2, and VEGF-1 expression were assessed by immunohistochemistry (IHC). Tumours with dMMR were those demonstrating loss of MMR protein expression, and tumours with high VEGF-1 expression were those showing moderate or strong cytoplasmic staining. The primary endpoint was the association between tumour hMLH1 or/and hMSH2-deficient and VEGF-1 expression; the relation between tumour MMR-status and IFL response rate was the secondary endpoint. Of the 67 mCRCs patients, 29 (43%) were hMLH1 or/and hMSH2-deficient and 15 (22%) were pMMR mCRCs. At diagnosis, patients with hMLH1 or/and hMSH2-deficient tumours expressed lower levels of VEGF-1 compared to pMMR tumour patients (p=0.01). More than half (n=17, 59%) of those with dMMR were chemosensitive to first-line IFL regimen, while just one-fifth (n=3, 20%) of those with pMMR were chemosensitive to the IFL regimen (p=0.045). Association between MMR-status and VEGF-1 expression predicts clinical outcome in mCRC patients.

Constitutional mismatch repair-deficiency: current problems and emerging therapeutic strategies.

Mismatch repair (MMR) proteins remove errors from newly synthesized DNA, improving the fidelity of DNA replication. A loss of MMR causes a mutated phenotype leading to a predisposition to cancer.In the last 20 years, an increasing number of patients have been described with biallelic MMR gene mutations in which MMR defects are inherited from both parents. This leads to a syndrome with recessive inheritance, referred to as constitutional mismatch repair-deficiency (CMMRD). CMMRD is a rare childhood cancer predisposition syndrome. The spectrum of CMMRD tumours is broad and CMMRD-patients possess a high risk of multiple cancers including hematological, brain and intestinal tumors. The severity of CMMRD is highlighted by the fact that patients do not survive until later life, emphasising the requirement for new therapeutic interventions.Many tumors in CMMRD-patients are hypermutated leading to the production of truncated protein products termed neoantigens. Neoantigens are recognized as foreign by the immune system and induce antitumor immune responses. There is growing evidence to support the clinical efficacy of neoantigen based vaccines and immune checkpoint inhibitors (collectively referred to as immunotherapy) for the treatment of CMMRD cancers. In this review, we discuss the current knowledge of CMMRD, the advances in its diagnosis, and the emerging therapeutic strategies for CMMRD-cancers.

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.

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.

Microsatellite instability in metaplasia-dysplasia-adenocarcinoma sequence of Barrett esophagus: a retrospective study.

AimTo analyze the loss of mismatch repair (MMR) system protein expression in metaplasia-dysplasia-adenocarcinoma sequence of Barrett esophagus (BE).MethodsThis study retrospectively analyzed the data from 70 patients with pathohistological diagnosis of BE or esophageal adenocarcinoma (EAC) treated at the Clinical Department of Pathology and Cytology, University Hospital Center Zagreb, from January 2009 to January 2011. Patients were divided into three groups: BE without dysplasia (22 patients), BE with dysplasia (37 patients), and EAC (11 patients). Immunohistochemical expression of MutL homologue 1 (MLH1), MutS homologue 2 (MSH2), postmeiotic segregation increased 2 (PMS2), and MutS homologue 6 (MSH6) of DNA MMR system was measured and compared with tumor protein p53 expression.ResultsA total of 81.8% and 81.8% patients with EAC, 32.4% and 35.1% patients with dysplasia, and 50% and 54.5% patients without dysplasia had loss of MLH1 and PMS2 expression, respectively. Patients with EAC and patients with dysplasia did not have loss of MSH2 and MSH6 expression, and 18.2% patients without dysplasia had loss of MSH2 and MSH6 expression. There was a strong positive correlation between MLH1 and PMS2 expression (Spearman ρ 0.97; P < 0.001) and between MSH2 and MSH6 expression (Spearman ρ 0.90, P < 0.001) in the entire sample and in all BE groups. No significant correlations of MLH1 and PMS2 with p53 expression were found, except in dysplasia group (φ 0.402, P = 0.030 for MSH1; φ 0.371, P = 0.042 for PMS2).ConclusionAlthough we demonstrated considerable loss of MLH1 and PMS2 expression in BE-associated carcinoma sequence, due to the retrospective study design and low number of patients we cannot conclude that MLH1 and PMS2 can be used as biomarkers for patient surveillance and therapy-making decisions.Oxford Centre for Evidence-based Medicine level of evidence: 3

Abstract P4-04-01: Loss of mismatch repair predicts resistance to endocrine therapy and sensitivity to CDK4/6 inhibitors in ER+ breast cancer

Abstract Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic resistance occurs in ˜1/3 of patients and acquired resistance in ˜1/5 of the remainder. While many resistance mechanisms have been explored, clinical trials testing associated therapeutics have had mixed outcomes. Understanding mechanisms of resistance and finding therapeutic strategies to target them, therefore, remain important challenges. In this study we discover an intriguing link between mismatch repair (MMR) deficiency, specifically of the MutL complex (MLH1/3, PMS1/2), and endocrine therapy resistance in ER+ disease. We find a direct role for MutL loss in inducing endocrine therapy resistance in vitro and in vivo by knocking down multiple MutL genes using CRISPR and stable shRNA approaches validated using standard rescue experiments. We identify the underlying mechanism: MutL deficiency in ER+ breast cancer abrogates Chk2-mediated feedback inhibition of CDK4/6 that appears necessary for endocrine therapy responsiveness. Consequently, pharmacological targeting of CDK4/6 in vitro and in vivo significantly inhibits growth of endocrine therapy resistant MutL-deficient ER+ breast cancer cells. These results are corroborated by data from a neoadjuvant clinical trial demonstrating that cell cycle regulation of MutL-mutant tumors is estrogen-independent but CDK4/6 dependent. The results of this study provide important biological and clinically relevant insights. 1) a novel role for MMR in endocrine therapy resistance 2) A mechanism underlying the effectiveness of CDK4/6 inhibitors in some de novo endocrine therapy resistant tumors. While there are currently no biomarkers to guide the use of CDK4/6 inhibitors for ER+ breast cancer, markers of MMR dysregulation could identify patients in whom CDK4/6 inhibition may, most effectively, prevent disease recurrence. Citation Format: Haricharan S, Punturi N, Singh P, Holloway KR, Anurag M, Schmelz J, Schmidt C, Lei JT, Suman V, Hunt K, Olson Jr JA, Hoog J, Li S, Huang S, Edwards DP, Kavuri SM, Bainbridge MN, Ma CX, Ellis MJ. Loss of mismatch repair predicts resistance to endocrine therapy and sensitivity to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-01.

Pattern of mismatch repair protein loss and its clinicopathological correlation in colorectal cancer in North India

To determine the mismatch repair (MMR) protein loss in colorectal cancer (CRC) in north Indian patients and its clinicopathological correlation. A prospective study on patients with colorectal cancer from a tertiary level hospital conducted between May 2014 and June 2015. MMR protein loss was determined using immunohistochemistry for MLH1, MSH2, PMS2 and MSH6. 52 patients (38 male and 14 females) of CRC, with median age of 52.5 years who underwent resection form the study group. 18 (35%) patients were < 50 years of age. Family history of malignancy was present in 3 (6 %) patients. A total of 15 (29%) patients had loss of MMR protein of which 7 (46%) were < 50 years. Most common MMR loss was combined loss of MSH2 + MSH6 [6 (11.5%)] followed by isolated loss of PMS2 [5 (9.6%)]. MMR protein loss was more frequent in patients with right side colon cancer [12 (42%)] compared to left [3 (13%)] (p = 0.033). MMR protein loss was seen in 11 (34%) out of 32 patients fulfilling the revised Bethesda criteria compared to 4 (20%) out of 20 patients who did not fulfil the criteria (p = 0.352). This study demonstrates high frequency of MMR protein loss in colorectal cancer in north Indian patients which was more common in right colon cancer. Many patients having MMR protein loss do not satisfy the revised Bethesda criteria and would have been missed if selective testing was done. Further research and larger studies are required to validate these findings and develop India specific clinical criteria.

Intratumoural evolutionary landscape of high-risk prostate cancer: the PROGENY study of genomic and immune parameters.

BackgroundIntratumoural heterogeneity (ITH) is well recognised in prostate cancer (PC), but its role in high-risk disease is uncertain. A prospective, single-arm, translational study using targeted multiregion prostate biopsies was carried out to study genomic and T-cell ITH in clinically high-risk PC aiming to identify drivers and potential therapeutic strategies.Patients and methodsForty-nine men with elevated prostate-specific antigen and multiparametric-magnetic resonance imaging detected PC underwent image-guided multiregion transperineal biopsy. Seventy-nine tumour regions from 25 patients with PC underwent sequencing, analysis of mutations, copy number and neoepitopes combined with tumour infiltrating T-cell subset quantification.ResultsWe demonstrated extensive somatic nucleotide variation and somatic copy number alteration heterogeneity in high-risk PC. Overall, the mutational burden was low (0.93/Megabase), but two patients had hypermutation, with loss of mismatch repair (MMR) proteins, MSH2 and MSH6. Somatic copy number alteration burden was higher in patients with metastatic hormone-naive PC (mHNPC) than in those with high-risk localised PC (hrlPC), independent of Gleason grade. Mutations were rarely ubiquitous and mutational frequencies were similar for mHNPC and hrlPC patients. Enrichment of focal 3q26.2 and 3q21.3, regions containing putative metastasis drivers, was seen in mHNPC patients. We found evidence of parallel evolution with three separate clones containing activating mutations of β-catenin in a single patient. We demonstrated extensive intratumoural and intertumoural T-cell heterogeneity and high inflammatory infiltrate in the MMR-deficient (MMRD) patients and the patient with parallel evolution of β-catenin. Analysis of all patients with activating Wnt/β-catenin mutations demonstrated a low CD8+/FOXP3+ ratio, a potential surrogate marker of immune evasion.ConclusionsThe PROGENY (PROstate cancer GENomic heterogeneitY) study provides a diagnostic platform suitable for studying tumour ITH. Genetic aberrations in clinically high-risk PC are associated with altered patterns of immune infiltrate in tumours. Activating mutations of Wnt/β-catenin signalling pathway or MMRD could be considered as potential biomarkers for immunomodulation therapies.Clinical Trials.gov IdentifierNCT02022371

Clinical Impact of Mismatch Repair Protein Testing on Outcome of Early Staged Colorectal Carcinomas.

Colorectal cancer is the third most common cancer in men and second most common in women globally. In the present study, we aimed to analyse the proportion of patients with loss of immunostaining for mismatch repair (MMR) proteins in all newly diagnosed stage II cases of colorectal cancer for the purpose of prognostication, for determination of further chemotherapeutic strategy and for familial screening. From January 2014 to December 2015, 62 consecutive newly diagnosed cases of stage II colorectal cancer were included in the study. Details of each patient related to their demographic profile and tumour profile were recorded. All the cases were grossed and staged according to College of American Pathologist (CAP) guidelines. The expression of MMR proteins (which was earlier validated on normal as well as tumour tissue) in FFPE tumour tissue using IHC for mut L homologue 1 (MLH1), mut S homologue 2 (MSH2), mut S homologue 6 (MSH6) and post-meiotic segregation increased 2 (PMS2) was studied. Information regarding stage, treatment, clinical outcome and overall survival was retrieved when available. Out of a total of 371 cases, 62 (16.7%) cases were of stage II CRC, out of which 43 (12%) were treatment naive. Among the selected 62 cases, 26 (41.9%) demonstrated loss of MMR proteins and 36 (58.0%) cases had intact nuclear expression. Out of the cases with MMR loss, 38.4% showed loss of MLH1 and PMS2, 30.7% showed loss of MSH2 and MSH6, 26.9% showed isolated loss of PMS2 and 3.8% showed isolated loss of MSH6. Right-sided location (57.6%) was more common than left-sided (19.2%) and transverse colon (23.0%). Majority of the cases were moderately differentiated (65.3%) in morphology. There was no intratumoural infiltrate in most of the cases (53.8%), and only 3.8% cases showed marked intratumoural infiltrate. Also, peritumoural lymphocytic infiltrate was mild to moderate in most of the cases (26.9%) and marked Crohn's-like infiltrate was seen in only 7.6% cases. Our study shows that the routine evaluation of MMR proteins is achievable and essential for the purpose of prognostication, planning of treatment strategies and ascertaining a hereditary basis of CRC. The incidence of MMR protein loss was quite high in our study compared to other studies probably due to a difference in ethnicity. Though a right-sided predominance was supported, none of the typical morphological features of microsatellite instability (MSI) tumours were substantiated by our study, highlighting the lack of importance of histology for predicting MSI, and emphasising the point that MSI testing should be done as a routine procedure in all stage II CRC. A short follow-up was done for all our cases and comparison between the survival of the chemotherapy treated MSI cases versus those which were treatment naïve was performed and revealed that chemotherapy (CT) did not provide additional benefit to survival; MSI tumours in general are a better prognostic category and do not require additional chemotherapy.

Somatic tumor profiling of DNA mismatch repair (MMR) deficient endometrial cancers (EC).

e17121 Background: Approximately 20% of EC have loss of MMR ( MSH2, MSH6, MLH1, PMS2) protein expression by immunohistochemistry (IHC). The majority have somatic MLH1/PMS2 loss, driven by MLH1 promoter hypermethylation. For the remaining patients (pts), germline testing for Lynch Syndrome (LS) is recommended. However, half do not have a corresponding germline mut. This is considered “Lynch-like syndrome” (LLS) &amp; clinical management is challenging. We sought to determine if tumor profiling could identify somatic mut potentially underpinning loss of protein expression. Methods: Per institutional standard, all EC, regardless of age or family history undergo reflex LS screening with IHC for MMR protein expression. Pts consented to IRB approved protocols. Tumor-normal sequencing was performed via custom next-generation sequencing panel (MSK-IMPACT). Electronic medical records were reviewed. Results: 16 pt have completed tumor sequencing, median age 53 (35-83), 6 (38%) &lt; 50 yrs at diagnosis. 2 had personal history of additional cancer (DCIS, ovary), none had first degree relative with colon or EC. A mix of EC histologies was represented: 10 endometrioid (all grades), 2 clear cell, 4 mixed. There were no serous cancers. There were median 58 mut (9-546), 14 (88%) had hyper or ultra mutated EC. 5 EC were driven by somatic POLE mut (3 known hotspot, 1 likely pathogenic), all ultra-mutated phenotype, resulting in multiple somatic MSH6 muts with isolated IHC MSH6 loss. 4 EC had MSH2/MSH6 IHC loss with corresponding double somatic mut in MSH2. 5 had one somatic mut corresponding to the MMR protein loss, assessment of LOH in these cases is pending. Two cases are unexplained: 40 yo with IHC MLH1 loss, 47mut; 69 yo, IHC MSH6 loss, 12 mut. In this cohort of LLS, somatic muts were frequently observed in ARID1A (13,81%), PTEN (10,63%) &amp; PIK3CA(9,56%), in keeping with non serous histologies. Conclusions: In line with our prior report that pt with LLS had benign personal &amp; family cancer histories compared with LS pts, we have identified that in 56% of LLS EC either POLE mut or double somatic MMR mut likely underpins the MMR IHC loss. As such, in these LLS EC cases, somatic tumor profiling may help to rule out LS. Further testing is ongoing to increase cohort size.

Clinical utility of Oncotype DX in early stage colon cancer.

e15076 Background: With the advent of colon cancer screening, patients with early stage colon cancer will be more common in our clinics. The evidence supporting the absolute benefit of chemotherapy in resected Stage II and (to a lesser extent) Stage IIIA disease is poor. Not all patients benefit from chemotherapy and toxicity is a problem. There is a need for validated biomarkers to assess individual patient recurrence risk and discriminate absolute treatment benefit. Several studies have validated the role of the OncotypeDX testing in Stage II/IIIA disease. Our objective is to characterize whether this test impacted oncologists’ decisions in treating patients with Stage II/IIIA in the adjuvant setting. Methods: :The Onco typeDX assay is a multi-gene reverse-transcriptase-polymerase-chain-reaction test that analyses the expression of 12 genes involved in key biologic pathways in colon cancer. Stage II and Stage IIIA colon cancers were studied in affiliated hospitals of our region in southwest Ireland. All data collected is prospective and each colon cancer was assigned a recurrence risk score. Oncologists were blinded to this score and the decision to prescribe adjuvant chemotherapy was recorded. After un-blinding the score, a second decision was recorded and comparisons made. Results: :From August 2015 to September 2016, 70 patients have been recruited with M: F of 2:1. Median age at diagnosis was 65 years. Most patients (80%) had stage II disease, 11 of whom had mismatch repair loss on IHC. OncotypeDX testing has been carried out and reported for 59 patients (85%), MMR intact. Recurrence scores: &lt; 30 in 46 patients (77.9%), 30-40 in 10 patients, and &gt; 40 in 3 patients. The treatment plan was altered in 16 patients (27%), of whom 12 patients (20%) received none or less intense chemotherapy. Conclusions: We have shown that the decision to prescribe adjuvant chemotherapy was changed in 27% of patients. This test has helped to define patients with low scores, where chemotherapy-related toxicity is a concern especially in older patients. Absolute benefit of adjuvant chemotherapy versus the risk of toxicity should be discussed. . Hospital managers may be interested in cost savings due to a reduction in chemotherapy use.

Distinct expression profile of key molecules in crawling-type early gastric carcinoma.

Gastric "crawling-type" adenocarcinoma (CRA) is a tumor histologically characterized by irregularly fused glands with low-grade cellular atypia that tends to spread laterally in the mucosa. To date, the expression characteristics of the key molecules involved in CRA, including receptor tyrosine kinases (RTKs), mismatch repair (MMR) proteins, phosphatase and tensin homolog (PTEN), as well as the Epstein-Barr virus (EBV) status, have yet to be uncovered. We constructed tissue microarrays of 94 CRAs, 72 conventional-type differentiated adenocarcinomas (CDAs), and 71 intramucosal poorly cohesive adenocarcinomas (PCAs) from early gastric cancers to evaluate and compare the pathological and expression profiles of potential key molecules for molecular classification (EBV; four MMR proteins-MLH1, MSH2, PMS2, and MSH6; three RTKs-HER2, MET, and EGFR; PTEN; and p53). None of the CRAs showed MMR deficiency (0.0% vs. 5.6%, CRA vs. CDA, p=0.036), HER2 overexpression (0.0% vs. 12.5%, p=0.001), or loss of PTEN expression (0.0% vs. 9.7%, p=0.003). Moreover, MET overexpression (4.4% vs. 19.4%, p=0.004), and a mutant p53 pattern (12.4% vs. 62.5%, p<0.001) were significantly less common in CRAs than in CDAs. However, clinicopathological features and all the profile of the molecules of CRAs were close to those of the PCA group. CRA demonstrated unique clinicopathological characteristics and showed a distinct expression profile of key molecules, which was close to that of a null phenotype. These results support the classification of CRA as a distinct subgroup of gastric adenocarcinoma.

Pathogenic germline MCM9 variants are rare in Australian Lynch-like syndrome patients

Lynch syndrome is a hereditary cancer syndrome caused by the autosomal dominant inheritance of loss-of-function mutations in DNA mismatch repair (MMR) genes. Approximately one quarter of clinically suspected cases have no identifiable germline mutation in any MMR gene, a condition known as Lynch-like syndrome (LLS). MCM9 was recently identified as the DNA helicase in the mammalian MMR complex and loss of helicase activity results in microsatellite instability. We hypothesized that pathogenic variants in MCM9 may account for LLS. The 5'UTR and coding region of MCM9 were sequenced in germline DNA of 109 Australian patients with LLS and variants were cross-referenced with three population-based databases (dbSNP144, 1000 Genomes, ExAC). The functional effect of variants was assessed in silico with PolyPhen-2, SIFT and CONDEL. Fifteen variants that included six common SNPs and nine variants of unknown significance (VUS) were identified. We conclude that VUS occur in MCM9 in a small proportion of LLS patients and MCM9 mutations are unlikely to explain most LLS cases.

Augmented HR Repair Mediates Acquired Temozolomide Resistance in Glioblastoma.

Augmented HR repair is a novel mechanism underlying acquired temozolomide resistance in GBM, and this raises the possibility of improving the therapeutic response to temozolomide by targeting HR with small-molecule inhibitors of CDK1/2. Mol Cancer Res; 14(10); 928-40. ©2016 AACR.