A feasibility study using a quantum cascade laser-based infrared microscope for the rapid and label-free classification of colorectal cancer tissues is presented. Infrared imaging is a reliable, robust, automated, and operator-independent tissue classification method that has been used for differential classification of tissue thin sections identifying tumorous regions. However, long acquisition time by the so far used FT-IR-based microscopes hampered the clinical translation of this technique. Here, the used quantum cascade laser-based microscope provides now infrared images for precise tissue classification within few minutes. We analyzed 110 patients with UICC-Stage II and III colorectal cancer, showing 96% sensitivity and 100% specificity of this label-free method as compared to histopathology, the gold standard in routine clinical diagnostics. The main hurdle for the clinical translation of IR-Imaging is overcome now by the short acquisition time for high quality diagnostic images, which is in the same time range as frozen sections by pathologists.

Since 2016, large nested urothelial carcinoma (LNUC) has been included within the WHO classification of urothelial tumors. Limited reports with mainly small case series have confirmed the malignant behavior of LNUC despite its bland morphological appearance. We evaluated, for the first time, markers for new immunooncological or targeted therapies including FGFR3 mutational status and PD-L1 status, the frequency of TERT-promoter mutations and the molecular subtype in a cohort of 25 LNUC using SNaPshot analysis and immunohistochemistry. Of the 25 cases, 17 were pure LNUC, with 13 showing an additional exophytic papillary/papillary-like component. Seven mixed LNUCs presented areas of classical nested variant urothelial carcinoma (NVUC) and one showed a component of conventional urothelial carcinoma. Of the 17 evaluable pure LNUCs, 16 were FGFR3-mutated with identical mutations in their concomitant papillary/papillary-like components. An FGFR3 mutation was found in 1/7 evaluable mixed LNUCs combined with NVUC. TERT-promoter mutations were detected in 86.7% pure and 83.3% mixed tumors. Immunohistochemistry revealed a luminal phenotype; PD-L1 was negative in the majority of tumor cells and tumor-associated immune cells. Pure LNUC is a prime example of a luminal, FGFR3-mutated, mostly PD-L1-negative tumor. In contrast, FGFR3 mutations seem to be rare in mixed LNUC, which may indicate a different pathway of tumor development.

Background: The EGFR (epithelial growth factor receptor) ligands amphiregulin (AREG) and epiregulin (EREG) have been considered as predictors for EGFR-antibody efficacy. The effect of AREG and EREG expression levels in primary tumor samples on the outcome of bevacizumab-treated patients is unknown.Patients and Methods: Formalin-fixed paraffin-embedded (FFPE) tumor samples from surgically removed primaries of the AIO KRK-0207 trial have been tested for AREG and EREG expression. The AIO KRK-0207 trial was a randomized phase-3 study to investigate the best maintenance strategy after oxaliplatin/fluoropyrimidine plus bevacizumab induction treatment in patients with mCRC. Association of AREG and EREG levels with outcome parameters were investigated, taking into account RAS and BRAF mutations.Results: A total of 331 tumor samples had measurable AREG and EREG tissue levels. In the total cohort using continuous expression levels, higher logAREG and logEREG levels were associated with a significant longer overall survival (OS) (HR 0.80; p = 0.003 and HR 0.78; p = 0.001, respectively). The subgroup of BRAF mutant tumors displayed significantly lower AREG and EREG levels compared to wild-type tumors. The prognostic effect of AREG and EREG expression was limited to the double wild-type subpopulation, whereas in the RAS mutant and BRAF mutant subgroups no prognostic effect was detected.Conclusion: Low logAREG and logEREG levels are associated with a shorter OS in oxaliplatin/fluoropyrimidine plus bevacizumab treated patients. As low AREG and EREG level are associated with BRAF mutations, the prognostic value of EREG and AREG levels is limited to the RAS and BRAF wild-type subpopulation.

No abstract

Introduction In a retrospective analysis of two randomized phase III trials in mCRC patients treated first line with oxaliplatin, fluoropyrimidine with and without Bevacizumab (the AIO KRK 0207 and R091 trials) we evaluated the association of high microsatellite instability (MSI-H), immunoscore (IS) and PD-L1 expression in relation to overall survival (OS). Methods In total, 550 samples were analysed. Immunohistochemical analysis of the MMR proteins and additionally fragment length analysis was performed, molecular examinations via allele-discriminating PCR in combination with DNA sequencing. Furthermore PD-L1 and IS were assessed. Results MSI-H tumors were more frequent in right sided tumors (13.66% vs. 4.14%) and were correlated with mutant BRAF (p = 0.0032), but not with KRAS nor NRAS mutations (MT). 3.1% samples were found to be PD-L1 positive, there was no correlation of PDL1 expression with MSI-H status, but in a subgroup analysis of MSI-H tumors the percentage of PD-L1 positive tumors was higher than in MSS tumors (9.75% vs. 2.55%). 8.5% of samples showed a positive IS, MSI-H was associated with a high IS. The mean IS of the pooled population was 0.57 (SD 0.97), while the IS of MSI-H tumors was significantly higher (mean of 2.4; SD 1.4; p =< 0.0001). Discussion Regarding OS in correlation with MSI-H, PD-L1 and IS status we did not find a significant difference. However, PD-L1 positive mCRC tended to exhibit a longer OS compared to PD-L1 negative cancers (28.9 vs. 22.1 months).

Colorectal cancer is one of the leading malignancies and still accounts for almost 25 000 deaths in Germany each year. Although there is accumulating data on the molecular basis, treatment and clinical outcome of patients within clinical trials evidence from the real-world setting is mostly lacking. We started the molecular registry trial Colopredict Plus in 2013 to collect clinical and molecular data from a real-world cohort of patients with early colon cancer stage II and III in 70 German colon cancer centers focusing on the prognostic impact of high microsatellite instability. In this interim report, we characterize a clinical cohort of 2615 patients, of whom 1787 tissue probes were analyzed. Microsatellite status was assessed using immunhistochemistry and fragment length analysis, with a concordance of 91.4 %. These established histopathological methods are sensitive and cost-effective. The median age was 72 years, significantly higher compared to clinical trial populations, with a median Charlson Comorbidity Index of 3. The stage-dependent incidence of microsatellite instability was 23.7 % and was associated with female gender, BRAF-mutation, UICC stage II and localization in the right colon. Survival calculated in disease free, relapse free and overall survival significantly differed between MSI-H and MSS, in favor of MSI-H patients. Multivariate age-adjusted analyses of relapse-free survival, disease-free survival, and overall survival highlighted microsatellite instability as a robust and positive prognostic marker for early colon cancer independent of age.

e15086 Background: High microsatellite instability (MSI-H) is a prognostic marker in resected colon cancer (CC) identified in post-hoc analysis of multiple trials. However, validation in real-life cohorts and its association with clinical and molecular markers is lacking. Methods: In Sep 2013 we started a platform trial in 49 German cancer centers recruiting patients with UICC stage II and III CC diagnosed between 2008 to 2016. MSI was tested by immunohistochemistry (IHC) of mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. In case of any loss of protein expression (IHC neg), fragment length analysis (FLA) was performed, defining MSI high tumors (MSI-H) and MS stable tumors (MSS). Besides, mutations in known prognostic factors for CC such as RAS, BRAF, PI3K and others were determined by next generation sequencing (NGS). Results: By end of 2016, 1249 patients have been recruited into the trial: median age 73yrs., stage II/III: 686/563 pts. So far, tissue was analysed in 512 pts. Of these, 182 pts. were IHC neg with 116 pts. tested MSI-H upon FLA (22.7%). Median age was 73yrs., female/male: 260/252 pts., stageII/III: 292/220 pts. Association of MS status with clinical factors is shown in Table 1. Upon NGS analysis we found 16.3% BRAF mutations, 39% KRAS mutations, 2.8% NRAS mutations and 22.6% PI3K mutations. MSI-H status was significantly associated with BRAF mutation and wildtype status of RAS. Conclusions: We found a higher percentage of MSI-H cancers in our registry compared to reported data from randomised trials, possibly related to a higher median age in this real-life cohort. MSI-H was associated with female sex, primary tumor site, and distinct molecular markers and should therefore be considered a heterogeneous subgroup of CC. Updated analysis including NGS data and survival will be presented at the meeting. Clinical trial information: AIO- KRK-0413, DKRS:00004305. [Table: see text]

3595 Background: MSI-H is an established prognostic marker in early colon cancer. Moreover, MSI-H, tumor immune cell infiltration and PD-L1 expression are also discussed as potential predictive biomarkers for immunotherapy. However, little is known about the prognostic value of these biomarkers and their association among each other in mCRC. Methods: We analyzed samples from pts. with mCRC uniformly treated with a FP and Ox within two randomized AIO trials (KRK 0207 and RO91). MS status was assessed by immunohistochemistry (IHC) of mismatch repair proteins and subsequent fragment length analysis in case of protein loss or incoherent results. PD-L1 expression was determined by IHC (1% expression threshold). Tumor lymphocytic infiltration (CD8 and CD45RO) was scored according to the immunoscore (IS) concept by Galon et al (J Transl Med 2016). Results: 41/550 cases (7.5%) displayed MSI-H. The mean IS of the total population was 0.57 (SD 0.97), the IS of MSI-H pts. was significantly higher (mean of 2.4; SD 1.4; p≤0.0001). 17 cases were PD-L1 positive (pos.) (3 %), only four of these were MSI-H. MSI-H status was significantly correlated with a higher IS, but not with PD-L1 expression (table 1). There was no difference in median overall survival (mOS) between MSI-H and MS stable (MSS) pts. (mOS MSI-H/MSS: 17.6/22.5 months (mos.), log rank: p=0.85), PD-L1 negative (neg.) and pos. pts. (mOS PD-L1 neg./pos.: 22.1/28.9 mos., log rank: p=0.49) and IS high or low pts. (mOS IS high/low: 21.1/22.1mo., log rank: p=0.25). Conclusions: In contrast to early stage colon cancer, none of these parameters was prognostic in mCRC patients. Panel-sequencing with a total of 35 genes including RAS, BRAF and POL-E on cases with PD-L1 expression, high IS or MSI-H status to further characterize these cases will be reported. Clinical trial information: AIO-R091, AIO-KRK-0207: EudraCT-Nr: 2008-007974-39. [Table: see text]