Deficient Mismatch Research Articles

Evaluating the clinical utility of universal screening to identify Lynch syndrome in stage III/III colorectal cancer patients: A prospective observational study in Japan.

41 Background: Universal screening for Lynch syndrome (LS) by identifying deficient DNA mismatch repair (MMR) in the tumor tissue of all new colorectal cancer (CRC) patients is widely accepted. The population prevalence of LS is approximately 3% in Western countries, whereas it is approximately 0.7% in Japan. In addition, the number of relatives diagnosed per proband is 3.6 in Western countries, whereas there are even fewer diagnoses per proband in Japan. To address the issue of LS remaining largely underdiagnosed in Japan, we prospectively evaluated the clinical utility of universal screening of LS in CRC patients. Methods: From March 2016 to August 2019, all consecutive new cases of stage II/III CRC underwent immunohistochemistry (IHC) screening for MMR using MLH1, MSH2, MSH6, and PMS2 antibodies. The patients negative for both MLH1 and PMS2 (MLH1-/PMS2-) were subjected to reflex testing for BRAF V600E mutation. Patients with both MLH1-/PMS2- and BRAF negative (cohort A, n = 14) and those with other IHC patterns (cohort B, n = 13) were referred for genetic counseling (GC) and genetic testing (GT). Furthermore, relatives of probands with confirmed LS were referred for GC/GT if they were willing. Results: Overall, 591 pts were enrolled in this study. Patient background were as follows: > 70 y/o, 35%; right-sided/left-sided colon/rectum, 24%/24%/53%; and cStage II/III, 65%/35%. Of 591 patients, 40 (6.8%) had MMR deficiency. Of 27 patients with MLH1-/PMS2-, 24 underwent BRAF reflex testing; only 10 of these patients tested positive for mutation. Of 27 patients recommended for GC, 25 were referred for GC and 22 for GT, which revealed 12 LS cases (2%, mutation genes:MLH1/PMS2/MSH2; 4/2/6). The frequency of LS diagnosis with respect to patient background was as follows: > 70/≤70 y, 1.0/2.6%; right-sided/left-sided colon/rectum, 5.8/0/1.3%; and cStage II/III, 2.6/1.0%. Interestingly, only 3 (25%) of 12 patients who underwent GC/GT in cohort A had LS compared with 9 (90%) of 10 patients in cohort B ( p= 0.004). Moreover, among 11 relatives of 5 families who were willing to undergo GC/GT, six (55%) had LS, of whom two were first-degree relatives (33%), one was a second-degree relative (50%), and three were third-degree relatives (100%). Conclusions: This study showed that universal screening of LS in CRC patients is significantly useful in Japan. Furthermore, implementing a reflex testing strategy demonstrated high adherence to guidelines and the appropriateness of our referrals for GC/GT.

Early change in peripheral CD4+ T cells associated with clinical outcomes of immunotherapy in gastrointestinal cancer.

Biomarkers for immune checkpoint inhibitors (ICIs) are limited in gastrointestinal cancer. Peripheral blood lymphocyte subset and associated dynamic changes were retrospectively analyzed in patients with gastrointestinal cancer treated with ICIs. Cox regression and Kaplan-Meier analyses were conducted for survival. A total of 80 patients were enrolled. Baseline CD4+/CD8+ T cells were lower in patients who experienced tumor progression by 6months than in patients who did not (1.160±0.652 vs 1.705±0.924, respectively; p=0.003). In multivariate analyses, decline in CD4+ T cells after the first dose of ICIs (CD4-C1-decline) was an independent prognostic factor for overall survival (hazard ratio: 13.00; 95% CI: 2.24-75.54; p=0.004). Furthermore, CD4-C1-decline was a preferable indicator for progression in patients with deficient mismatch repair/microsatellite instability-high (p=0.027). Early change in CD4+ T cell counts in peripheral blood may act as a prognostic biomarker for gastrointestinal cancer patients treated with ICIs.

Identification of immune landscape signatures associated with clinical and prognostic features of hepatocellular carcinoma.

While cancer immunotherapy has been remarkably successful in some malignancies, some cancers derive limited benefit from current immunotherapies. Here, we combined immune landscape signatures with hepatocellular carcinoma clinical and prognostic features to classify them into distinct subtypes. The immunogenomic profiles, stromal cell features and immune cell composition of the subtypes were then systematically analyzed. Two independent prognostic indexes were established based on 6 immune-related genes and 17 differentially expressed genes associated with stromal cell content. These indexes were significantly correlated with tumor mutation burden, deficient DNA mismatch repair and microsatellite instability. In addition, tumor-infiltrating lymphocytes, including activated NK cells, resting memory CD4 T-cells, eosinophils, and activated mast cells were significantly correlated with hepatocellular carcinoma survival. In conclusion, we have comprehensively described the immune landscape signatures and identified prognostic immune-associated biomarkers of hepatocellular carcinoma. Our findings highlight potential novel avenues for improving responses to immunotherapy.

Clinical Application of Targeted Deep Sequencing in Metastatic Colorectal Cancer Patients: Actionable Genomic Alteration in K-MASTER Project.

PurposeNext-generation sequencing (NGS) can facilitate precision medicine approaches in metastatic colorectal cancer (mCRC) patients. We investigated the molecular profiling of Korean mCRC patients under the K-MASTER project which was initiated in June 2017 as a nationwide precision medicine oncology clinical trial platform which used NGS assay to screen actionable mutations.Materials and MethodsAs of 22 January 2020, total of 994 mCRC patients were registered in K-MASTER project. Targeted sequencing was performed using three platforms which were composed of the K-MASTER cancer panel v1.1 and the SNUH FIRST Cancer Panel v3.01. If tumor tissue was not available, cell-free DNA was extracted and the targeted sequencing was performed by Axen Cancer Panel as a liquid biopsy.ResultsIn 994 mCRC patients, we found 1,564 clinically meaningful pathogenic variants which mutated in 71 genes. Anti-EGFR therapy candidates were 467 patients (47.0%) and BRAF V600E mutation (n=47, 4.7%), deficient mismatch repair/microsatellite instability–high (n=15, 1.5%), HER2 amplifications (n=10, 1.0%) could be incorporated with recently approved drugs. The patients with high tumor mutation burden (n=101, 12.7%) and DNA damaging response and repair defect pathway alteration (n=42, 4.2%) could be enrolled clinical trials with immune checkpoint inhibitors. There were more colorectal cancer molecular alterations such as PIK3CA, KRAS G12C, atypical BRAF, and HER2 mutations and even rarer but actionable genes that approved or ongoing clinical trials in other solid tumors.ConclusionK-MASTER project provides an intriguing background to investigate new clinical trials with biomarkers and give therapeutic opportunity for mCRC patients.

Abstract 2964: Saffron has enhanced anti-proliferative effect against colorectal cancer cells with deficient DNA mismatch repair

Abstract Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. A subtype of CRC is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy, however, these roles in the context of of dMMR have not been explored. In this study, we aimed to study the effects of saffron in CRC cell lines with dMMR. Methods: Saffron crude extracts and specific compounds (crocin, crocetin and safranal) were used in colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MH3) and HCT116+3+5 (Inserted MLH1 and MSH3). CDC25b, p-H.H2AX, TPDP1, and GAPDH were analyzed by Western blot. Cell cycle was analyzed by FACS. Proliferation and cytotoxicity were analyzed by MTT. Wound and migration assays were also performed. Results: Saffron crudes restricted the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. Wound healing assay indicates that deficient MMR cells are doing better than proficient MMR cells. The expression of CDC25b and TDP1 were upregulated in proficient MMR cell compared to deficient MMR cell, while H2AX was significantly upregulated in both cells type particularly at >10 mg/ml saffron in a concentration-dependent manner (after normalization with GADPH as control).The reduction in cellular proliferation and motility might be due to cell cycle arrest at G2-phase rather than to apoptosis. The major active saffron compounds, safranal and crocin reproduced total saffron crudes effects but with lesser outcome. Conclusion: Saffron's anti-proliferative and healing effects is significant in cells with deficient MMR MLH1 and MSH3 genes. This effect may have a therapeutic implications and benefits for the CRC MSI tumors that are generally not recommended for 5FU-based treatment. Citation Format: Amr Amin, Akbar Soleimani, Chandraprabha Murali, Lucie Beresova, Hamid Shoraka, Hassan Brim, Hassan Ashktorab. Saffron has enhanced anti-proliferative effect against colorectal cancer cells with deficient DNA mismatch repair [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2964.

AtezoTRIBE: a randomised phase II study of FOLFOXIRI plus bevacizumab alone or in combination with atezolizumab as initial therapy for patients with unresectable metastatic colorectal cancer

BackgroundImmune checkpoint inhibitors (ICIs) reported remarkable achievements in several solid tumours. However, in metastatic colorectal cancer (mCRC) promising results are limited to patients with deficient mismatch repair/microsatellite instability-high (dMMR/MSI-high) tumours due to their immune-enriched microenvironment. Combining cytotoxic agents and bevacizumab in mCRC with proficient mismatch repair/microsatellite stability (pMMR/MSS) could make ICIs efficacious by increasing the exposure of neoantigens, especially with highly active chemotherapy regimens, inducing immunogenic cell death, increasing the tumoral infiltration of CD8+ T-cells and reducing tumour-associated myeloid-derived suppressor cells. VEGF-blockade also plays an immunomodulatory role by inhibiting the expansion of T regulatory lymphocytes.Consistently with this rationale, a phase Ib study combined the anti-PDL-1 atezolizumab with FOLFOX/bevacizumab as first-line treatment of mCRC, irrespective of microsatellite status, and reported interesting activity and efficacy results, without safety concerns.Phase III trials led to identify FOLFOXIRI plus bevacizumab as an upfront therapeutic option in selected mCRC patients. Drawing from these considerations, the combination of atezolizumab with an intensified upfront treatment (FOLFOXIRI) and bevacizumab could be worthy of investigation.MethodsAtezoTRIBE is a prospective, open label, phase II, comparative trial in which initially unresectable and previously untreated mCRC patients, irrespective of microsatellite status, are randomized in a 1:2 ratio to receive up to 8 cycles of FOLFOXIRI/bevacizumab alone or in combination with atezolizumab, followed by maintenance with bevacizumab plus 5-fluoruracil/leucovorin with or without atezolizumab according to treatment arm until disease progression. The primary endpoint is PFS. Assuming a median PFS of 12 months for standard arm, 201 patients should be randomized in a 1:2 ratio to detect a hazard ratio of 0.66 in favour of the experimental arm. A safety run-in phase including the first 6 patients enrolled in the FOLFOXIRI/bevacizumab/atezolizumab arm was planned, and no unexpected adverse events or severe toxicities were highlighted by the Safety Monitoring Committee.DiscussionThe AtezoTRIBE study aims at assessing whether the addition of atezolizumab to an intensified chemotherapy plus bevacizumab might be an efficacious upfront strategy for the treatment of mCRC, irrespective of the microsatellite status.Trial registrationAtezoTRIBE is registered at Clinicaltrials.gov (NCT03721653), October 26th, 2018 and at EUDRACT (2017–000977-35), Februray 28th, 2017.

Duration of FOLFOX adjuvant chemotherapy in high-risk stage II and stage III colon cancer with deficient DNA mismatch repair.

4075 Background: In the IDEA collaboration, noninferiority was not confirmed for 3 months versus 6 months of FOLFOX adjuvant chemotherapy among patients with high-risk stage II and stage III colon cancer (CC). Patients with deficient DNA mismatch repair (dMMR) have a good prognosis, but for whom, whether limiting the duration of adjuvant therapy will compromise oncologic outcomes remains undefined. We evaluated the impact of 3 months of FOLFOX adjuvant chemotherapy or surgery alone in comparison with 6 months of FOLFOX on disease-free survival (DFS) in dMMR CC patients. Methods: This retrospective study included all consecutive patients who underwent curative surgical resection for high-risk stage II or III dMMR CC between May, 2011 and July, 2019. Prognostic factors were analyzed using Cox models, and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated. Results: A total of 242 dMMR CC patients were included (43.4% high-risk stage II, 56.6% stage III). The patients received 6 months of FOLFOX adjuvant chemotherapy (n = 66; median cycles [rang] = 12 [10-12]), 3 months of FOLFOX (n = 87; median cycles [rang] = 6 [4-8]), or surgery alone (n = 89). Three groups were generally well balanced, although more patients with stage III were in the 6-month therapy group (74.2%), compared with the 3-month therapy group (57.5%) and the surgery alone group (42.7%). As compared with 6 months of FOLFOX adjuvant chemotherapy in the overall population, 3 months therapy reduced DFS in multivariable analysis (HR, 2.78; 95CI, 1.18 to 6.47; P = 0.02), similar to surgery alone (HR, 2.30; 95CI, 0.99 to 5.38; P = 0.05). In the subgroup analysis, a therapy duration of 6 months was statistically superior to a duration of 3 months only in the patients with stage III, with a 3-year rate of DFS of 86.2% versus 70.8% (HR, 3.06; 95% CI, 1.14 to 8.19; P = 0.026). Conclusions: This study supports the 6-month duration of FOLFOX adjuvant chemotherapy in stage III dMMR CC.

Japan society of clinical oncology/Japanese society of medical oncology-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors, cooperated by the Japanese society of pediatric hematology/oncology

BackgroundThe development of novel antitumor agents and accompanying biomarkers has improved survival across several tumor types. Previously, we published provisional clinical opinion for the diagnosis and use of immunotherapy in patients with deficient DNA mismatch repair tumors. Recently, efficacy of tropomyosin receptor kinase inhibitors against neurotrophic receptor tyrosine kinase (NTRK) fusion gene-positive advanced solid tumors have been established as the second tumor-agnostic treatment, making it necessary to develop the guideline prioritized for these patients.MethodsClinical questions regarding medical care were formulated for patients with NTRK-positive advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO) and Japanese Society of Medical Oncology (JSMO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and Japanese Society of Pediatric Hematology/Oncology, and the public comments among all Societies’ members was done.ResultsThe current guideline describes 3 clinical questions and 15 recommendations for whom, when, and how NTRK fusion should be tested, and what is recommended for patients with NTRK fusion-positive advanced solid tumors.ConclusionIn the NTRK guideline, the committee proposed 15 recommendations for performing NTRK testing properly to select patients who are likely to benefit from tropomyosin receptor kinase inhibitors.

PD-L1 expression, DNA mismatch repair genes, and HPV types in cervical squamous cell carcinoma.

5533 Background: Previous studies observed low PD-L1 expression in cervical cancer, and that high PD-L1 expression is associated with deficient DNA mismatch repair genes and poor prognosis. No study has investigated the PD-L1 expression differences among HPV types in cervical cancer. We aim to determine PD-L1 expression in our patient population and its clinical significance. Methods: A total of 198 patients with SCC were identified and 60 had evaluable tumor specimens and clinical data. Immunohistochemistry on MMR genes and PD-L1 expression using tissue microarrays was performed. HPV analysis for 15 high-risk types was done by multiplex PCR and gel electrophoresis. Correlation between PD-L1 expression, MMR status, HPV types and other clinical parameters was analyzed using χ2 or Fisher's exact test. Cumulative 5-year survival was analyzed by Kaplan-Meier curves and log rank test. Results: Of the 60 patients, 90% were black, 55% between ages of 30-55 and 40% older than 55. 33 patients had poorly, 20 moderately, and 7 well-differentiated tumors. At 5-year follow up, 11 had recurrence, 14 patients died of disease and 7 lost to follow up. 93.3% of tumors had positive staining for PD-L1 with 56.7% showing high expression. Patients aged 30-55 showed a higher rate of PD-L1 expression. Majority had intact MMR (91.7%). High-risk HPV DNA was extracted in 41 specimens (68.3%), 11 of these were infected with ≥2 or more types, identifying 6 high-risk types not included in vaccine. Correlation analysis showed that there is significant correlation between age and PD-L1 expression on tumor cells (p = 0.047). No correlation between MMR status and PD-L1 expression. Mean disease free survival (DFS) was 43.7, 36.5, and 6.3 months for high, low and negative PD-L1 expression, respectively (p = 0.002). In patients with stage I-II disease, mean DFS was 52.2, 42.6 and 7.6 months for high, low and negative PD-L1 expression, respectively (p = 0.001). Similar direct relationship is observed with overall survival. Cases with mixed HPV infection are associated with late clinical stage at diagnosis (p = 0.037) and lower PD-L1 expression. Conclusions: Contrary to previous studies, our results showed remarkably high rate of PD-L1 expression in cervical SCC, which is associated with longer DFS and OS, especially in early stage tumors. Mixed HPV infection is associated with late clinical stage at diagnosis and lower PD-L1 expression.

Comparison of phosphoproteomic profiles in left- and right-sided colorectal cancers.

582 Background: Left- and right-sided colorectal cancers (CRCs) are different in terms of prognosis, treatment response and underlying molecular mechanisms. The aim of this study was to unravel the phosphoproteomic profiles of left- and right-sided primary CRCs and to identify potential drug targets for both primary tumor sites. Methods: In total phosphoproteomic profiles of 69 fresh-frozen biopsies, including 34 left-sided and 35 right-sided primary CRCs, were obtained by LC-MS/MS after cell lysis, digestion and phosphopeptide enrichment using anti-phosphotyrosine antibodies. MS/MS spectra were searched against a UniProt human reference proteome FASTA file using MaxQuant software. Fold changes between normalized intensities were calculated. Group comparison was performed using the R package Limma. P values below 0.01 and fold changes > two were considered significant and biologically relevant. Results: In total 2,840 phosphopeptides were detected in at least 5% of the samples. Of these, 36 phosphopeptides were significantly upregulated and 14 were significantly downregulated in right-sided compared to left-sided CRCs using our stringent selection criteria. Since BRAF mutations and deficient DNA mismatch repair (dMMR) were more frequently observed in right-sided CRC (p < 0.01), group comparison was repeated with MMR proficient CRCs only, resulting in 21 phosphopeptides up- and one downregulated in left- versus right-sided CRC. The identified phosphoproteins from the phosphopeptides included three FDA approved drug targets. Conclusions: Differences in phosphoproteomic profiles were detected between left- and right-sided CRCs, besides molecular differences such as BRAF mutation and MMR status. These results indicate that the molecular biology of left-and right-sided CRCs may explain their differences in clinical behavior and response to treatment.

The role of the deficient DNA mismatch repair system as a factor of sensitivity to targeted therapy for glioblastoma multiforme

The role of the deficient DNA mismatch repair system as a factor of sensitivity to targeted therapy for glioblastoma multiforme

Clinical Targeted Next-Generation Sequencing Shows Increased Mutational Load in Endometrioid-type Endometrial Adenocarcinoma With Deficient DNA Mismatch Repair.

A subset of endometrial adenocarcinomas (EACs) exhibit microsatellite instability and have deficient DNA mismatch repair (dMMR). The overall aim of the study was to compare the spectrum of mutations in endometrioid-type EAC with and without dMMR by using a clinically validated next-generation sequencing assay. We retrospectively identified 19 EACs with known mismatch repair status that had undergone targeted sequencing of a panel of cancer-related genes. The mismatch repair status was ascertained by immunohistochemistry against MLH1, PMS2, MSH2, and MSH6 mismatch proteins. Somatic mutations in EAC with dMMR were compared against those in cases with proficient MMR (pMMR). The dMMR EAC showed a normalized mean of 66.6 mutations/Mb per case compared with pMMR EAC with a mean of 26.2 (P<0.05). The most commonly mutated genes were PTEN (89% of dMMR, 50% of pMMR), PIK3CA (67% vs. 40%), ATM (89% vs. 40%), and FLT3 (67% vs. 50%). The transition/transversion ratio was 4.7 versus 2.8 for the dMMR and pMMR cohorts, respectively (P<0.05). Copy number variant analysis did not demonstrate significant differences between the dMMR and pMMR cohorts and was not correlated with histologic grade of EAC. In conclusion, tumorigenesis of EAC in the context of dMMR demonstrated heavier mutational burdens and higher transition/transversion ratio. The spectrum of genetic alterations can potentially help identify cases with microsatellite instability phenotype using next-generation sequencing data from a targeted cancer gene panel.

Abstract 2483: Molecular mechanisms of dianhydrogalactitol (VAL-083) in overcoming chemoresistance in glioblastoma

Abstract Standard treatments for glioblastoma (GBM) include surgery, radiation and chemotherapy with temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%, largely due to chemoresistance. Evidence shows that cancer cells utilize DNA damage repair pathways to overcome cytotoxic effects of chemotherapy. GBM tumors expressing O6-methylguanine-DNA-methyltransferase (MGMT) display intrinsic chemoresistance to TMZ and nitrosoureas, while a deficient DNA mismatch repair system (MMR) confers chemoresistance to TMZ and platinum agents. Alterations in p53, particularly gain-of-function mutations, are correlated with increased MGMT-expression and poor prognoses in GBM. Second line anti-angiogenic treatment with bevacizumab has not improved overall survival and has been shown to induce intratumor hypoxia and increased chemoresistance. VAL-083 is a bi-functional alkylating agent that readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has demonstrated activity against GBM in prior NCI-sponsored clinical trials. VAL-083 induces interstrand cross-links at guanine-N7 causing DNA double-strand breaks and cancer cell death. VAL-083 is equiactive against GBM cancer stem cells (CSCs) and non-CSCs independent of MGMT and p53 status, in vitro. We recently showed that VAL-083 leads to irreversible S/G2-phase cell cycle arrest, proposing synergy with S-phase specific chemotherapeutics, including topoisomerase and PARP inhibitors. VAL-083 further showed persistent activation of the homologous recombination (HR) DNA repair pathway and its potency was increased when HR was impaired, demonstrating that VAL-083-induced lesions are repaired via HR suggesting increased VAL-083 potency in HR-impaired tumors. Further, hypoxic cancer cells are known to downregulate their HR pathway, proposing increased VAL-083 potency in hypoxic tumors. Bevacizumab treatment increases hypoxia in tumor cells, presumably impairing HR, proposing VAL-083 as a treatment option in HR-deficient or hypoxic cancers following, or as part of a combination treatment with, bevacizumab. Here, VAL-083 cytotoxicity and DNA damage response was evaluated by crystal violet assays, western blot and flow cytometry. VAL-083’s ability to overcome MMR-related chemoresistance was investigated using lentiviral MLH1 and MSH2 vectors in HCT116 and LoVo cancer cells. We report synergy between VAL-083 and etoposide or camptothecin in A549 and PC3 cancer cell lines. We also investigated the potency of VAL-083 in GBM under hypoxia either in vitro or in vivo as part of a combination treatment with bevacizumab. Our results demonstrate a distinct anti-cancer mechanism for VAL-083, resulting in the ability to overcome resistance to TMZ and nitrosoureas, increased activity in cancers with impaired HR and synergy with etoposide or camptothecin. Citation Format: Beibei Zhai, Anna Gobielewska, Anne Steino, Jeffrey A. Bacha, Dennis M. Brown, Simone Niclou, Mads Daugaard. Molecular mechanisms of dianhydrogalactitol (VAL-083) in overcoming chemoresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2483. doi:10.1158/1538-7445.AM2017-2483

Abstract A006: Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers

Abstract Background: Deficient DNA mismatch repair (MMR) boosts the accumulation of frameshift mutations in genes encompassing coding microsatellites (cMS). This results in translation of proteins with mutation-induced frameshift peptide (FSP) neoantigens rendering MMR-deficient microsatellite-unstable (MSI) cancers highly immunogenic. Patients with MSI cancers and healthy individuals affected by Lynch syndrome, an inherited prediposition for MSI cancers, develop specific immune responses against these neoantigens. MSI cancers are unique in tumor immunology, because they express a defined set of long neoantigens that result from functionally relevant driver mutations and therefore are shared by the majority of MSI cancers. Consequently, MSI cancers in Lynch syndrome are an ideal model to evaluate the concept of cancer vaccines, which, with the increasing knowledge about mutational antigens in a wide variety of cancer types, can potentially be applied to many human cancer types. We here report the results of a clinical phase I/IIa trial as the first step to translate this concept into the clinical application. Methods: The vaccination protocol comprised 3 cycles of 4 subcutaneous applications of FSP antigens (frameshift variants of the coding microsatellite-containing genes AIM2, HT001, TAF1B) mixed with Montanide ISA-51 VG over a 6 month period. Inclusion criteria were history of MSI-H colorectal cancer (UICC stage III or IV) and completion of standard chemotherapy. Phase I of the trial evaluated safety and toxicity as the primary endpoint (6 patients), phase IIa addressed the induction of cellular and humoral immune responses (16 patients). Results: Significant induction of FSP-specific immune responses against one or more FSP antigens was observed in all patients vaccinated per protocol. No vaccination-induced systematic severe adverse events occurred. Few patients had stage IV disease and were evaluable according to RECIST. One heavily pretreated patient with bulky metastases showed a stable disease and stable CEA levels over 7 months under the study treatment. Conclusions: Vaccination with FSPs is well tolerated and leads to the induction of humoral and cellular immune responses. FSP vaccination represents a promising novel approach for treatment of MSI cancer patients and for tumor prevention in Lynch syndrome, allowing the evaluation of the concept of preventive cancer vaccines in an ideal model scenario of a defined high-risk patient population. Note: This abstract was not presented at the conference. Citation Format: Magnus von Knebel Doeberitz, Matthias Kloor, Miriam Reuschenbach, Claudia Pauligk, Mohammad-Reza Rafiyan, Salah-Eddin Al Batran, Julia Karbach, Mirjam Tariverdian, Elke Jaeger. Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A006.

Morphological, molecular and genetic study of MMR-deficient colorectal cancer: Contribution of somatic tools in diagnosis of Lynch syndrome.

e14629 Background: Lynch syndrome (LS) is the most frequent inherited colorectal cancer (CRC) syndrome, due to germline mutations in one of the mismatch repair (MMR) genes. Out of the 15% of CRCs associated with deficient DNA mismatch repair system (dMMR) that are characterized by high levels of microsatellite instability (MSI-H), only 3% are attributable to Lynch syndrome. The other 12% are sporadic, with acquired dMMR phenotype caused in tumors by hypermethylation of the promoter of gene hMLH1, often associated with BRAF V600E mutation. A probably underestimated group does not respond to any of these definitions and is called Lynch-like syndrome (LLS). The aim of the study was to compare the pathological and clinical features of the LS, LLS, and sporadic CRCs and to evaluate the contribution of somatic tools to the screening of potential LS patients. Methods: Two hundred and twelve patients with a dMMR CRC were included. The clinical, surgical, pathological and genetic data were retrieved. A complete MM...

914 Colon Carcinomas Arising From Sessile Serrated Polyps Are Disproportionately Interval Cancers With Deficient DNA Mismatch Repair

914 Colon Carcinomas Arising From Sessile Serrated Polyps Are Disproportionately Interval Cancers With Deficient DNA Mismatch Repair

Should mismatch repair status be determined in all stage II and III colon cancer patients?

SUMMARY Microsatellite instability constitutes an important oncogenic molecular pathway in colon cancer (12–15%). Microsatellite instability is a molecular marker of deficient DNA mismatch repair (MMR), which occurs from somatic hypermethylation of the MLH1 promoter or less frequently as a result of germline mutations in MMR genes. The excellent prognosis of patients with stage II deficient MMR colon cancer today, makes this information mandatory in order to decide or not on adjuvant chemotherapy. MMR deficiency may also predict tumor chemoresistance to 5-fluorouracil adjuvant treatment. However, when looking at very recent data on 5-fluorouracil plus oxaliplatin-based adjuvant regimens, it seems that the benefit of this adjuvant therapy is similar between deficient and proficient MMR patients.

Prognostic impact of KRAS and BRAFV600E mutations stratified by tumor site in resected stage III colon cancer patients treated with adjuvant mFOLFOX6 with or without cetuximab: NCCTG N0147 (Alliance).

3523 Background: The association of KRAS or BRAFV600E mutations with prognosis in colon cancers has been inconsistent. Since primary tumor site may influence outcome, we analyzed KRAS and BRAFV600E stratified by tumor site in resected stage III colon cancers from a randomized adjuvant trial of mFOLFOX6 + cetuximab chemotherapy (N= 2,686) where no survival difference by treatment was found. Methods: 2,580 tumors were analyzed for mutations in BRAFV600E (exon 15) or KRAS (codons 12, 13), and for deficient DNA mismatch repair (dMMR). Cox models were used, adjusting for mutation status, age, sex, treatment, T-stage, histologic grade, nodal status, tumor site, and MMR. After study initiation, eligibility was restricted to patients (pts) with KRAS wild-type (WT) tumors. At a median follow-up 4.1 yrs, 83% of pts are alive. Results: KRAS and BRAFV600E mutations were detected in 716 (28%) and 346 (14%) tumors, respectively; dMMR was found in 314 (12%). Proximal (to splenic flexure) tumors (50%) were associated with older age and were more likely to be high grade (33 vs 18%), T-stage3,4 (88 vs 82%), mutated for KRAS (33 vs 23%) or BRAFV600E (23 vs 4%), and dMMR (21 vs 3%)[all p &lt;0.02]. Pts with distal vs proximal tumors showed improved disease-free survival (DFS)[ HR 0.7, 95% CI (0.6-0.9); p&lt;0.01 unadjusted for KRAS or BRAF;]. Mutant KRAS [HR=1.5 (1.2-1.7); p&lt;.0001] or mutant BRAF [HR=1.4 (1.1-1.7); p=0.02] were each independently associated with worse DFS. The KRAS and tumor site interaction was significant (unadjusted pinteraction=0.02; adjusted pinteraction=0.07), with poorer DFS among distal tumors having mutant KRAS [HR 1.7 (1.3-2.2); p&lt;.0001]. No interaction was observed for BRAFV600E and tumor site, despite worse DFS for mutant BRAFV600E among proximal tumors [HR 1.3 (1-1.8); p=0.04]. Conclusions: The adverse prognostic impact of KRAS, but not BRAFV600E, mutations was confined to the distal colon. This finding underscores the importance of tumor site in the interpretation of the prognostic impact of KRAS status, and motivates similar analyses in pts with advanced disease.

Microsatellite Instability in Sarcoma: Fact or Fiction?

Microsatellite instability (MSI) is a unique molecular abnormality, indicative of a deficient DNA mismatch repair (MMR) system. Described and characterized in the colorectal cancer literature, the MSI-positive phenotype is predictive of disease susceptibility, pathogenesis, and prognosis. The clinical relevance of MSI in colorectal cancer has inspired similar inquisition within the sarcoma literature, although unfortunately, with very heterogeneous results. Evolving detection techniques, ill-defined sarcoma-specific microsatellite loci and small study numbers have hampered succinct conclusions. The literature does suggest that MSI in sarcoma is observed at a frequency similar to that of sporadic colorectal cancers, although there is little evidence to suggest that MSI-positive tumors share distinct biological attributes. Emerging evidence in Ewing sarcoma has demonstrated an intriguing mechanistic role of microsatellite DNA in the activation of key EWS/FLI-target genes. These findings provide an alternative perspective to the biological implications of microsatellite instability in sarcoma and warrant further investigation using sophisticated detection techniques, sensitive microsatellite loci, and appropriately powered study designs.

Abstract 5018: Genetic and epigenetic investigation of breast carcinoma arising in Lynch syndrome families

Abstract Breast carcinoma is the most common cancer type in females, but its incidence is generally not increased in Lynch syndrome (LS). Deficient DNA mismatch repair (MMR) is a hallmark of LS tumors that are unequivocal manifestations of the syndrome (e.g., colorectal and breast carcinoma). Because of controversial study results, it is unclear at present whether or not breast carcinoma is specifically associated with LS. We addressed this question by determining the genetic and epigenetic profiles for all available breast carcinomas from LS families from a nation-wide registry, including 24 tumors from MMR gene mutation carriers and 17 tumors from non-carriers. The average age at breast carcinoma diagnosis was similar in mutation carriers and non-carriers (56 years for each). Among breast carcinomas from mutation carriers, 43% showed loss of MMR protein corresponding to the germline mutation, and high-degree microsatellite instability (MSI-H) was present in 36%. Moreover, in mutation carriers, the age at diagnosis was lower for breast carcinomas that were MMR-deficient (by absent MMR protein and/or MSI present) compared to MMR proficient tumors (51 vs. 60 years, p= 0.031). Breast carcinomas showed frequent inactivation of tumor suppressor genes (TSG) by promoter methylation. The average number of methylated TSGs out of 24 per tumor was 3.1 for mutation carriers and 2.8 for non-carriers. The gene-specific distribution of methylation was also similar between the two breast tumor sets. In mutation carriers, the most frequently methylated TSGs were RASSF1 (77%), APC (45%), CDH13 (45%), GSTP1 (32%) and CDKN2B (27%). In non-carriers, the respective frequencies were 75%, 47%, 59%, 35%, and 29%. When compared to tumors arising in other organs from LS mutation carriers, methylation of CDKN2B was seen as a particular characteristic of breast tumorigenesis. Our investigation shows that TSG promoter methylation is a common characteristic of breast carcinomas arising in LS and affects mutation carriers and non-carriers with comparable overall rates and gene-specific distributions. Analysis of MMR protein expression and MSI revealed that while many breast carcinomas from LS mutation carriers follow MMR-driven tumorigenesis characteristic of tumors of the LS spectrum in general, a significant subset of breast tumors also exists that appears to arise and progress along separate pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5018. doi:1538-7445.AM2012-5018