The Complexity of Shapes: How the Circularity of Tumor Nodules Affects Prognosis in Colorectal Cancer.

The current stratification of tumor nodules in colorectal cancer (CRC) staging is subjective and leads to high interobserver variability. In this study, the objective assessment of the shape of lymph node metastases (LNMs), extranodal extension (ENE), and tumor deposits (TDs) was correlated with outcomes. A test cohort and a validation cohort were included from 2 different institutions. The test cohort consisted of 190 cases of stage III CRC. Slides with LNMs and TDs were annotated and processed using a segmentation algorithm to determine their shape. The complexity ratio was calculated for every shape and correlated with outcomes. A cohort of 160 stage III CRC cases was used to validate findings. TDs showed significantly more complex shapes than LNMs with ENE, which were more complex than LNMs without ENE (P < .001). In the test cohort, patients with the highest sum of complexity ratios had significantly lower disease-free survival (P < .01). When only the nodule with the highest complexity was considered, this effect was even stronger (P < .001). This maximum complexity ratio per patient was identified as an independent prognostic factor in the multivariate analysis (hazard ratio, 2.47; P < .05). The trends in the validation cohort confirmed the results. More complex nodules in stage III CRC were correlated with significantly worse disease-free survival, even if only based on the most complex nodule. These results suggest that more complex nodules reflect more invasive tumor biology. As most of the more complex nodules were diagnosed as TDs, we suggest providing a more prominent role for TDs in the nodal stage and include an objective complexity measure in their definition.

Approach for Phased Sequence-Based Genotyping of the Critical Pharmacogene Dihydropyrimidine Dehydrogenase (DPYD).

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.

Cancer and left atrial enlargement in patients with ischemic stroke.

BACKGROUND: Cancer is associated with an increased risk of atrial fibrillation. Whether cancer is also associated with atrial cardiopathy, another atrial pathology associated with heightened ischemic stroke risk, is uncertain. METHODS: We conducted a retrospective cross-sectional study among consecutive patients hospitalized with acute ischemic stroke at a quaternary care center in New York, United States from 2011 through 2016. The study exposure was active cancer. The study outcome was atrial cardiopathy, defined as a left atrial volume index ≥35 mL/m2 on echocardiography. We used multivariable logistic regression, adjusting for baseline characteristics, to evaluate the relationship between cancer (active or historical) and atrial cardiopathy. We performed a subgroup analysis among patients with embolic stroke of undetermined source (ESUS). RESULTS: The final cohort included 1104 patients with acute ischemic stroke, of whom 10 % had active cancer and 47 % had atrial cardiopathy. Patients with atrial cardiopathy, compared to those without, were older (median age, 77 versus 68 years), and more frequently had hypertension, coronary disease, and atrial fibrillation. Active cancer was present in 9.6 % of patients with atrial cardiopathy (n = 50/520) and 10.4 % of patients without (n = 61/584). There was no association between active cancer and atrial cardiopathy among the overall ischemic stroke cohort (adjusted odds ratio [OR], 0.91; 95 % confidence interval [CI], 0.60-1.37) nor in patients with ESUS (aOR, 0.64; 95 % CI, 0.30-1.36). When the cancer exposure was broadened to include any history of cancer (n = 236, 21.4 %), there still was no significant association with atrial cardiopathy (aOR, 0.93; 95 % CI, 0.68-1.25). CONCLUSIONS: When defining atrial cardiopathy by left atrial volume, we did not find an association between cancer and atrial cardiopathy in patients with ischemic stroke, including among those with ESUS. Future studies, evaluating other atrial cardiopathy biomarkers and settings, are needed to further investigate any potential link between cancer and atrial cardiopathy.

Mortality in acute ischemic stroke patients with new cancer diagnosed during the index hospitalization versus after discharge.

BACKGROUND: Early diagnosis of previously unknown cancer (i.e., occult cancer) after an acute ischemic stroke (AIS) could result in faster initiation of cancer therapy and potentially improve clinical outcomes. Our study aimed to compare mortality rates between AIS patients with occult cancer diagnosed during the index stroke hospitalization versus those diagnosed after hospital discharge. METHODS: Among consecutive AIS patients treated at our stroke center from 2015 through 2020, we identified new cancer diagnoses made within the year after the AIS. We used multivariable Cox regression analyses to evaluate the association between the timing of occult cancer diagnosis (during the AIS hospitalization versus after discharge) and long-term survival. RESULTS: Of 3894 AIS patients with available long-term follow-up data, 59 (1.5 %) were diagnosed with a new cancer within one year after index stroke. Of these, 27 (46 %) were diagnosed during the index hospitalization and 32 (54 %) were diagnosed after discharge. During a median follow-up of 406 days (interquartile range, 89-1073), 70 % (n = 19) of patients whose cancer was diagnosed during hospitalization had died, compared to 63 % (n = 20) of patients whose cancer was diagnosed after discharge (p= 0.58). In our main multivariable model, there was no difference in long-term mortality between patient groups (adjusted hazard ratio, 1.16; 95 % confidence interval, 0.53-2.52; p= 0.71). CONCLUSIONS: In this analysis, timing of a new cancer diagnosis after AIS did not seem to influence patients' long-term survival. Given the fairly small number of included patients with previously occult cancer, larger multicenter studies are needed to confirm our results.

A combined spatial score of granzyme B and CD68 surpasses CD8 as an independent prognostic factor in TNM stage II colorectal cancer.

BACKGROUND: Previous assessments of peritumoral inflammatory infiltrate in colorectal cancer (CRC) have focused on the role of CD8+ T lymphocytes. We sought to compare the prognostic value of CD8 with downstream indicators of active immune cell function, specifically granzyme B (GZMB) and CD68 in the tumour microenvironment. METHODS: Immunohistochemical (IHC) staining was performed for CD8, GZMB, CD68 and CD163 on next-generation tissue microarrays (ngTMAs) in a primary cohort (n = 107) and a TNM stage II validation cohort (n = 151). Using digital image analysis, frequency of distinct immune cell types was calculated for tumour proximity (TP) zones with varying radii (10 µm-100 µm) around tumour cells. RESULTS: Associations notably of advanced TNM stage were observed for low density of CD8 (p = 0.002), GZMB (p < 0.001), CD68 (p = 0.034) and CD163 (p = 0.011) in the primary cohort. In the validation cohort only low GZMB (p = 0.036) was associated with pT4 stage. Survival analysis showed strongest prognostic effects in the TP25µm zone at the tumour centre for CD8, GZMB and CD68 (all p < 0.001) in the primary cohort and for CD8 (p = 0.072), GZMB (p = 0.035) and CD68 (p = 0.004) in the validation cohort with inferior prognostic effects observed at the tumour invasive margin. In a multivariate survival analysis, joint analysis of GZMB and CD68 was similarly prognostic to CD8 in the primary cohort (p = 0.007 vs. p = 0.002) and superior to CD8 in the validation cohort (p = 0.005 vs. p = 0.142). CONCLUSION: Combined high expression of GZMB and CD68 within 25 µm to tumour cells is an independent prognostic factor in CRC and of superior prognostic value to the well-established CD8 in TNM stage II cancers. Thus, assessment of antitumoral effect should consider the quality of immune activation in peritumoral inflammatory cells and their actual proximity to tumour cells.

Development of a Score for Prediction of Occult Malignancy in Stroke Patients (Occult-5 Score).

BACKGROUND AND PURPOSE: Malignancy associated acute ischemic stroke (AIS) requires specific diagnostic work-up, treatment and prevention to improve outcome. This study aimed to develop a biomarker-based score for prediction of occult malignancy in AIS patients. METHODS: Single-center cross-sectional study including consecutive AIS patients treated between July 2017 and November 2018. Patients with active malignancy at presentation, or diagnosed within 1 year thereafter and patients free of malignancy, were included and malignancy associated biomarkers were assessed. LASSO analyses of logistic regression were performed to determine biomarkers predictive of active malignancy. Predictors were derived from a predictive model for active malignancy. A comparison between known and unknown (=occult) malignancies when the index stroke occurred was used to eliminate variables not associated with occult malignancy. A predictive score (OCCULT-5 score) for occult malignancy was developed based on the remaining variables. RESULTS: From 1001 AIS patients, 61 (6%) presented an active malignancy. Thirty-nine (64%) were known and 22 (36%) occult. Five variables were included in the final OCCULT-5 score: age ≥ 77 years, embolic stroke of undetermined source, multi-territorial infarcts, D-dimer levels ≥ 820 µ/gL, and female sex. A score of ≥ 3 predicted an underlying occult malignancy with a sensitivity of 64%, specificity of 73%, positive likelihood ratio of 2.35 and a negative likelihood ratio of 0.50. CONCLUSIONS: The OCCULT-5 score might be useful to identify patients with occult malignancy. It may thus contribute to a more effective and timely treatment and thus lead to a positive impact on overall outcome.

Role of enterocyte-specific gene polymorphisms in response to adjuvant treatment for stage III colorectal cancer.

OBJECTIVES: The enterocyte subtype of colorectal cancer (CRC) responds favorably to oxaliplatin-based adjuvant treatment for stage III CRC. We examined the clinical significance of single-nucleotide polymorphisms (SNPs) in enterocyte-related genes MS4A12 and CDX2 in response to adjuvant treatment for stage III CRC. PATIENTS AND METHODS: A total of 350 patients with stage III CRC were included: 274 received adjuvant treatment with surgical resection (discovery cohort) and 76 received surgery alone (control cohort). In the discovery cohort, 68 patients received FOLFOX and 206 received oral fluoropyrimidine. SNPs were analyzed by PCR-based direct sequencing. RESULTS: In the discovery cohort, the MS4A12 rs4939378 G/G variant was associated with lower 5-year survival than any A allele [70% vs. 90%, univariate: hazard ratio (HR) 2.29, 95% confidence interval (CI) 1.03-5.06, P = 0.035; multivariate: HR 2.58, 95% CI 1.15-5.76, P = 0.021]. Patients with the CDX2 rs3812863 G/G variant had better overall survival than those with any A allele, although this was not significant in multivariate analysis (5 year-survival: 95% vs. 82%, univariate: HR 0.34, 95% CI 0.12-0.97, P = 0.034; multivariate: HR 0.39, 95% CI 0.13-1.11, P = 0.078). The SNPs did not show significant association with overall survival in the control cohort, and significant interaction was observed between MS4A12 genotypes and groups (P = 0.007). CONCLUSIONS: Our findings suggest that MS4A12 and CDX2 gene polymorphisms may predict outcome in stage III CRC. However, the clinical significance of SNPs for response to oxaliplatin may differ by tumor stage.

Clinical significance of enterocyte-specific gene polymorphisms as candidate markers of oxaliplatin-based treatment for metastatic colorectal cancer.

Colorectal cancer (CRC) can be classified into subtypes based on gene expression signatures. Patients with stage III enterocyte subtype of the CRC Assigner classifier have been shown to benefit from oxaliplatin adjuvant therapy. Here, we investigated whether single nucleotide polymorphisms (SNPs) in two enterocyte subtype-related genes, MS4A12 and CDX2, could predict the efficacy of oxaliplatin in first-line treatment for patients with metastatic CRC (mCRC). Three cohorts of patients were included: a discovery cohort receiving FOLFOX ± bevacizumab (BEV) (n = 146), a validation cohort receiving FOLFOXIRI + BEV (n = 230), and a control cohort receiving FOLFIRI + BEV (n = 228). SNPs were analyzed by PCR-based direct sequencing. In the discovery cohort, MS4A12 rs4939378 and CDX2 rs3812863 were identified as potential markers of efficacy. In the validation cohort, any G allele of MS4A12 rs4939378 was associated with longer progression-free survival (PFS) than the A/A variant in both univariate analysis (12.4 vs. 10.9 months, hazard ratio [HR] 0.70, 95% confidence interval [CI] 0.49-0.99, P = 0.033) and multivariable analysis (HR 0.65, 95%CI 0.44-0.97, P = 0.035) in patients expressing wild-type KRAS, but not mutant KRAS. In contrast, longer PFS was observed for patients expressing the CDX2 rs3812863 G/G variant than any A allele in univariate analysis (32.3 vs. 10.3 months, HR 0.39, 95%CI 0.19-0.81, P = 0.004) only in patients expressing mutant KRAS. These findings were not observed in the control cohort. Thus, MS4A12 and CDX2 SNPs may have utility as predictive biomarkers of response to oxaliplatin-based treatment in mCRC patients.

Impact on survival through consolidation radiotherapy for diffuse large B-cell lymphoma: a comprehensive meta-analysis.

Rituximab has improved response rates and overall survival in B-cell lymphoma (DLBCL). Radiotherapy is an effective treatment modality for lymphomas, but there is uncertainty on its use as consolidation after chemo-immunotherapy mainly in advanced stages. We evaluated its efficacy with a comprehensive meta-analysis and a systematic search of Pubmed, Embase, Cochrane, and abstracts from ASCO, ASH, ESMO and ASTRO published from June 1966 and December 2018. We identified 11 trials that evaluated consolidation radiotherapy following chemotherapy in a randomized fashion in 4'584 patients. The primary endpoint of this meta-analysis was PFS. As three of the eleven trials were retracted, this data is based on 2414 patients. For the primary endpoint (PFS), we found a hazard ratio (HR) of 0.77 (0.51 to 1.17, pooled (tau2: 0.25; I2: 85%), and a HR of 0.80 (0.53 to 1.21, pooled (bivariate meta-analysis). For overall survival, the HR is 0.93 (0.61 to 1.40; pooled (tau2: 0.25; I2: 74%) and 0.86 (0.58 to 1.27) in a bivariate meta-analysis. The lack of benefit did not change over time (p-value: 0.95 (tau2: 0.32; I2: 88%), and was also absent for PFS when stratifying for chemotherapy, the use of Rituximab, age, the dose of radiotherapy, application to patients in complete remission and with bulky disease. None of the trials used a PET-guided approach. This meta-analysis revealed no survival benefit when consolidation radiotherapy is given to unselected DLBCL patients following chemotherapy. These results need to be considered in future trials in the PET-CT era.

Epidermal growth factor receptor mRNA expression: A potential molecular escape mechanism from regorafenib.

Regorafenib has improved the survival of patients with refractory metastatic colorectal cancer (mCRC), yet the mechanisms of inherited or acquired resistance are not well understood. A total of 50 patients with refractory mCRC were enrolled. Circulating tumor cell (CTC) enumeration was carried out at baseline, day 21 after initiation of regorafenib, and at the time of progression of disease (PD) using the CellSearch System (Veridex LLC, NJ, USA). Poly(A) mRNA was extracted from CTCs, and gene expression of epithelial and epithelial-mesenchymal transition markers was analyzed by a multiplex-PCR based DNA Chip. Patients with fewer than 3 CTCs at baseline and day 21 had a longer progression-free survival than those with 3 or more CTCs (3.3 vs 2.0 months, P = .008 and 3.3 vs 2.0 months, P = .004, respectively). Patients with fewer than 3 CTCs at baseline and day 21 had a longer overall survival (OS) than those with 3 or more CTCs (10.0 vs 4.6 months, P < .001 and 8.7 vs 3.8 months, P = .003, respectively). In multivariable analysis, CTC counts remained significantly associated with OS at baseline and day 21 (P = .019 and P = .028). Circulating tumor cell EGFR gene expression was upregulated at day 21 and/or PD in 64% of patients. Patients had significantly increased EGFR expression at PD compared to baseline (P = .041) and at day 21 and/or PD compared to baseline (P = .004). Our findings suggest that CTC count and EGFR expression could be useful markers of regorafenib efficacy and outcomes. Upregulation of CTC EGFR expression might be a molecular escape mechanism under regorafenib therapy.

Tumour budding and its clinical implications in gastrointestinal cancers.

Tumour budding in colorectal cancer has become an important prognostic factor. Represented by single cells or small tumour cell clusters at the invasion front of the tumour mass, these tumour buds seem to reflect cells in a 'hybrid' state of epithelial-mesenchymal transition, and evidence indicates that the presence of these entities is associated with lymph node metastasis, local recurrence and distant metastatic disease. The International Tumour Budding Consensus Conference (ITBCC) has highlighted a scoring system for the reporting of tumour budding in colorectal cancer, as well as different clinical scenarios that could affect patient management. Other organs are not spared: tumour budding has been described in numerous gastrointestinal and non-gastrointestinal cancers. Here, we give an update on ITBCC validation studies in the context of colorectal cancer and the clinical implications of tumour budding throughout the upper gastrointestinal and pancreatico-biliary tract.

Preservation of Epstein-Barr virus status and mismatch repair protein status along the metastatic course of gastric cancer.

AIMS: Epstein-Barr virus (EBV) in-situ hybridisation and mismatch repair (MMR) protein immunohistochemistry identifies two subgroups of gastric cancer (GC) with high immunogenicity and likelihood for response to immune check-point inhibition. As tumour biology may change during the metastatic course, which can negatively influence the success of therapeutic decisions made on primary tissue, we investigated the consistency of GC EBV and MMR status within primary tumours and metastases. METHODS AND RESULTS: We investigated a cohort of 415 primary resected GC, including 111 cases with corresponding distant metastases and 297 cases with lymph node metastases. Tumours were analysed by EBV in-situ hybridisation and MLH1, PMS2, MSH2 and MSH6 immunohistochemistry using tissue microarray technique. Primary tumours were grouped as EBV-positive MMR-proficient, EBV-negative MMR-deficient and EBV-negative MMR-proficient. Eleven of 415 (2.7%) of primary tumours were EBV-positive MMR-proficient, whereas 49 of 415 (11.8%) of tumours were EBV-negative MMR-deficient. EBV and MMR protein status showed full concordance with that of the primary tumours. MMR-deficient tumours were of lower pT-category (P < 0.001), had fewer lymph node metastases [24 of 49 (49%) versus 273 of 361 (75.6%) cases; P < 0.001] and a lower rate of distant metastases [six of 49 (12.2%) versus 105 of 366 (28.7%) cases; P = 0.015]. CONCLUSION: We demonstrate a strong correlation of EBV and MMR status between primary tumours, lymph node and distant metastases in a large series of primary resected GC. The cases showed the expected frequency of EBV-positive MMR-deficient and EBV-negative MMR-proficient tumours. We conclude that tissue testing for molecular subtyping for therapeutic decision-making can be reliably performed on primary tumours and metastases in GC.

Simple acute phase protein score to predict long-term survival in patients with acute myeloid leukemia.

High levels of acute phase reactants can be associated with adverse outcome in patients with various solid tumor types. For patients with acute myeloid leukemia (AML), this correlation is unknown. We retrospectively investigated the prognostic value of pretreatment acute phase protein levels in 282 consecutive newly diagnosed AML patients undergoing at least one cycle of intensive induction chemotherapy. We applied a new score integrating pre-treatment C-reactive protein (CRP), fibrinogen, and albumin levels termed the CFA ratio, and we stratified patients into two groups: Patients with a CFA ratio below 3.06 had decisively better progression-free (26.2 vs 7.7 months; P < .001), disease-free (56.4 vs 8.7 months; P < .001), and overall survival (61.2 vs 13.8 months; P < .001). Results remained significant when adjusting for confounders including European Leukemia Network risk group. Early mortality also tended to be lower in the low CFA ratio group. Finally, patients with lower modified Glasgow prognostic score (mGPS) similarly had better outcome. In conclusion, our data suggest that an elevated CFA ratio as well as a high mGPS are associated with adverse outcome in patients with newly diagnosed AML undergoing intensive induction. These parameters should undergo prospective evaluation for their contribution to risk profiling in AML patients.

Colorectal cancer: epigenetic alterations and their clinical implications.

Colorectal cancer (CRC) is a heterogeneous disease with distinct molecular and clinical features, which reflects the wide range of prognostic outcomes and treatment responses observed among CRC patients worldwide. Our understanding of the CRC epigenome has been largely developed over the last decade and it is now believed that among thousands of epigenetic alterations present in each tumor, a small subgroup of these may be considered as a CRC driver event. DNA methylation profiles have been the most widely studied in CRC, which includes a subset of patients with distinct molecular and clinical features now categorized as CpG island methylator phenotype (CIMP). Major advances have been made in our capacity to detect epigenetic alterations, providing us with new potential biomarkers for diagnostic, prognostic and therapeutic purposes. This review aims to summarize our current knowledge about epigenetic alterations occurring in CRC, underlying their potential future clinical implications in terms of diagnosis, prognosis and therapeutic strategies for CRC patients.

Genetic variants in CCL5 and CCR5 genes and serum VEGF-A levels predict efficacy of bevacizumab in metastatic colorectal cancer patients.

Early VEGF-A reduction (EVR) by targeting abundant VEGF-A is a potential predictive marker of bevacizumab (BEV). The CCL5/CCR5 axis modulates VEGF-A production via endothelial progenitor cells migration. We tested whether genetic polymorphisms in the CCL5/CCR5 pathway could predict efficacy of BEV in patients with metastatic colorectal cancer (mCRC) in a first-line setting. Genomic DNA was extracted from 215 samples from three independent cohorts: 61 patients receiving FOLFOX+BEV (evaluation cohort); 83 patients receiving FOLFOX (control cohort); 71 patients receiving FOLFOX/XELOX+BEV (exploratory cohort) for validation and serum biochemistry assay (n = 48). Single nucleotide polymorphisms of genes in the CCL5/CCR5 pathway were analyzed by PCR-based direct sequencing. Considering the unbalanced distribution of patient baseline characteristics between the evaluation and control cohorts, propensity score matching analysis was performed. Serum VEGF-A levels during treatment were measured using ELISA. Among the evaluation and control cohorts, patients with any CCL5 rs2280789 G allele had longer progression-free survival (PFS) and overall survival (OS) when receiving FOLFOX+BEV than FOLFOX (PFS: 19.8 vs. 11.0 months, HR 0.44, 95%CI: 0.24-0.83, p = 0.004; OS: 41.8 vs. 24.5 months, HR: 0.50, 95%CI: 0.26-0.95, p = 0.024). No significant difference was shown in patients with the A/A variant. In the exploratory cohort, CCL5 rs2280789 G alleles were associated with higher VEGF-A levels at baseline and a greater decrease in VEGF-A levels at day 14 compared to the A/A variant. CCL5 and CCR5 impact the angiogenic environment, and the genotypes in CCL5/CCR5 genes may identify specific populations who will benefit from BEV in first-line treatment for mCRC.

AMPK variant, a candidate of novel predictor for chemotherapy in metastatic colorectal cancer: A meta-analysis using TRIBE, MAVERICC and FIRE3.

AMP-activated protein kinase (AMPK) is a key sensor of energy homeostasis and regulates cell metabolism, proliferation and chemotherapy/radiotherapy sensitivities. This study aimed to explore the relationship between the AMPK pathway-related single nucleotide polymorphisms (SNPs) and clinical outcomes in patients with metastatic colorectal cancer (mCRC). We analyzed a total of 884 patients with mCRC enrolled in three randomized clinical trials (TRIBE, MAVERICC and FIRE-3: where patients were treated with FOLFIRI, mFOLFOX6 or FOLFOXIRI combined with bevacizumab or cetuximab as the first-line chemotherapy). The association between AMPK pathway-related SNPs and clinical outcomes was analyzed across the six treatment cohorts, using a meta-analysis approach. Our meta-analysis showed that AMPK pathway had significant associations with progression-free survival (PFS; p < 0.001) and overall survival (OS; p < 0.001), but not with tumor response (TR; p = 0.220): PRKAA1 rs13361707 was significantly associated with favorable PFS (log HR = -0.219, SE = 0.073, p = 0.003), as well as PRKAA1 rs10074991 (log HR = -0.215, SE = 0.073, p = 0.003), and there were suggestive associations of PRKAG1 rs1138908 with unfavorable OS (log HR = 0.170, SE = 0.083, p = 0.041), and of UBE2O rs3803739 with unfavorable PFS (log HR = 0.137, SE = 0.068, p = 0.042) and OS (log HR = 0.210, SE = 0.077, p = 0.006), although these results were not significant after false discovery rate adjustment. AMPK pathway-related SNPs may be predictors for chemotherapy in mCRC. Upon validation, our findings would provide novel insight for selecting treatment strategies.

International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study.

BACKGROUND: The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS: An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance. FINDINGS: Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0·97 for colon tumour; r=0·97 for invasive margin; p<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 [8%] patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio [HR] for high vs low Immunoscore 0·20, 95% CI 0·10-0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21-0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system. INTERPRETATION: The Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer. FUNDING: French National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.

Potential role of PIN1 genotypes in predicting benefit from oxaliplatin-based and irinotecan-based treatment in patients with metastatic colorectal cancer.

PIN1-mediated substrate isomerization plays a role in the repair of DNA double-strand breaks. We hypothesized that genetic polymorphisms in PIN1-related pathways may affect tumor sensitivity to oxaliplatin or irinotecan in metastatic colorectal cancer (mCRC) patients. We analyzed genomic DNA from five cohorts of mCRC patients (total 950) treated with different first-line treatments: oxaliplatin cohorts 1 (n = 146) and 2 (n = 70); irinotecan cohorts 1 (n = 228), and 2 (n = 276); and combination cohort (n = 230). Single nucleotide polymorphisms of candidate genes were analyzed by PCR-based direct sequencing. In the oxaliplatin cohort 1, patients carrying any PIN1 rs2233678 C allele had shorter progression-free survival (PFS) and overall survival (OS) than the G/G variant (PFS, 7.4 vs. 15.0 months, hazard ratio [HR] 3.24, P < 0.001; OS, 16.9 vs. 31.5 months, HR: 2.38, P = 0.003). In contrast, patients with C allele had longer median PFS than patients with G/G (11.9 vs. 9.4 months, HR: 0.64, 95%CI: 0.45-0.91, P = 0.009) in the irinotecan cohort 1. No significant differences were observed in the combination cohort. In comparison between the irinotecan cohort 1 and combination cohort, the patients carrying the G/G variant benefit greatly from the combination compared with irinotecan-based regimen (PFS, 11.6 vs. 9.4 months, HR 0.61, 95%CI: 0.47-0.78, P < 0.001; OS, 30.6 vs. 24.0 months, HR 0.79, 95%CI: 0.62-1.02, P = 0.060), while no significant difference was shown in any C allele. Germline PIN1 polymorphisms may predict clinical outcomes in mCRC patients receiving oxaliplatin-based or irinotecan-based therapy, and identify specific populations favorable to oxaliplatin plus irinotecan combination therapy.

Predictive value of TLR7 polymorphism for cetuximab-based chemotherapy in patients with metastatic colorectal cancer.

The TLR7 and TLR9 signalings are implicated in the regulation of the immune system through type-I interferon induction. Preclinical studies have demonstrated the immunomodulatory and antitumor effects of TLR7 and TLR9 agonists in combination with cetuximab. We tested the hypothesis that genetic variations in TLR7 and TLR9 and their downstream molecules IRF5 and IRF7 were associated with outcomes in metastatic colorectal cancer (mCRC) patients receiving cetuximab-based chemotherapy. Six single nucleotide polymorphisms (SNPs) in TLR7, TLR9, IRF5 and IRF7 were tested for the association with RR, PFS, and OS in KRAS-wild type mCRC patients. Patients treated with FOLFIRI + cetuximab or FOLFIRI + bevacizumab in the FIRE-3 trial served as a discovery set (FIRE3-Cet, n = 244) or a control set (FIRE3-Bev, n = 246), respectively. Patients treated with FOLFOX or SOX + cetuximab in the JACCRO-CC05/06 trial served as a validation set (JACCRO, n = 76). Genomic DNA isolated from tumor tissue samples was analyzed by PCR-based direct sequencing. In the discovery cohort, patients with the TLR7 rs3853839 G/G variant showed a trend toward longer PFS than those with any C variants (median 10.0 vs. 11.8 months, HR 1.39, p = 0.092). This preliminary association was confirmed in the validation cohort, and those with the G/G genotype showed a PFS benefit compared with others (univariate: 9.1 vs. 11.6 months, HR 2.04, p = 0.005, multivariate: HR 2.02, 95% CI: 1.14-3.55, p = 0.015). This association was not observed in the control cohort. Our findings suggest that TLR7 rs3853839 predicts the outcome of cetuximab-based chemotherapy in mCRC patients.

Single nucleotide polymorphisms in the IGF-IRS pathway are associated with outcome in mCRC patients enrolled in the FIRE-3 trial.

The Insulin-like growth factor (IGF)/IGF-receptor pathway with its scaffolding proteins Insulin Receptor Substrate (IRS)1 and IRS2 are crucial regulators of metabolism and progression in metastatic colorectal cancer (mCRC). The goal of the study was the identification of predictive and prognostic markers among IRS1, IRS2, IGF1 and IGF-1R SNPs in mCRC patients enrolled in the FIRE-3 trial. Four SNPs of IRS (IRS1 rs1801278, rs1801123; IRS2 rs1805097, rs2289046) and four SNPs of IGF1-IGFR1 (rs6214, rs6220, rs2946834, rs2016347) were analyzed by PCR/direct-sequencing in the FIRE-3 trial. The relation of SNPs with PFS and OS was evaluated through Kaplan-Meier method and log-rank test in the overall population and in subgroup according to RAS status and treatment arm. In the overall population IRS1 rs1801123 C/- carriers (N= 105) achieved significantly worse OS compared to T/T (N = 464) in univariate (HR = 1.32 [95%CI 1.03-1.70], p = 0.029) and in multivariable. Similar results were observed among RAS wild type. Patients with IGF1 rs2946834 T/- variant (N= 280) achieved improved PFS compared to C/C (N = 257) in univariate (HR = 0.77 [95%CI 0.64-0.92], p = 0.004) and in multivariable. In the RAS wild-type subgroup IGF1 rs2946834 T/- carriers showed better PFS and OS compared to C/C (univariate HR for PFS = 0.65 [95%CI 0.51-0.81], p < 0.001; multivariable HR for PFS = 0.63 [95%CI 0.50-0.81], p < 0.001). IRS1 rs1801123 SNP was identified as a new prognostic marker for mCRC. IGF1 rs2946834 was confirmed as prognostic factor in the overall population and in RAS wild type patients. Our findings underline the importance of IGF downstream signaling pathway in RAS wild-type mCRC patient.