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The dysplasia grading of Barrett’s esophagus (BE), based on the histomorphological assessment of formalin-fixed, paraffin-embedded (FFPE) tissue, suffers from high interobserver variability leading to an unsatisfactory prediction of cancer risk. Thus, pre-analytic preservation of biological molecules, which could improve risk prediction in BE enabling molecular and genetic analysis, is needed. We aimed to evaluate such a molecular pre-analytic fixation tool, PAXgene-fixed paraffin-embedded (PFPE) biopsies, and their suitability for histomorphological BE diagnostics in comparison to FFPE. In a ring trial, 9 GI pathologists evaluated 116 digital BE slides of non-dysplastic BE (NDBE), low-grade dysplasia (LGD), high-grade dysplasia (HGD), and esophageal adenocarcinomas (EAC) using virtual microscopy. Overall quality, cytological and histomorphological parameters, dysplasia criteria, and diagnosis were analyzed. PFPE showed better preservation of nuclear details as chromatin and nucleoli, whereas overall quality and histomorphologic parameters as visibility of basal lamina, goblet cells, and presence of artifacts were scored as equal to FFPE. The interobserver reproducibility with regard to the diagnosis was best for NDBE and EAC (κF = 0.72–0.75) and poor for LGD and HGD (κF = 0.13–0.3) in both. In conclusion, our data suggest that PFPE allows equally confident histomorphological diagnosis of BE and EAC, introducing a novel tool for molecular analysis and parallel histomorphological evaluation.

PDF file - 83K, Baseline characteristics of 52 patients with RCC lung metastasis. * determined by Heng et al.

PDF file - 90K, Baseline characteristics of 140 patients with CRC lung metastasis. The stage was determined by pathological examination at the time of diagnosis.

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Purpose: If immune cells are involved in tumor surveillance and have a prognostic impact in most primary tumors, little is known about their significance in metastases. Because patients' survival is heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the control of metastases. We therefore characterized the tumor immune microenvironment and its prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary tumors.Experimental Design: We analyzed by immunohistochemistry (n = 192) and qPCR (n = 32) the immune environments of colorectal carcinoma and RCC lung metastases.Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher densities of DC-LAMP+ mature dendritic cells (P < 0.0001) and lower densities of NKp46+ NK cells (P < 0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells were similar. High densities of CD8+ and DC-LAMP+ cells correlated with longer overall survival (OS) in colorectal carcinoma (P = 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were associated with improved survival in RCC (P = 0.002) but not in colorectal carcinoma. Densities of immune cells correlated significantly from primary to relapsing metastases for the same patient. A TH1 orientation was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found in RCC metastases.Conclusions: Our results show a major prognostic value of the immune pattern (CD8+/DC-LAMP+ cell densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin Cancer Res; 19(15); 4079–91. ©2013 AACR.

Purpose: Adding oxaliplatin to adjuvant 5-fluorouracil (5-FU) chemotherapy improves 3-year disease-free survival (DFS) after resection of stage III colon cancer. Several studies suggest that patients with tumors exhibiting defective mismatch repair (MMR) do not benefit from adjuvant 5-FU chemotherapy, but there are few data on 5-FU–oxaliplatin (FOLFOX) adjuvant chemotherapy in this setting. The aim of this study was to evaluate the prognostic value of MMR status for DFS in patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy.Experimental Design: MMR status was determined by microsatellite instability testing or immunohistochemistry in 303 unselected patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy in 9 centers. Cox proportional hazards models were used to examine the association between MMR status and 3-year DFS.Results: The 3-year DFS rate was significantly higher in the 34 patients (11.2% of the study population) with defective MMR tumors (90.5%) than in patients with proficient MMR tumors (73.8%; log-rank test; HR = 2.16; 95% CI, 1.09–4.27; P = 0.027). In multivariate analysis, MMR status remained an independent significant prognostic factor for DFS (HR = 4.48; 95% CI, 1.34–14.99; P = 0.015).Conclusion: MMR status is an independent prognostic biomarker for DFS in patients with stage III colon cancer receiving adjuvant FOLFOX chemotherapy. Clin Cancer Res; 17(23); 7470–8. ©2011 AACR.

PDF file - 101K, Influence of pre-operative patient treatments on the distribution of CD8+, DC-LAMP+ and NKp46+ immune cells in CRC (a, b and c) and RCC lung metastases (d, e and f). 63/140 patients with CRC lung metastases have been treated with neo-adjuvant chemotherapy and 9/52 patients with RCC lung metastases have been treated with IL2/IFN. Whiskers length represents 10-90 percentile. ns, not significant (Mann-Whitney test).

PDF file - 88K, Gene expression in lung metastases from CRC and RCC. Expression of genes related to (a) immune cell populations, (b) Th1/Th2 orientations, (c) inflammation and angiogenesis, (d) immunosuppression, (e) cytotoxicity, (f) chemokines/chemokine receptors in lung metastases from CRC (grey bars) and RCC (white bars). Expression levels of genes were determined using threshold cycle (Ct) values normalized to actin B ACTB (ΔCt). Whiskers length represents 10-90 percentile. ns, not significant; *P

PDF file - 107K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of CRC lung metastases. Statistical comparison was performed by the log-rank test.

PDF file - 56K, P values corresponding to different cutoff (minimum P value, first, second and third quartile) in CRC and RCC lung metastases. {section sign}P values were corrected by the formula proposed by Altman et al.

PDF file - 148K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of RCC lung metastases. Representation of the Kaplan-Meier curves for the duration of OS according to the presence of NKp46+ cells in the CT (d), in the IM (e) and in combined region (CT+IM) (f) of RCC lung metastases. Statistical comparison was performed by the log-rank test.

Purpose: Pathologic TNM staging is currently the best prognostic factor for non‐small cell lung carcinoma (NSCLC). However, even in early-stage NSCLC, the recurrence rates after surgery range from 25% to 50%. The preoperative detection of circulating tumor cells (CTC) could be useful to tailor new therapeutic strategies in NSCLC. We assessed the presence of CTC in NSCLC patients undergoing surgery, using cytologic analyses, after their isolation by size of epithelial tumor cells (ISET method). The presence and the number of CTCs were considered and correlated with clinicopathologic parameters including patient follow-up.Experimental Design: Of the 247 blood samples tested, 208 samples were from patients with resectable NSCLC and 39 from healthy subjects. The mean follow-up was 24 months. An image of detected cells with presumably nonhematologic features [initially defined as “circulating nonhematologic cells” (CNHC)] was recorded. The presence of CNHC was assessed blindly and independently by 10 cytopathologists, using cytologic criteria of malignancy on stained filters. The count of detected CNHCs was made for each filter.Results: One hundred two of 208 (49%) patients showed CNHCs corresponding to CNHC with malignant cytopathologic features in 76 of 208 (36%) cases. CNHCs were not detected in the control group. A level of 50 or more CNHCs corresponding to the third quartile was associated with shorter overall and disease-free-survival, independently of disease staging, and with a high risk of recurrence and death in early-stage I + II-resectable NSCLC.Conclusion: A high percentage of NSCLC patients show preoperative detection of CNHC by the ISET method. The presence and level of 50 or more CNHCs are associated with worse survival of patients with resectable NSCLC. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

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Supplementary Figures S1-S2; Supplementary Table S1.

Perioperative chemotherapy is increasingly used in combination with surgery for the treatment of patients with locally advanced, resectable gastric cancer. Histologic tumor regression grade (TRG) has emerged as an important prognostic factor; however, a common standard for its evaluation is lacking. Moreover, the clinical significance of regressive changes in metastatic lymph nodes (LNs) remains unclear. We conducted an international study to examine the interobserver agreement of a TRG system that is based on the Becker system for the primary tumors and additionally incorporates regression grading in LNs. Twenty observers at different levels of experience evaluated the TRG in 60 histologic slides (30 primary tumors and 30 LNs) based on the following criteria: for primary tumors, grade 1 represented complete response (no residual tumor), grade 2 represented 50% residual tumor, as described by Becker et al. For LNs, grade "a" represented complete, grade "b" represented partial, and grade "c" represented no regression. The interobserver agreement was estimated using the Kendall's coefficient of concordance (W). Regarding primary tumors, agreement was good irrespective of the level of experience, reaching a W-value of 0.70 overall, 0.71 among subspecialized, and 0.71 among nonsubspecialized observers. Regarding LNs, interobserver agreement was moderate to good, with W-values of 0.52 overall, 0.64 among subspecialized, and 0.45 among nonsubspecialized observers. These findings indicate that the combination of the Becker TRG system with a three-tiered grading of regression in LNs generates a system that is reproducible. Future studies should investigate whether the additional information of TRG in LNs adds to the prognostic value of histologic regression grading in gastric cancer specimens.

Tumors with microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). To date however, tools for predicting efficacy of these new therapies are lacking. Here we combined high-throughput DNA and RNA sequencing of tumors from 117 patients with MSI mCRC treated with anti-PD-1 +/- anti-CTLA-4 and enrolled into two cohorts, i.e., the NIPICOL clinical trial (NCT03350126) and the ImmunoMSI prospective cohort that were used as discovery and validation cohorts in this ancillary study, respectively. All analyses based on previously suggested DNA/RNA biomarkers of resistance failed to identify robust predictors of treatment response in patients, e.g., the level of MSI, mutational burden, the presence of specific somatic DNA variants as assessed by exome-sequencing and the activity of canonical signaling pathways or the presence of specific cellular contingents within the tumor bulk as estimated by RNA-sequencing. By contrast, resistance to ICI was found to depend both on a small subset of somatic DNA variants located in microsatellite-containing genes with very diverse biological functions and the expression of a stromal-oriented RNA component. Testing of these predictors in 5 large additional independent retrospective and prospective cohorts (IDEA and MOSAIC clinical trials) regrouping 446 patients with nonmetastatic or metastatic MSI CRC untreated with ICI allowed us to validate the specificity of the predictive nature of these indicators regarding ICI-treated MSI mCRC patients. The use of these DNA/RNA predictors will help to refine the ICI-based precision therapy of patients with metastatic MSI mCRC.

Background & Aims Next-generation sequencing (NGS) was recently approved by the United States Food and Drug Administration to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) before treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI. Methods CRC samples used in this post hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex polymerase chain reaction and immunohistochemistry. Whole-exome sequencing (WES) was used to evaluate MSISensor, the Food and Drug Administration-approved and NGS-based method for assessment of MSI. This was performed in (1) a prospective, multicenter cohort of 102 patients with mCRC (C1; 25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (2) an independent retrospective, multicenter cohort of 113 patients (C2; 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), and (3) a publicly available series of 118 patients with CRC from The Cancer Genome Atlas (C3; 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared with MSISensor in C1, C2, and C3 at the exome level or after downsampling sequencing data to the MSK-IMPACT gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 patients with new CRC (137 dMMR/MSI) enriched in tumors deficient in MSH6 (n = 35) and PMS2 (n = 9) after targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG). Results At the exome level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4 of 25; sensitivity, 84%; 95% confidence interval [CI], 63.9%–95.5%), 32% of mCRC (8 of 25; sensitivity, 68%; 95% CI, 46.5%–85.1%), and 9.1% of non-mCRC from C2 (8 of 88; sensitivity, 90.9%; 95% CI, 82.9%–96%), and 9.8% of CRC from C3 (5 of 51; sensitivity, 90.2%; 95% CI, 78.6%–96.7%). Misdiagnosis included 4 mCRCs treated with ICI, of which 3 showed an overall response rate without progression at this date. At the exome level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status among C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-IMPACT gene panel (sensitivity, 72.5%; 95% CI, 64.2%–79.7%), particularly in the MSH6-deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare after targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity, 99.3%; 95% CI, 96%–100%; specificity, 100%). Conclusions In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI polymerase chain reaction, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may rapidly become a reference method for NGS-based testing of MSI in CRC, especially in mCRC, where accurate MSI status is required before the prescription of ICI.