Identification of biomarkers predictive of metastasis development in early-stage colorectal cancer using network-based regularization.

Colorectal cancer (CRC) is the third most common cancer and the second most deathly worldwide. It is a very heterogeneous disease that can develop via distinct pathways where metastasis is the primary cause of death. Therefore, it is crucial to understand the molecular mechanisms underlying metastasis. RNA-sequencing is an essential tool used for studying the transcriptional landscape. However, the high-dimensionality of gene expression data makes selecting novel metastatic biomarkers problematic. To distinguish early-stage CRC patients at risk of developing metastasis from those that are not, three types of binary classification approaches were used: (1) classification methods (decision trees, linear and radial kernel support vector machines, logistic regression, and random forest) using differentially expressed genes (DEGs) as input features; (2) regularized logistic regression based on the Elastic Net penalty and the proposed iTwiner-a network-based regularizer accounting for gene correlation information; and (3) classification methods based on the genes pre-selected using regularized logistic regression. Classifiers using the DEGs as features showed similar results, with random forest showing the highest accuracy. Using regularized logistic regression on the full dataset yielded no improvement in the methods' accuracy. Further classification using the pre-selected genes found by different penalty factors, instead of the DEGs, significantly improved the accuracy of the binary classifiers. Moreover, the use of network-based correlation information (iTwiner) for gene selection produced the best classification results and the identification of more stable and robust gene sets. Some are known to be tumor suppressor genes (OPCML-IT2), to be related to resistance to cancer therapies (RAC1P3), or to be involved in several cancer processes such as genome stability (XRCC6P2), tumor growth and metastasis (MIR602) and regulation of gene transcription (NME2P2). We show that the classification of CRC patients based on pre-selected features by regularized logistic regression is a valuable alternative to using DEGs, significantly increasing the models' predictive performance. Moreover, the use of correlation-based penalization for biomarker selection stands as a promising strategy for predicting patients' groups based on RNA-seq data.

International consensus to standardise histopathological scoring for small bowel strictures in Crohn's disease.

OBJECTIVE: Effective medical therapy and validated trial outcomes are lacking for small bowel Crohn's disease (CD) strictures. Histopathology of surgically resected specimens is the gold standard for correlation with imaging techniques. However, no validated histopathological scoring systems are currently available for small bowel stricturing disease. We convened an expert panel to evaluate the appropriateness of histopathology scoring systems and items generated based on panel opinion. DESIGN: Modified RAND/University of California Los Angeles methodology was used to determine the appropriateness of 313 candidate items related to assessment of CD small bowel strictures. RESULTS: In this exercise, diagnosis of naïve and anastomotic strictures required increased bowel wall thickness, decreased luminal diameter or internal circumference, and fibrosis of the submucosa. Specific definitions for stricture features and technical sampling parameters were also identified. Histopathologically, a stricture was defined as increased thickness of all layers of the bowel wall, fibrosis of the submucosa and bowel wall, and muscularisation of the submucosa. Active mucosal inflammatory disease was defined as neutrophilic inflammation in the lamina propria and any crypt or intact surface epithelium, erosion, ulcer and fistula. Chronic mucosal inflammatory disease was defined as crypt architectural distortion and loss, pyloric gland metaplasia, Paneth cell hyperplasia, basal lymphoplasmacytosis, plasmacytosis and fibrosis, or prominent lymphoid aggregates at the mucosa/submucosa interface. None of the scoring systems used to assess CD strictures were considered appropriate for clinical trials. CONCLUSION: Standardised assessment of gross pathology and histopathology of CD small bowel strictures will improve clinical trial efficiency and aid drug development.

Histologic Features of Colon Biopsies (Geboes Score) Associated With Progression of Ulcerative Colitis for the First 36 Months After Biopsy.

BACKGROUND & AIMS: In addition to findings from endoscopy, histologic features of colon biopsies have been associated with outcomes of patients with ulcerative colitis (UC). We investigated associations between Geboes scores (a system to quantify structural changes and inflammatory activity in colon biopsies) and UC progression, and the time period over which this association is valid. METHODS: We analyzed data from 399 asymptomatic patients with UC enrolled in the ACERTIVE study, followed at 13 inflammatory bowel disease (IBD) centers in Portugal through 31 December 2019. Blood and stool samples were collected and analyzed, and all patients underwent sigmoidoscopy within 24 h of sample collection. We assessed baseline endoscopic status (Mayo endoscopic subscore), histologic features of 2 sigmoid and 2 rectal biopsies (Geboes score), and concentration of fecal calprotectin (FC). The primary outcome was UC progression (surgical, pharmacologic, and clinical events). We generated survival curves for 36 months or less and more than 36 months after biopsy according to Geboes score using the Kaplan-Meier method and compared findings with those from a log rank test. Cox regression was adjusted for Mayo endoscopic subscore, Geboes score, and level of FC; results were expressed as adjusted hazard ratios (HR) with 95% CIs. RESULTS: Patients with Geboes scores >2B.0, Geboes scores >3.0, or Geboes scores >4.0 had a higher frequency of, and a shorter time to UC progression, than patients with Geboes scores ≤2B.0, Geboes scores ≤3.0, or Geboes score ≤4.0 (P < .001). Disease progression occurred earlier in patients with Geboes scores >2B.0, Geboes scores >3.0, or Geboes scores >4.0 compared with patients with Geboes scores ≤2B.0 (HR, 2.021; 95% CI, 1.158-3.526), Geboes scores ≤3.0 (HR, 2.007; 95% CI, 1.139-3.534), or Geboes scores ≤4.0 (HR, 2.349; 95% CI, 1.269-4.349), respectively, in the first 36 months after biopsy. Similar results were found for patients with concentrations of FC below 150 µg/g. CONCLUSIONS: We found histologic features of colon biopsies (Geboes score) to be an independent risk factor for progression of UC in the first 36 months after biopsy.

Histopathology Scoring Systems of Stenosis Associated With Small Bowel Crohn's Disease: A Systematic Review.

BACKGROUND & AIMS: Stenosis is a common complication of Crohn's disease (CD) that has no effective medical therapy. Development of antifibrotic agents will require testing in randomized controlled trials. Computed tomography enterography- and magnetic resonance enterography-based technologies might be used to measure outcomes in these trials. These approaches have been validated in studies of patients with symptomatic strictures who underwent imaging evaluations followed by resection with histopathologic grading of the intestinal tissue for inflammation and/or fibrosis (the reference standard). Imaging findings have correlated with findings from quantitative or semiquantitative histologic evaluation of the degree of fibromuscular stenosis and/or inflammation on the resection specimen. However, it is not clear whether histologic findings are an accurate reference standard. We performed a systematic review of all published histologic scoring systems used to assess stenosing CD. METHODS: We performed a comprehensive search of Embase and MEDLINE of studies through March 13, 2019, that used a histologic scoring system to characterize small bowel CD and assessed inflammatory and fibrotic alterations within the same adult individual. All scores fitting the criteria were included in our analysis, independent of the presence of stricturing disease, as long as inflammation and fibrosis were evaluated separately but in the same scoring system. RESULTS: We observed substantial heterogeneity among the scoring systems, which were not derived from modern principles for evaluative index development. None had undergone formal validity or reliability testing. None of the existing indices had been constructed according to accepted methods for the development of evaluative indices. Basic knowledge regarding their operating properties were lacking. Specific indices for evaluating the important pathologic component of myofibroblast hypertrophy or hyperplasia have not been proposed. CONCLUSIONS: In a systematic review of publications, we found a lack of validated histopathologic scoring systems for assessment of fibromuscular stenosis. Data that describe the operating properties of existing cross-sectional imaging techniques for stenosing CD should be questioned. Development and validation of a histopathology index is an important research priority.

Comparison of different histological indexes in the assessment of UC activity and their accuracy regarding endoscopic outcomes and faecal calprotectin levels.

OBJECTIVE: Histological remission is being increasingly acknowledged as a therapeutic endpoint in patients with UC. The work hereafter described aimed to evaluate the concordance between three histological classification systems-Geboes Score (GS), Nancy Index (NI) and RobartsHistopathologyIndex (RHI), as well as to evaluate their association with the endoscopic outcomes and the faecal calprotectin (FC) levels. DESIGN: Biopsy samples from 377 patients with UC were blindly evaluated using GS, NI and RHI. The results were compared with the patients' Mayo Endoscopic Score and FC levels. RESULT: GS, NI and RHI have a good concordance concerning the distinction between patients in histological remission or activity. RHI was particularly close to NI, with 100% of all patients classified as being in remission with NI being identified as such with RHI and 100% of all patients classified as having activity with RHI being identified as such with NI. These scores could also predict the Mayo Endoscopic Score and the FC levels, with their sensitivity and specificity levels depending on the chosen cut-offs. Moreover, higher FC levels were statistically associated with the presence of neutrophils in the epithelium, as well as with ulceration or erosion of the intestinal mucosa. CONCLUSIONS: GS, NI and RHI histopathological scoring systems are comparable in what concerns patients' stratification into histological remission/activity. Additionally, FC levels are increased when neutrophils are present in the epithelium and the intestinal mucosa has erosions or ulcers. The presence of neutrophils in the epithelium is, indeed, the main marker of histological activity.

Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue.

BACKGROUND: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. METHODS: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. RESULTS: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. CONCLUSIONS: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.

Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.

Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

Case report: Primary CDK4/6 inhibitor and endocrine therapy in locally advanced breast cancer and its effect on gut and intratumoral microbiota.

Locally advanced breast cancer poses significant challenges to the multidisciplinary team, in particular with hormone receptor (HR) positive, HER2-negative tumors that classically yield lower pathological complete responses with chemotherapy. The increasingly significant use of CDK 4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) in different breast cancer settings has led to clinical trials focusing on this strategy as a primary treatment, with promising results. The impact of the microbiota on cancer, and vice-versa, is an emerging topic in oncology. The authors report a clinical case of a postmenopausal female patient with an invasive breast carcinoma of the right breast, Luminal B-like, staged as cT4cN3M0 (IIIB). Since the lesion was considered primarily inoperable, the patient started letrozole and ribociclib. Following 6 months of systemic therapy, the clinical response was significant, and surgery with curative intent was performed. The final staging was ypT3ypN2aM0, R1, and the patient started adjuvant letrozole and radiotherapy. This case provides important insights on primary CDK4/6i plus ET in locally advanced unresectable HR+/HER2- breast cancer and its potential implications in disease management further ahead. The patient's gut microbiota was analyzed throughout the disease course and therapeutic approach, evidencing a shift in gut microbial dominance from Firmicutes to Bacteroidetes and a loss of microbial diversity following 6 months of systemic therapy. The analysis of the intratumoral microbiota from the surgical specimen revealed high microbial dissimilarity between the residual tumor and respective margins.

Corrigendum: Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

[This corrects the article DOI: 10.3389/fmolb.2023.1082915.].

Sialyl LewisX/A and Cytokeratin Crosstalk in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) encompasses multiple entities and is generally highly aggressive and metastatic. We aimed to determine the clinical and biological relevance of Sialyl-Lewis X and A (sLeX/A)-a fucosylated glycan involved in metastasis-in TNBC. Here, we studied tissues from 50 TNBC patients, transcripts from a TNBC dataset from The Cancer Genome Atlas (TCGA) database, and a primary breast cancer cell line. All 50 TNBC tissue samples analysed expressed sLeX/A. Patients with high expression of sLeX/A had 3 years less disease-free survival than patients with lower expression. In tissue, sLeX/A negatively correlated with cytokeratins 5/6 (CK5/6, which was corroborated by the inverse correlation between fucosyltransferases and CK5/6 genes. Our observations were confirmed in vitro when inhibition of sLeX/A remarkably increased expression of CK5/6, followed by a decreased proliferation and invasion capacity. Among the reported glycoproteins bearing sLeX/A and based on the STRING tool, α6 integrin showed the highest interaction score with CK5/6. This is the first report on the sLeX/A expression in TNBC, highlighting its association with lower disease-free survival and its inverse crosstalk with CK5/6 with α6 integrin as a mediator. All in all, sLeX/A is critical for TNBC malignancy and a potential prognosis biomarker and therapeutic target.

Correction: Association of Tumor-Infiltrating Lymphocytes With Survival in Stages II and III Colorectal Cancer.

[This corrects the article DOI: 10.7759/cureus.31144.].

Expression of NORAD correlates with breast cancer aggressiveness and protects breast cancer cells from chemotherapy.

The recently discovered human lncRNA NORAD is induced after DNA damage in a p53-dependent manner. It plays a critical role in the maintenance of genomic stability through interaction with Pumilio proteins, limiting the repression of their target mRNAs. Therefore, NORAD inactivation causes chromosomal instability and aneuploidy, which contributes to the accumulation of genetic abnormalities and tumorigenesis. NORAD has been detected in several types of cancer, including breast cancer, which is the most frequently diagnosed and the second-leading cause of cancer death in women. In the present study, we confirmed upregulated NORAD expression levels in a set of human epithelial breast cancer cell lines (MDA-MB-231, MDA-MB-436, and MDA-MB-468), which belong to the most aggressive subtypes (triple-negative breast cancer). These results are in line with previous data showing that high NORAD expression levels in basal-like tumors were associated with poor prognosis. Here, we demonstrate that NORAD downregulation sensitizes triple-negative breast cancer cells to chemotherapy, through a potential accumulation of genomic aberrations and an impaired capacity to signal DNA damage. These results show that NORAD may represent an unexploited neoadjuvant therapeutic target for chemotherapy-unresponsive breast cancer.

Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

Background: Around 40% of ER+/HER2-breast carcinomas (BC) present mutations in the PIK3CA gene. Assessment of PIK3CA mutational status is required to identify patients eligible for treatment with PI3Kα inhibitors, with alpelisib currently the only approved tyrosine kinase inhibitor in this setting. U-PIK project aimed to conduct a ring trial to validate and implement the PIK3CA mutation testing in several Portuguese centers, decentralizing it and optimizing its quality at national level. Methods: Eight Tester centers selected two samples of patients with advanced ER+/HER2- BC and generated eight replicates of each (n = 16). PIK3CA mutational status was assessed in two rounds. Six centers used the cobas® PIK3CA mutation test, and two used PCR and Sanger sequencing. In parallel, two reference centers (IPATIMUP and the Portuguese Institute of Oncology [IPO]-Porto) performed PIK3CA mutation testing by NGS in the two rounds. The quality of molecular reports describing the results was also assessed. Testing results and molecular reports were received and analyzed by U-PIK coordinators: IPATIMUP, IPO-Porto, and IPO-Lisboa. Results: Overall, five centers achieved a concordance rate with NGS results (allele frequency [AF] ≥5%) of 100%, one of 94%, one of 93%, and one of 87.5%, considering the overall performance in the two testing rounds. NGS reassessment of discrepancies in the results of the methods used by the Tester centers and the reference centers identified one probable false positive and two mutations with low AF (1-3%, at the analytical sensitivity threshold), interpreted as subclonal variants with heterogeneous representation in the tissue sections processed by the respective centers. The analysis of molecular reports revealed the need to implement the use of appropriate sequence variant nomenclature with the identification of reference sequences (HGVS-nomenclature) and to state the tumor cell content in each sample. Conclusion: The concordance rates between the method used by each tester center and NGS validate the use of the PIK3CA mutational status test performed at these centers in clinical practice in patients with advanced ER+/HER2- BC.

Assessment of a Large-Scale Unbiased Malignant Pleural Effusion Proteomics Study of a Real-Life Cohort.

BACKGROUND: Pleural effusion (PE) is common in advanced-stage lung cancer patients and is related to poor prognosis. Identification of cancer cells is the standard method for the diagnosis of a malignant PE (MPE). However, it only has moderate sensitivity. Thus, more sensitive diagnostic tools are urgently needed. METHODS: The present study aimed to discover potential protein targets to distinguish malignant pleural effusion (MPE) from other non-malignant pathologies. We have collected PE from 97 patients to explore PE proteomes by applying state-of-the-art liquid chromatography-mass spectrometry (LC-MS) to identify potential biomarkers that correlate with immunohistochemistry assessment of tumor biopsy or with survival data. Functional analyses were performed to elucidate functional differences in PE proteins in malignant and benign samples. Results were integrated into a clinical risk prediction model to identify likely malignant cases. Sensitivity, specificity, and negative predictive value were calculated. RESULTS: In total, 1689 individual proteins were identified by MS-based proteomics analysis of the 97 PE samples, of which 35 were diagnosed as malignant. A comparison between MPE and benign PE (BPE) identified 58 differential regulated proteins after correction of the p-values for multiple testing. Furthermore, functional analysis revealed an up-regulation of matrix intermediate filaments and cellular movement-related proteins. Additionally, gene ontology analysis identified the involvement of metabolic pathways such as glycolysis/gluconeogenesis, pyruvate metabolism and cysteine and methionine metabolism. CONCLUSION: This study demonstrated a partial least squares regression model with an area under the curve of 98 and an accuracy of 0.92 when evaluated on the holdout test data set. Furthermore, highly significant survival markers were identified (e.g., PSME1 with a log-rank of 1.68 × 10-6).

Portuguese Consensus Recommendations for Next-Generation Sequencing of Lung Cancer, Rare Tumors, and Cancers of Unknown Primary Origin in Clinical Practice.

Next-generation sequencing (NGS) has been implemented in clinical oncology for diagnosis, prognosis, and therapeutic guidance. Among the various NGS applications in molecular oncology, we focused on the following topics: laboratory standards for targeted gene panels (somatic mutations) and therapeutic guidance based on NGS of lung cancer and rare cancers, namely sarcomas and cancers of unknown primary. Multiple quality control checkpoints should be addressed in the pre-analytical phase for good quality and interpretation of the NGS results. It includes tumor size and cellularity, tissue processing and decalcification, tumor fraction, tumor viability, fixatives, and staining. Communication between clinicians and laboratory support is also essential. In lung cancer, all patients with non-squamous non-small cell lung cancer should be tested with a NGS panel, and it should include not only genes with approved targeted therapies (ALK, BRAF, EGFR, MET, NTRK, RET, and ROS1) but also genes with potentially actionable genomic alterations (HER2 and KRAS). Since there is a lack of extensive knowledge regarding the use of NGS in rare tumors performing comprehensive genomic profiling, NGS panels to better manage the disease are recommended. Moreover, other patients with other incurable solid tumors may benefit from being included in biomarker-driven clinical trials. Multidisciplinary tumor boards with the participation of experts with the ability to integrate genomic profiling data are essential to tailor the best strategy for each patient. Considering that there are no national guidelines, this article aims to guide laboratory and clinical practice for the use of NGS in the context of lung cancer, rare tumors, and cancer of unknown primary in Portugal.

Na área da oncologia clínica, a sequenciação de nova geração (NGS) foi implementada com o objetivo de contribuir para o diagnóstico, prognóstico e orientação terapêutica. A utilização de NGS em oncologia molecular é vasta, focalizando-se estas recomendações nas: normas laboratoriais para painéis genéticos direcionados (mutações somáticas) e na orientação terapêutica baseada em NGS de cancro do pulmão e cancros raros, nomeadamente sarcomas e cancros de origem desconhecida. Para que sejam obtidos resultados de NGS com a qualidade que permita a sua correta interpretação, devem ser abordados múltiplos controlos de qualidade na fase pré-analítica que disponibilizem informação sobre o tamanho e celularidade do tumor, processamento e descalcificação de tecidos, fração tumoral, viabilidade do tumor, fixadores e coloração utilizados. A comunicação entre os diferentes intervenientes no processo, em particular entre os clínicos e o laboratório também contribui, de forma inequívoca, para a interpretação dos resultados de NGS. Todos os doentes com cancro do pulmão de não pequenas células não escamoso devem ser testados com um painel de NGS, que deve incluir não só genes com terapias dirigidas aprovadas (ALK, BRAF, EGFR, MET, NTRK, RET e ROS1), mas também genes com alterações genómicas identificadas como potenciais alvos terapêuticos (HER2 e KRAS). Dada a escassez de evidência científica sobre a utilização de NGS em tumores raros, recomenda-se a realização de painéis genómicos abrangentes que poderão contribuir para uma melhor gestão da doença. Adicionalmente, outros doentes, com outros tumores sólidos incuráveis, podem beneficiar da inclusão em ensaios clínicos orientados por biomarcadores. A realização de reuniões multidisciplinares com a participação de diferentes especialistas capazes de integrar dados dos perfis genómicos são fundamentais para a escolha da melhor estratégia para cada doente. Considerando que não existem recomendações nacionais, este artigo visa orientar a prática laboratorial e clínica para a utilização de NGS em tumores do pulmão, raros e cancros de origem primária desconhecida em Portugal.

Phenotype-Genotype Correlation in Colorectal Cancer: A Real-Life Study.

BACKGROUND AND AIMS: Colorectal cancer (CRC) is a heterogeneous disease with distinctive genetic pathways, such as chromosomal instability, microsatellite instability and methylator pathway. Our aim was to correlate clinical and genetic characteristics of CRC patients in order to understand clinical implications of tumour genotype. METHODS: Single-institution retrospective cohort of patients who underwent curative surgery for CRC, from 2012 to 2014. RAS and BRAF mutations were evaluated with the real-time PCR technique Idylla®. Mismatch repair deficiency (dMMR) was characterized by absence of MLH1, MSH6, MSH2 and/or PMS2 expression, evaluated by tissue microarrays. Overall survival (OS) and disease-free survival (DFS) were assessed using survival analysis. RESULTS: Overall, 242 patients were included (males 57.4%, age 69.3 ± 12.9 years; median follow-up 49 months). RAS-mutated tumours were associated with reduced DFS (p = 0.02) and OS (p = 0.045) in stage I-III CRC. BRAF-mutated tumours were more predominant in females and in the right colon, similarly to dMMR tumours. BRAF status did not influence OS (4 years)/DFS (3.5 years) in stage I-III disease. However, after relapse, length of survival was 3.5 months in BRAF-mutated tumours in contrast to 18.6 months in BRAF wild-type tumours (p = NS). No germline mutations in mismatch repair genes were so far identified in the patients with dMMR tumours. Molecular phenotype (RAS, BRAF and MMR) did not influence OS in metastatic patients. Our small sample size may be a limitation of the study. CONCLUSION: In our cohort, RAS-mutated tumours were associated with worse DFS and OS in early-stage CRC, whereas the remaining molecular variables had no prognostic influence.

INTRODUÇÃO: O cancro colo-rectal (CCR) é uma doença heterogénea, com vias genéticas distintas, nomeadamente instabilidade cromossómica, instabilidade de microssatélites e via metiladora. O nosso objetivo foi correlacionar as características clínicas e genéticas dos doentes com CCR e, deste modo, conhecer as implicações na prática clínica do genótipo tumoral. MÉTODOS: Estudo de coorte retrospectivo unicêntrico de doentes diagnosticados com CCR e submetidos a cirurgia com intuito curativo, entre 2012 e 2014. As mutações RAS e BRAF foram avaliadas pela técnica de real time PCR Idylla®. A deficiência de mismatch repair (MMR) foi avaliada pela técnica de tissue microarrays e definida pela ausência de expressão de MLH1, MSH6, MSH2 e/ou PMS2. A sobrevivência global (SG) e a sobrevivência livre de doença (SLD) foram avaliadas por análise de sobrevivência. RESULTADOS: No total, foram incluídos 242 doentes (homens 57.4%, idade 69.3 ± 12.9 anos, mediana de seguimento de 49 meses). Os tumores RAS-mutados associaram-se a menor SLD (p = 0.02) e SG (p = 0.045) em doentes com CCR estadio I­III. Os tumores BRAF-mutados foram mais frequentes em mulheres e nos tumores do cólon direito, assim como os tumores com deficiência para MMR. O status BRAF não influenciou a SG (4 anos)/SLD (3.5 anos) nos estadio I­III. Contudo, após a recidiva, o tempo de sobrevivência foi de 3.5 meses nos tumores BRAF-mutados, em comparação com 18.6 meses nos tumores sem esta mutação (p = NS). Não se identificaram mutações germinativas nos genes de mismatch repair nos doentes com tumores deficientes para estas proteinas (dMMR). O perfil molecular (RAS, BRAF e MMR) não influenciou a sobrevivência global dos doentes com metástases ao diagnóstico. O tamanho da amostra pode ser uma limitação do estudo. CONCLUSÃO: Na nossa coorte, os tumores RASmutados associaram-se a pior SLD e SG nos estádios precoces de CCR. Os restantes marcadores moleculares não influenciaram o prognóstico dos doentes.

Association of Tumor-Infiltrating Lymphocytes With Survival in Stages II and III Colorectal Cancer.

The tumor microenvironment is crucial in tumourigenesis, response to therapy, and elimination of tumor cells. Tumor-infiltrating lymphocytes (TILs) promote the host immune response and are associated with a better prognosis in colorectal cancer (CRC). This multicentric retrospective study evaluated the relationship between the presence and intensity of TILs and survival outcomes. A total of 651 patients from four Portuguese oncological centers who underwent surgical resection for stages II or III colorectal adenocarcinoma between 2016 and 2019 were included in this study. The mean age of the study population was 70 years; 58.2% were males. The median overall survival was 58.03 ± 1.29 months (95% confidence interval (CI) 55.50 - 60.56), and the median disease-free survival (DFS) was 53.02 ± 1.39 months (95% CI 50.29 - 55.74). Patients with high infiltrate (including those with moderate, abundant, or Crohn-like infiltrate) had significantly longer DFS i.e., 58.48 ± 1.84 months (95% CI 54.87 - 62.09 months) vs 49.22 ± 1.75 months (95% CI 45.79 - 52.64 months) in the group with absent or minimal infiltrate; p = 0.003. Assessing the side of the tumor, high infiltrate was associated with higher DFS (59.86 ± 2.36 months (95% CI 55.23 - 64.50 months) vs 49.60 ± 2.40 months (95% CI 44.90 - 54.29 months), p = 0.011). This work reinforces the importance of research into possible prognostic and predictive factors in patients with CRC.

Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer.

PURPOSE: Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. EXPERIMENTAL DESIGN: We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. RESULTS: In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. CONCLUSIONS: Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

Genetic and microenvironmental intra-tumor heterogeneity impacts colorectal cancer evolution and metastatic development.

Colorectal cancer (CRC) is a highly diverse disease, where different genomic instability pathways shape genetic clonal diversity and tumor microenvironment. Although intra-tumor heterogeneity has been characterized in primary tumors, its origin and consequences in CRC outcome is not fully understood. Therefore, we assessed intra- and inter-tumor heterogeneity of a prospective cohort of 136 CRC samples. We demonstrate that CRC diversity is forged by asynchronous forms of molecular alterations, where mutational and chromosomal instability collectively boost CRC genetic and microenvironment intra-tumor heterogeneity. We were able to depict predictor signatures of cancer-related genes that can foresee heterogeneity levels across the different tumor consensus molecular subtypes (CMS) and primary tumor location. Finally, we show that high genetic and microenvironment heterogeneity are associated with lower metastatic potential, whereas late-emerging copy number variations favor metastasis development and polyclonal seeding. This study provides an exhaustive portrait of the interplay between genetic and microenvironment intra-tumor heterogeneity across CMS subtypes, depicting molecular events with predictive value of CRC progression and metastasis development.

Next-Generation Sequencing in Breast Cancer Management: A Case Report of Genomic Tumour Evolution over Time.

The clinicopathological breast cancer subtypes are used in clinical practice to better anticipate biological behaviour and guide systemic treatment strategy. In the adjuvant setting, genomic assay recurrence scores became widely available for luminal-like disease. Recently, next-generation sequencing (NGS) platforms have been used, essentially, in more advanced disease setting, in situations refractory to conventional treatment, or even in rare cancers for which there are no established treatment guidelines. Moreover, subpopulations of cancer cells with unique genomes within the same patient may exist across different regions of a tumour or evolve over time, which is called intratumoural heterogeneity. We herein report a case of a 38-year-old woman with breast cancer whose primary and metastatic disease exhibited discordant expression of hormone receptors, with the former being positive and the latter negative. Furthermore, the NGS analysis revealed slight and dynamic changes of mutational profiles between different metastatic lesions, potentially impacting breast cancer management and prognosis. These alterations may reflect tissular and temporal changes in tumour subclones and may also be due to the selective pressure caused by antineoplastic treatment. The use of genomic analyses in order to improve cancer treatment has been studied prospectively with encouraging results. The widespread use of NGS tests in clinical practice also creates new challenges. The most relevant may be to know which genomic alterations detected should be valued and how they should be targeted.