What Is New about the Semimembranosus Distal Tendon? Ultrasound, Anatomical, and Histological Study with Clinical and Therapeutic Application.

The semimembranosus muscle inserts into several tendons that are associated with some pathologies. Although ultrasound is useful for studying, diagnosing, and managing these pathologies, the correct interpretation of any images requires a clear knowledge of the related anatomical structures and the inter-related functions. We studied 38 cryopreserved non-paired knees from adult anatomical specimens and 4 non-paired knees from 29 to 38-week-old fetuses. The semimembranosus muscle and its tendons were located, observed, and injected under ultrasound guidance. The macroscopic anatomy was studied using dissection and anatomical cuts and the tendons were analyzed histologically. Measurements of muscle were taken 10 cm from the medial epicondyle and just before the tendon divided. The ultrasound facilitated the identification of the different divisions of the tendon of semimembranosus muscle and the rotation of the muscle and tendon from medial to posterior. An anatomical study confirmed this rotation and revealed an average width, thickness, and diameter of 38.29 mm, 14.36 mm, and 112.64 mm, respectively. Important relationships were observed between the divisions of the main tendons and the medial collateral ligament, the posterior side of the knee and popliteus muscle. This information can help to explain knee pathologies and facilitate rehabilitation after surgery.

Dermatofibrosarcoma protuberans. A study of 148 patients.

INTRODUCTION: Dermatofibrosarcoma protuberans (DFSP) is the most common sarcoma of the skin. Although distant metastases are infrequent, DFSP is highly aggressive locally with frequent local recurrences. It has been reported that the presence within the tumour of areas histopathologically mimicking fibrosarcoma may increase the risk of recurrence. OBJECTIVE: To review the clinical features of our patients with DFSP and the factors associated with recurrence of the tumour, focusing on the presence of fibrosarcomatous areas. METHODS: Retrospective study of patients with DFSP diagnosed in 1990-2021 in a tertiary university hospital. The medical records were reviewed to obtain the following data: age, sex, tumour location, diameter, and evolution time, presence of fibrosarcomatous areas, development of recurrence, and follow-up. Factors possibly associated with disease-free survival were analyzed with Kaplan-Meier method and multivariate Cox regression. RESULTS: 148 patients (74 women/74 men, mean age 46.28 years, SD 14.431) were included in the study. Tumours involved head and neck in 15 cases, thorax in 31, abdomen in 16, upper back in 43, lower back in 10, upper extremities in 10, and lower extremities in 23. Fibrosarcoma-like areas were observed in 16 tumours (10.81%) . In 17 patients (11.49%) recurrences were observed (13 local recurrences, 3 lung metastasis, and 1 local recurrence with lung metastasis). Fibrosarcomatous DFSP recurred more frequently than classic DFSP (50% vs. 6.82%, respectively), and its disease free survival was significantly lower (p<0.001). In multivariate Cox regression the presence of fibrosarcomatous areas was the only factor influencing disease free survival. CONCLUSIONS: It is important to identify the fibrosarcomatous variant since it recurs more frequently and has lower recurrence-free survival. Distant metastases, mainly in the lung, are also more frequent in fibrosarcomatous DFSP.

Identification of a Twelve-microRNA Signature with Prognostic Value in Stage II Microsatellite Stable Colon Cancer.

We aimed to identify and validate a set of miRNAs that could serve as a prognostic signature useful to determine the recurrence risk for patients with COAD. Small RNAs from tumors of 100 stage II, untreated, MSS colon cancer patients were sequenced for the discovery step. For this purpose, we built an miRNA score using an elastic net Cox regression model based on the disease-free survival status. Patients were grouped into high or low recurrence risk categories based on the median value of the score. We then validated these results in an independent sample of stage II microsatellite stable tumor tissues, with a hazard ratio of 3.24, (CI95% = 1.05-10.0) and a 10-year area under the receiver operating characteristic curve of 0.67. Functional analysis of the miRNAs present in the signature identified key pathways in cancer progression. In conclusion, the proposed signature of 12 miRNAs can contribute to improving the prediction of disease relapse in patients with stage II MSS colorectal cancer, and might be useful in deciding which patients may benefit from adjuvant chemotherapy.

Possible Points of Ulnar Nerve Entrapment in the Arm and Forearm: An Ultrasound, Anatomical, and Histological Study.

BACKGROUND: Ulnar nerve entrapment is one of the most common entrapment neuropathies, usually occurring in the cubital tunnel of the elbow and in Guyon's canal of the wrist. However, it can also occur at other anatomical locations. PURPOSE: Our aim was to review other possible locations of ulnar nerve entrapment in an ultrasound and anatomical study. MATERIAL AND METHODS: Eleven upper limbs from eight adult corpses were ultrasonographically examined and subsequently dissected in a dissection laboratory. Four specific anatomical points were analysed, and any anatomical variations were documented. Moreover, six samples of the nerve were taken for histological analysis. RESULTS: Distinct anatomical relationships were observed during ultrasound and dissection between the ulnar nerve and the medial intermuscular septum, the triceps aponeurosis, Osborne's fascia at the elbow, the arcuate ligament of Osborne and the intermuscular aponeurosis between the flexor carpi ulnaris and the flexor digitorum superficialis muscles. A statistical study showed that these locations are potential areas for ulnar nerve compression. In addition, a fourth head of the triceps brachii muscle was found in some specimens. CONCLUSION: Results demonstrate that ultrasound is a good tool to investigate ulnar nerve entrapment neuropathy and to identify other anatomical points where the nerve can remain compressed.

Lessons learnt from the implementation of a colorectal cancer screening programme for lynch syndrome in a tertiary public hospital.

BACKGROUND: Lynch syndrome (LS) is the first cause of inherited colorectal cancer (CRC), being responsible for 2-4% of all diagnoses. Identification of affected individuals is important as they have an increased lifetime risk of multiple CRC and other neoplasms, however, LS is consistently underdiagnosed at the population level. We aimed to evaluate the yield of LS screening in CRC in a single-referral centre and to identify the barriers to its effective implementation. METHODS: LS screening programme included individuals with CRC < 70 years, multiple CRC, or endometrial cancer at any age. Mismatch repair (MMR) protein immunohistochemistry (IHC) analysis was performed in routine practice on the surgical specimen and, if MLH1 IHC was altered, MLH1 gene promoter methylation was analysed. Results were collected in the CRC multidisciplinary board database. LS suspected individuals (altered MMR IHC without MLH1 promoter methylation) were referred to the Cancer Genetic Counselling Unit (CGCU). If accepted, a genetic study was performed. Two checkpoints were included: review of the pathology data and verification of patient referral by a genetic counsellor. RESULTS: Between 2016 and 2019, 381 individuals were included. MMR IHC analysis was performed in 374/381 (98.2 %) CRC cases and MLH1 promoter methylation in 18/21 (85.7 %). Seventeen of the 20 LS suspected individuals were invited for referral at the CGCU. Two cases were not invited and the remaining patient died of cancer before completion of tumour screening. Fifteen individuals attended and a genetic analysis was performed in 15/20 (75 %) LS suspected individuals. Ten individuals were diagnosed with LS, in concordance with the IHC profile (2.7 % of the total cohort). This led to cascade testing in 58/75 (77.3 %) of the available adult relatives at risk, identifying 26 individuals with LS. CONCLUSIONS: Establishing a standardized institutional LS screening programme with checkpoints in the workflow is key to increasing the yield of LS identification.

COLONOMICS - integrative omics data of one hundred paired normal-tumoral samples from colon cancer patients.

Colonomics is a multi-omics dataset that includes 250 samples: 50 samples from healthy colon mucosa donors and 100 paired samples from colon cancer patients (tumor/adjacent). From these samples, Colonomics project includes data from genotyping, DNA methylation, gene expression, whole exome sequencing and micro-RNAs (miRNAs) expression. It also includes data from copy number variation (CNV) from tumoral samples. In addition, clinical data from all these samples is available. The aims of the project were to explore and integrate these datasets to describe colon cancer at molecular level and to compare normal and tumoral tissues. Also, to improve screening by finding biomarkers for the diagnosis and prognosis of colon cancer. This project has its own website including four browsers allowing users to explore Colonomics datasets. Since generated data could be reuse for the scientific community for exploratory or validation purposes, here we describe omics datasets included in the Colonomics project as well as results from multi-omics layers integration.

Adoptive NK Cell Transfer as a Treatment in Colorectal Cancer Patients: Analyses of Tumour Cell Determinants Correlating With Efficacy In Vitro and In Vivo.

Background: Colorectal cancer (CRC) is a heterogeneous disease with variable mutational profile and tumour microenvironment composition that influence tumour progression and response to treatment. While chemoresistant and poorly immunogenic CRC remains a challenge, the development of new strategies guided by biomarkers could help stratify and treat patients. Allogeneic NK cell transfer emerges as an alternative against chemoresistant and poorly immunogenic CRC. Methods: NK cell-related immunological markers were analysed by transcriptomics and immunohistochemistry in human CRC samples and correlated with tumour progression and overall survival. The anti-tumour ability of expanded allogeneic NK cells using a protocol combining cytokines and feeder cells was analysed in vitro and in vivo and correlated with CRC mutational status and the expression of ligands for immune checkpoint (IC) receptors regulating NK cell activity. Results: HLA-I downmodulation and NK cell infiltration correlated with better overall survival in patients with a low-stage (II) microsatellite instability-high (MSI-H) CRC, suggesting a role of HLA-I as a prognosis biomarker and a potential benefit of NK cell immunotherapy. Activated allogeneic NK cells were able to eliminate CRC cultures without PD-1 and TIM-3 restriction but were affected by HLA-I expression. In vivo experiments confirmed the efficacy of the therapy against both HLA+ and HLA- CRC cell lines. Concomitant administration of pembrolizumab failed to improve tumour control. Conclusions: Our results reveal an immunological profile of CRC tumours in which immunogenicity (MSI-H) and immune evasion mechanisms (HLA downmodulation) favour NK cell immunosurveillance at early disease stages. Accordingly, we have shown that allogeneic NK cell therapy can target tumours expressing mutations conferring poor prognosis regardless of the expression of T cell-related inhibitory IC ligands. Overall, this study provides a rationale for a new potential basis for CRC stratification and NK cell-based therapy.

Identifying causal models between genetically regulated methylation patterns and gene expression in healthy colon tissue.

BACKGROUND: DNA methylation is involved in the regulation of gene expression and phenotypic variation, but the inter-relationship between genetic variation, DNA methylation and gene expression remains poorly understood. Here we combine the analysis of genetic variants related to methylation markers (methylation quantitative trait loci: mQTLs) and gene expression (expression quantitative trait loci: eQTLs) with methylation markers related to gene expression (expression quantitative trait methylation: eQTMs), to provide novel insights into the genetic/epigenetic architecture of colocalizing molecular markers. RESULTS: Normal mucosa from 100 patients with colon cancer and 50 healthy donors included in the Colonomics project have been analyzed. Linear models have been used to find mQTLs and eQTMs within 1 Mb of the target gene. From 32,446 eQTLs previously detected, we found a total of 6850 SNPs, 114 CpGs and 52 genes interrelated, generating 13,987 significant combinations of co-occurring associations (meQTLs) after Bonferromi correction. Non-redundant meQTLs were 54, enriched in genes involved in metabolism of glucose and xenobiotics and immune system. SNPs in meQTLs were enriched in regulatory elements (enhancers and promoters) compared to random SNPs within 1 Mb of genes. Three colorectal cancer GWAS SNPs were related to methylation changes, and four SNPs were related to chemerin levels. Bayesian networks have been used to identify putative causal relationships among associated SNPs, CpG and gene expression triads. We identified that most of these combinations showed the canonical pathway of methylation markers causes gene expression variation (60.1%) or non-causal relationship between methylation and gene expression (33.9%); however, in up to 6% of these combinations, gene expression was causing variation in methylation markers. CONCLUSIONS: In this study we provided a characterization of the regulation between genetic variants and inter-dependent methylation markers and gene expression in a set of 150 healthy colon tissue samples. This is an important finding for the understanding of molecular susceptibility on colon-related complex diseases.

Ki-67 immunoexpression and radiological assessment of necrosis improves accuracy of conventional and modified core biopsy systems in predicting the final grade assigned to adult-soft tissue sarcomas. An international collaborative study.

Based on the French Federation Nationale des Centers de Lutte Contre le Cancer (FNCLCC) grading system, this study assesses the accuracy of conventional and modified core biopsy (CB) systems in predicting the final grade (low vs high) assigned to the resected specimen. Substituting Ki-67 immunoexpression for mitotic count, and radiological for histological assessment of necrosis, we used two modified FNCLCC CB grading systems: (1) Ki-67 immunoexpression alone, and (2) Ki-67 plus radiological assessment of necrosis. We graded 199 soft tissue sarcomas (STS) from nine centers, and compared the results for the conventional (obtained from local histopathology reports) and modified CB systems with the final FNCLCC grading of the corresponding resected specimens. Due to insufficient sample quality or lack of available radiologic data, five cases were not evaluated for Ki67 or radiological assessment of necrosis. The conventional FNCLCC CB grading system accurately identified 109 of the 130 high-grade cases (83.8%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (87.9%) of the 199 resected tumors; overestimating the final grade in three cases and underestimating in 21 cases. Modified system 1 (Ki-67) accurately identified 117 of the 130 high-grade cases (90.0%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (89.7%) of the 195 evaluated cases; overestimating seven and underestimating 13 cases. Modified system 2 (Ki-67 plus radiological necrosis) accurately identified 120 of the 130 high-grade cases (92.3%). This last matched the final FNCLCC grading (low vs high) in 177 (91.2%) of the 194 evaluated cases; overestimating seven and underestimating 10 cases. Modified system 2 obtained highest area under ROC curves, although not statistically significant. Underestimated CB grades did not correlate with histological subtypes, although many of the discrepant cases were myxoid tumors (myxofibrosarcomas or myxoid liposarcomas), leiomyosarcomas or undifferentiated pleomorphic/spindle cell sarcomas. Using modified FNCLCC CB grading systems to replace conventional mitotic count and histologic assessment of necrosis may improve the distinction between low and high-grade STS on CB. Our study confirms that classifying grade 1 as low grade and grades 2 and 3 as high grade improves correlation between CB and final grade by up to 21%, irrespective of CB system used. A higher than expected Ki-67 score in a low-grade sarcoma diagnosed on CB should raise concern that a higher-grade component may not have been sampled. Furthermore, correlation of all clinicopathological and radiological findings at multidisciplinary meetings is essential to assess the histological grade on CB as accurately as possible.

Stimulated emission depletion (STED) super resolution imaging of RNA- and protein-containing domains in fixed cells.

Super resolution microscopy has changed our capability to visualize and understand spatial arrangements of RNA- and protein-containing domains in individual cells. In a previous study, we described a novel lncRNA, Tumor-associated NBL2 transcript (TNBL), which originates from a primate specific macrosatellite repeat. We aimed to describe several aspects of TNBL lncRNA, with one focus being pinpointing its precise location in the nucleus, as well as visualizing its interactions with proteins to deduce its functionality. Using a combination of STimulated Emission Depletion (STED) super resolution microscopy, single molecule RNA (smRNA) FISH against TNBL, and immunofluorescence against SAM68 perinucleolar body, we resolved the spatial complexity of the interaction between TNBL aggregates and SAM68 bodies at the perinucleolar region. Here, we describe protocols for a step-by-step optimized smRNA FISH/IF and STED imaging, detailing parameter settings, and three-dimensional data analysis of spatial positioning of subnuclear structures. These protocols can be employed for single-cell imaging of complex nuclear RNA-protein structures.

Lymphocytic infiltration in stage II microsatellite stable colorectal tumors: A retrospective prognosis biomarker analysis.

BACKGROUND: Identifying stage II patients with colorectal cancer (CRC) at higher risk of progression is a clinical priority in order to optimize the advantages of adjuvant chemotherapy while avoiding unnecessary toxicity. Recently, the intensity and the quality of the host immune response in the tumor microenvironment have been reported to have an important role in tumorigenesis and an inverse association with tumor progression. This association is well established in microsatellite instable CRC. In this work, we aim to assess the usefulness of measures of T-cell infiltration as prognostic biomarkers in 640 stage II, CRC tumors, 582 of them confirmed microsatellite stable. METHODS AND FINDINGS: We measured both the quantity and clonality index of T cells by means of T-cell receptor (TCR) immunosequencing in a discovery dataset (95 patients with colon cancer diagnosed at stage II and microsatellite stable, median age 67, 30% women) and replicated the results in 3 additional series of stage II patients from 2 countries. Series 1 and 2 were recruited in Barcelona, Spain and included 112 fresh frozen (FF, median age 69, 44% women) and 163 formalin-fixed paraffin-embedded (FFPE, median age 67, 39% women) samples, respectively. Series 3 included 270 FFPE samples from patients recruited in Haifa, Northern Israel, as part of a large case-control study of CRC (median age 73, 46% women). Median follow-up time was 81.1 months. Cox regression models were fitted to evaluate the prognostic value of T-cell abundance and Simpson clonality of TCR variants adjusting by sex, age, tumor location, and stage (IIA and IIB). In the discovery dataset, higher TCR abundance was associated with better prognosis (hazard ratio [HR] for ≥Q1 = 0.25, 95% CI 0.10-0.63, P = 0.003). A functional analysis of gene expression on these tumors revealed enrichment in pathways related to immune response. Higher values of clonality index (lower diversity) were not associated with worse disease-free survival, though the HR for ≥Q3 was 2.32 (95% CI 0.90-5.97, P = 0.08). These results were replicated in an independent FF dataset (TCR abundance: HR = 0.30, 95% CI 0.12-0.72, P = 0.007; clonality: HR = 3.32, 95% CI 1.38-7.94, P = 0.007). Also, the association with prognosis was tested in 2 independent FFPE datasets. The same association was observed with TCR abundance (HR = 0.41, 95% CI 0.18-0.93, P = 0.03 and HR = 0.56, 95% CI 0.31-1, P = 0.042, respectively, for each FFPE dataset). However, the clonality index was associated with prognosis only in the FFPE dataset from Israel (HR = 2.45, 95% CI 1.39-4.32, P = 0.002). Finally, a combined analysis combining all microsatellite stable (MSS) samples demonstrated a clear prognosis value both for TCR abundance (HR = 0.39, 95% CI 0.26-0.57, P = 1.3e-06) and the clonality index (HR = 2.13, 95% CI 1.44-3.15, P = 0.0002). These associations were also observed when variables were considered continuous in the models (HR per log2 of TCR abundance = 0.85, 95% CI 0.78-0.93, P = 0.0002; HR per log2 or clonality index = 1.16, 95% CI 1.03-1.31, P = 0.016). LIMITATIONS: This is a retrospective study, and samples had been preserved with different methods. Validation series lack complete information about microsatellite instability (MSI) status and pathology assessment. The Molecular Epidemiology of Colorectal Cancer (MECC) study had information about overall survival instead of progression-free survival. CONCLUSION: Results from this study demonstrate that tumor lymphocytes, assessed by TCR repertoire quantification based on a sequencing method, are an independent prognostic factor in microsatellite stable stage II CRC.

Lung metastases share common immune features regardless of primary tumor origin.

BACKGROUND: Only certain disseminated cells are able to grow in secondary organs to create a metastatic tumor. Under the hypothesis that the immune microenvironment of the host tissue may play an important role in this process, we have categorized metastatic samples based on their immune features. METHODS: Gene expression data of metastatic samples (n=374) from four secondary sites (brain, bone, liver and lung) were used to characterize samples based on their immune and stromal infiltration using gene signatures and cell quantification tools. A clustering analysis was done that separated metastatic samples into three different immune categories: high, medium and low. RESULTS: Significant differences were found between the immune profiles of samples metastasizing in distinct organs. Metastases in lung showed a higher immunogenic score than metastases in brain, liver or bone, regardless of their primary site of origin. Also, they preferentially clustered in the high immune group. Samples in this cluster exhibited a clear inflammatory phenotype, higher levels of immune infiltrate, overexpression of programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) pathways and upregulation of genes predicting clinical response to programmed cell death protein 1 (PD-1) blockade (T-cell inflammatory signature). A decision tree algorithm was used to select CD74 as a biomarker that identify samples belonging to this high-immune subtype of metastases, having specificity of 0.96 and sensitivity of 1. CONCLUSIONS: We have found a group of lung-enriched metastases showing an inflammatory phenotype susceptible to be treated with immunotherapy.

Investigating the concordance in molecular subtypes of primary colorectal tumors and their matched synchronous liver metastasis.

To date, no systematic analyses are available assessing concordance of molecular classifications between primary tumors (PT) and matched liver metastases (LM) of metastatic colorectal cancer (mCRC). We investigated concordance between PT and LM for four clinically relevant CRC gene signatures. Twenty-seven fresh and 55 formalin-fixed paraffin-embedded pairs of PT and synchronous LM of untreated mCRC patients were retrospectively collected and classified according to the MSI-like, BRAF-like, TGFB activated-like and the Consensus Molecular Subtypes (CMS) classification. We investigated classification concordance between PT and LM and association of TGFBa-like and CMS classification with overall survival. Fifty-one successfully profiled matched pairs were used for analyses. PT and matched LM were highly concordant in terms of BRAF-like and MSI-like signatures, (90.2% and 98% concordance, respectively). In contrast, 40% to 70% of PT that were classified as mesenchymal-like, based on the CMS and the TGFBa-like signature, respectively, lost this phenotype in their matched LM (60.8% and 76.5% concordance, respectively). This molecular switch was independent of the microenvironment composition. In addition, the significant change in subtypes was observed also by using methods developed to detect cancer cell-intrinsic subtypes. More importantly, the molecular switch did not influence the survival. PT classified as mesenchymal had worse survival as compared to nonmesenchymal PT (CMS4 vs CMS2, hazard ratio [HR] = 5.2, 95% CI = 1.5-18.5, P = .0048; TGFBa-like vs TGFBi-like, HR = 2.5, 95% CI = 1.1-5.6, P = .028). The same was not true for LM. Our study highlights that the origin of the tissue may have major consequences for precision medicine in mCRC.

Pathologist Experience and Concordance in the Diagnosis of Dysplasia in Long-standing Inflammatory Bowel Disease.

Surveillance colonoscopies focused to detect dysplasia are recommended to prevent colorectal cancer in patients with long-standing colonic inflammatory bowel disease (IBD). To date, histologic diagnosis and gradation of IBD-related dysplasia has been challenged by a high variability among pathologists. We aimed to analyze the observer characteristics that are correlated with concordance deviations in this diagnosis. Eight pathologists evaluated a set of 125 endoscopic biopsy samples with a representative distribution of nondysplastic and dysplastic lesions from long-standing IBD patients. Two rounds of diagnosis were carried out during a period of 18 months. The κ test was applied to analyze concordance. Pathologists were grouped on the basis of their experience. A subanalysis was performed by eliminating the highly prevalent nondysplastic samples, as well as an analysis after observers' grouping. Overall interobserver agreement was good (κ=0.73), with an even higher pairwise value (κ=0.86) as well as the intraobserver agreement values (best κ=0.85). After eliminating the highly prevalent nondysplastic samples, the interobserver agreement was still moderate to good (best overall κ=0.50; best paired κ=0.72). Notable differences were seen between the pathologists with a high-volume and low-volume practice (best overall κ=0.61 and 0.41, respectively). The agreement in the diagnosis of dysplasia in IBD endoscopic biopsies may have been undervalued over time. This is the first study evaluating pathologists' diagnostic robustness in this field. The results suggest that examining a large volume of samples is the key factor to increase the consistency in the diagnosis and gradation of IBD-related dysplasia.

New Methylation Biomarker Panel for Early Diagnosis of Dysplasia or Cancer in High-Risk Inflammatory Bowel Disease Patients.

Background: The risk of developing colorectal cancer (CRC) is increased in patients with inflammatory bowel disease (IBD) of the colon. The aim of the study was to evaluate the effectiveness of selected methylation gene panel for the early detection of CRC in high-risk IBD patients. Methods: In a discovery phase, 73 biopsies of 48 IBD patients (associated or not to CRC) were analyzed from genome-wide DNA methylation analysis using the Illumina Human Methylation 450K BeadChip. The panel of 5 genes selected (EYA4, SLIT2, FLI1, USP44, and SND1) was validated prospectively using methylation-specific melting curve analysis in biopsies of diseased and adjacent healthy tissue of 203 patients: 38 with IBD and associated neoplasia, 81 patients with IBD (25 of them with high risk), 48 with sporadic CRC, and 36 healthy controls. Results: The prevalence of methylation was higher in patients with IBD and associated neoplasia (both in diseased and adjacent healthy tissue, 71% and 52%, respectively) than in healthy controls (2/36, 6%; P = 6.72E-05). Methylation in IBD patients at high risk of dysplasia or cancer was more frequently detected than in patients at low risk (92% vs 57%; odds ratio, 8.63; P = 0.001). EYA4 and SLIT2 were the markers most frequently methylated. Differences in methylation levels were more evident in healthy mucosa (82% vs 15% high vs low risk, respectively; P = 1.25E-05). Conclusions: Analysis of this panel of methylation markers may help in the early identification of colorectal dysplasia or cancer in high-risk IBD patients.

Telomere length alterations in microsatellite stable colorectal cancer and association with the immune response.

Telomeres are repetitive sequences (TTAGGG) located at the end of chromosomes. Telomeres progressively shorten with each cell replication cycle, ultimately leading to chromosomal instability and loss of cell viability. Telomere length anomaly appears to be one of the earliest and most prevalent genetic alterations in malignant transformation. Here we aim to estimate telomere length from whole-exome sequencing data in colon tumors and normal colonic mucosa, and to analyze the potential association of telomere length with clinical factors and gene expression in colon cancer. Reads containing at least five repetitions of the telomere sequence (TTAGGG) were extracted from the raw sequences of 42 adjacent normal-tumor paired samples. The number of reads from the tumor sample was normalized to build the Tumor Telomere Length Ratio (TTLR), considered an estimation of telomere length change in the tumor compared to the paired normal tissue. We evaluated the associations between TTLR and clinical factors, gene expression and copy number (CN) aberrations measured in the same tumor samples. Colon tumors showed significantly shorter telomeres than their paired normal samples. No significant association was observed between TTLR and gender, age, tumor location, prognosis, stromal infiltration or molecular subtypes. The functional gene set enrichment analysis showed pathways related to immune response significantly associated with TLLR. By extracting a relative measure of telomere length from whole-exome sequencing data, we have assessed that colon tumor cells predominantly shorten telomeres, and this alteration is associated with expression changes in genes related to immune response and inflammation in tumor cells.

Phase II Study of Gemcitabine Plus Sirolimus in Previously Treated Patients with Advanced Soft-Tissue Sarcoma: a Spanish Group for Research on Sarcomas (GEIS) Study.

BACKGROUND: Gemcitabine plus sirolimus enhances apoptosis in vitro and increases anti-tumor efficacy in vivo in soft-tissue sarcoma (STS) models. OBJECTIVE: The objective of this study was to evaluate the activity and toxicity of the combination of gemcitabine plus sirolimus in patients with STS after failure of standard chemotherapy. PATIENTS AND METHODS: Advanced STS patients, previously treated with doxorubicin and/or ifosfamide, were included in this single-arm phase II study. Patients received gemcitabine 800 mg/m2 intravenously (iv) at 10 mg/m2/min on days 1 and 8 every 3 weeks plus sirolimus 5 mg daily orally (po). After enrolment of the first 12 patients, the study protocol was amended due to toxicity and the starting dose of sirolimus was reduced to 3 mg daily po. Archival tumor samples were analyzed for extracellular signal-regulated kinase 1 and 2 (ERK1/2) expression and correlated with outcome. The primary endpoint was progression-free rate (PFR) at 3 months. RESULTS: From May 2012 to May 2013, 28 patients were enrolled at eight centers. PFR at 3 and 6 months was 44% and 20%, respectively, with 12 patients being free of progression at 3 months. Median progression-free survival (PFS) was 1.85 months (95% confidence interval [CI] 0.73-2.97) and median overall survival (OS) was 9.2 months (95% CI 5.8-12.5). No responses were observed. The most common grade 3-4 hematologic toxicities were neutropenia (48%) and leukopenia (41%) and the most frequent grade 3 non-hematologic toxicities were infection (18.5%), transaminitis (15%), fatigue (11%), and pneumonitis (11%). ERK1/2 expression was significantly correlated with PFS (p = 0.026). CONCLUSIONS: The combination of gemcitabine and sirolimus is an active treatment in STS. Further investigation is warranted. ClinicalTrials.gov identifier: NCT01684449.

Optimization of RAS/BRAF Mutational Analysis Confirms Improvement in Patient Selection for Clinical Benefit to Anti-EGFR Treatment in Metastatic Colorectal Cancer.

In metastatic colorectal cancer (mCRC), recent studies have shown the importance to accurately quantify low-abundance mutations of the RAS pathway because anti-EGFR therapy may depend on certain mutation thresholds. We aimed to evaluate the added predictive value of an extended RAS panel testing using two commercial assays and a highly sensitive and quantitative digital PCR (dPCR). Tumor samples from 583 mCRC patients treated with anti-EGFR- (n = 255) or bevacizumab- (n = 328) based therapies from several clinical trials and retrospective series from the TTD/RTICC Spanish network were analyzed by cobas, therascreen, and dPCR. We evaluated concordance between techniques using the Cohen kappa index. Response rate, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutant allele fraction (MAF). Concordance between techniques was high when analyzing RAS and BRAF (Cohen kappa index around 0.75). We observed an inverse correlation between MAF and response in the anti-EGFR cohort (P < 0.001). Likelihood ratio analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value. PFS and OS were significantly longer in RAS/BRAF wild-type patients, independently of the technique. However, the predictability of both PFS and OS were higher when we considered a threshold of 1% in the RAS scenario (HR = 1.53; CI 95%, 1.12-2.09 for PFS, and HR = 1.9; CI 95%, 1.33-2.72 for OS). Although the rate of mutations observed among techniques is different, RAS and BRAF mutational analysis improved prediction of response to anti-EGFR therapy. Additionally, dPCR with a threshold of 1% outperformed the other platforms. Mol Cancer Ther; 16(9); 1999-2007. ©2017 AACR.

Elucidating the molecular basis of MSH2-deficient tumors by combined germline and somatic analysis.

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.