Intratumor morphologic and transcriptomic heterogeneity in V600EBRAF-mutated metastatic colorectal adenocarcinomas.

BACKGROUND: Intratumor heterogeneity (ITH) is described as the presence of various clones within one tumor, each with their own unique features in terms of morphology, inflammation, genetics or transcriptomics. Heterogeneity provides the fuel for drug resistance; therefore, an accurate assessment of tumor heterogeneity is essential for the development of effective therapies. The purpose of this study was to dissect morphologic and molecular ITH in colorectal adenocarcinoma. MATERIALS AND METHODS: A series of 120 V600EBRAF-mutated (V600EBRAFmt) consecutive metastatic colorectal adenocarcinomas was assessed for morphologic heterogeneity. The two heterogeneous components of each specimen underwent a histopathological, immunohistochemical and molecular characterization to evaluate: histologic variant, grading, tumor-infiltrating lymphocytes (TILs), mismatch repair proteins' expression, KRAS/BRAF/NRAS mutations, microsatellite instability (MSI) status and consensus molecular subtype (CMS). RESULTS: Thirty-one out of 120 (25.8%) V600EBRAFmt primary colorectal adenocarcinomas presented a heterogeneous morphology. Among these, eight cases had adequate material for molecular profiling. Five out of the eight (62.5%) cases resulted instable at MSI testing. The majority (62.5%) of the samples showed a CMS4 phenotype based on gene expression profiling. Heterogeneity in CMS classification was observed in four out of eight cases. One out of eight cases presented significant heterogeneity in the number of TILs between the two components of the tumor. CONCLUSIONS: Although the distribution of the immune infiltrate appears relatively conserved among heterogeneous areas of the same tumor, changes in gene expression profile and CMS occur in 50% of V600EBRAFmt adenocarcinoma cases in our small series and might contribute to variability in response to anticancer therapy and clinical outcomes. Assessment of morphological and molecular ITH is needed to improve colorectal cancer classification and to tailor anticancer treatments and should be included in the pathology report.

Prognostic and predictive impact of consensus molecular subtypes and CRCAssigner classifications in metastatic colorectal cancer: a translational analysis of the TRIBE2 study.

INTRODUCTION: The consensus molecular subtypes (CMS) demonstrated prognostic value in metastatic colorectal cancer (mCRC). Similarly, a prognostic impact was suggested for the pre-consensus CRCAssigner (CRCA) classifier in early stages. The potential predictive role of these classifiers with regard to the choice of the first-line therapy has not been established. We investigated the prognostic and predictive impact of CMS and CRCA subtypes among mCRC patients treated in the TRIBE2 study. METHODS: Among 679 randomized patients, 426 and 428 (63%) samples were profiled according to CMS and CRCA classifications, respectively. The prognostic and predictive impact of both CMS and CRCA subtypes was investigated with univariate and multivariate analyses for progression-free survival (PFS), PFS 2 (PFS2), and overall survival (OS). RESULTS: Significant associations of CMS and CRCA subtypes with PFS, PFS2, and OS were demonstrated; the CMS classifier confirmed its independent prognostic value in the multivariable model (P value for PFS/PFS2/OS = 0.01/0.07/0.08). The effect of treatment intensification was independent of CMS subtypes (P value for interaction for PFS/PFS2/OS = 0.88/0.75/0.55). A significant interaction effect between CRCA subtypes and treatment arm was demonstrated in PFS (P = 0.02), PFS2 (P = 0.01), and OS (P = 0.008). The benefit of FOLFOXIRI seemed more relevant in the stem-like (PFS, hazard ratio = 0.60; P = 0.03) and mixed subtypes (hazard ratio = 0.44; P = 0.002). These findings were confirmed in a subgroup of patients of the previous TRIBE study. CONCLUSIONS: We confirmed the independent prognostic role of CMS classification in mCRC independently of RAS/BRAF status. CRCA classification may help identifying subgroups of patients who may derive more benefit from FOLFOXIRI/bevacizumab.

Immunological combination treatment holds the key to improving survival in pancreatic cancer.

Advances in surgery, peri-operative care and systemic chemotherapy have not significantly improved the prognosis of pancreatic cancer for several decades. Early clinical trials of immunotherapy have yielded disappointing results proposing other means by which the tumour microenvironment serves to decrease the immune response. Additionally, the emergence of various subtypes of pancreatic cancer has emerged as a factor for treatment responses with immunogenic subtypes carrying a better prognosis. Herein we discuss the reasons for the poor response to checkpoint inhibitors and outline a rationale why combination treatments are likely to be most effective. We review the therapies which could provide optimal synergistic effects to immunotherapy including chemotherapy, agents targeting the stroma, co-stimulatory molecules, vaccinations and methods of immunogenic tumour priming including radiofrequency ablation. Finally, we discuss reasons why peri-operative and in particular neoadjuvant combination treatments are likely to be most effective and should be considered for early clinical trials.

Context matters-consensus molecular subtypes of colorectal cancer as biomarkers for clinical trials.

The Colorectal Cancer Subtyping Consortium identified four gene expression consensus molecular subtypes, CMS1 (immune), CMS2 (canonical), CMS3 (metabolic), and CMS4 (mesenchymal), using multiple microarray or RNA-sequencing datasets of primary tumor samples mainly from early stage colon cancer patients. Consequently, rectal tumors and stage IV tumors (possibly reflective of more aggressive disease) were underrepresented, and no chemo- and/or radiotherapy pretreated samples or metastatic lesions were included. In view of their possible effect on gene expression and consequently subtype classification, sample source and treatments received by the patients before collection must be carefully considered when applying the classifier to new datasets. Recently, several correlative analyses of clinical trials demonstrated the applicability of this classification to the metastatic setting, confirmed the prognostic value of CMS subtypes after relapse and hinted at differential sensitivity to treatments. Here, we discuss why contexts and equivocal factors need to be taken into account when analyzing clinical trial data, including potential selection biases, type of platform, and type of algorithm used for subtype prediction. This perspective article facilitates both our clinical and research understanding of the application of this classifier to expedite subtype-based clinical trials.

A seven-Gene Signature assay improves prognostic risk stratification of perioperative chemotherapy treated gastroesophageal cancer patients from the MAGIC trial.

Background: Following neoadjuvant chemotherapy for operable gastroesophageal cancer, lymph node metastasis is the only validated prognostic variable; however, within lymph node groups there is still heterogeneity with risk of relapse. We hypothesized that gene profiles from neoadjuvant chemotherapy treated resection specimens from gastroesophageal cancer patients can be used to define prognostic risk groups to identify patients at risk for relapse. Patients and methods: The Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial (n = 202 with high quality RNA) samples treated with perioperative chemotherapy were profiled for a custom gastric cancer gene panel using the NanoString platform. Genes associated with overall survival (OS) were identified using penalized and standard Cox regression, followed by generation of risk scores and development of a NanoString biomarker assay to stratify patients into risk groups associated with OS. An independent dataset served as a validation cohort. Results: Regression and clustering analysis of MAGIC patients defined a seven-Gene Signature and two risk groups with different OS [hazard ratio (HR) 5.1; P < 0.0001]. The median OS of high- and low-risk groups were 10.2 [95% confidence interval (CI) of 6.5 and 13.2 months] and 80.9 months (CI: 43.0 months and not assessable), respectively. Risk groups were independently prognostic of lymph node metastasis by multivariate analysis (HR 3.6 in node positive group, P = 0.02; HR 3.6 in high-risk group, P = 0.0002), and not prognostic in surgery only patients (n = 118; log rank P = 0.2). A validation cohort independently confirmed these findings. Conclusions: These results suggest that gene-based risk groups can independently predict prognosis in gastroesophageal cancer patients treated with neoadjuvant chemotherapy. This signature and associated assay may help risk stratify these patients for post-surgery chemotherapy in future perioperative chemotherapy-based clinical trials.

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation.

BACKGROUND: Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins. METHODS AND RESULTS: In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5A-ECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules - interleukin-8 and vascular endothelial growth factor. CONCLUSION: Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases.

CXCR1 and CXCR2 enhances human melanoma tumourigenesis, growth and invasion.

The aggressiveness of malignant melanoma is associated with differential expression of CXCL-8 and its receptors, CXCR1 and CXCR2. However, the precise functional role of these receptors in melanoma progression remains unclear. In this study, we investigate the precise functional role of CXCR1 and CXCR2 in melanoma progression. CXCR1 or CXCR2 were stably overexpressed in human melanoma cell lines, SBC-2 (non-tumourigenic) and A375P (low-tumourigenic) exhibiting low endogenous expression of receptors. Functional assays were performed to study the resulting changes in cell proliferation, motility and invasion, and in vivo tumour growth using a mouse xenograft model. Our data demonstrated that CXCR1- or CXCR2-overexpressing SBC-2 and A375P melanoma cells had enhanced proliferation, chemotaxis and invasiveness in vitro. Interestingly, CXCR1 or CXCR2 overexpression in SBC-2 cells induced tumourigenicity, and A375P cells significantly enhanced tumour growth as examined in vivo. Immunohistochemical analyses showed significantly increased tumour cell proliferation and microvessel density and reduced apoptosis in tumours generated from CXCR1- or CXCR2-overexpressing melanoma cells. CXCR1- or CXCR2-induced modulation of melanoma cell proliferation and migration was observed to be mediated through the activation of ERK1/2 phosphorylation. Together, these studies demonstrate that CXCR1 and CXCR2 play essential role in growth, survival, motility and invasion of human melanoma.

Nitrosomethylurea induced streptomycin resistance in Lycopersicon esculentum Mill.

High frequency of streptomycin resistant variants of Lycopersicon esculentum were isolated on selective shoot regeneration medium supplemented with IAA (0.5 mg/L), zeatin (1.5 mg/L) and streptomycin sulphate (500 mg/L). Nonmutagenized (controls) and NMU treated cotyledons were placed on shoot regeneration medium supplemented with antibiotic streptomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. Shoot regeneration occurred from the nodular tissues developed at the cut ends of cotyledons. Resistant shoots developed into complete plantlets on rooting medium containing selective concentration of antibiotic. Stability of streptomycin resistance was confirmed by leaf assay and reciprocal crosses between streptomycin-resistant and sensitive plants.

Induction of lincomycin and streptomycin resistance by nitrosomethylurea and ethyl methanesulphonate in Capsicum annuum L.

A high frequency of plastid-encoded antibiotic-resistant variants of Capsicum annuum were isolated on selective media following treatment by ethyl methanesulphonate (EMS) and nitrosomethylurea (NMU). Seeds and explants were mutagenized with 0.1% EMS and 5 mM NMU separately. Non-mutagenized cotyledons (controls), mutagenized cotyledons from EMS-treated seedlings and NMU-treated cotyledons were placed on regeneration medium supplemented with the antibiotics streptomycin or lincomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. The stability of streptomycin and lincomycin resistance was confirmed by leaf assay. NMU-treated cotyledons gave a higher frequency of variants than cotyledons from EMS-treated seedlings. The mutagenic effect of EMS was more pronounced using whole seeds rather than cotyledons; in contrast, NMU was more effective in inducing variations in cotyledons than in seeds.

EMS-induced streptomycin resistance in Solanum melongena.

Streptomycin-resistant mutations were induced in Solanum melongena by exposing seeds to ethyl methane sulphonate (EMS). Seed mutagenesis resulted in a high frequency of chlorophyll-deficient mutations and a low frequency of resistant shoots, both of which retained their resistance on subsequent testing. Reciprocal crosses between streptomycin-resistant and -sensitive plants showed a non-Mendelian transmission of the resistance trait. Streptomycin resistance is the first selectable and maternally inherited organelle marker described in brinjal.

A controlled trial of ivermectin and diethylcarbamazine in lymphatic filariasis.

Ivermectin is a new antifilarial drug that can be given in a single oral dose. To compare the efficacy and side effects of ivermectin with those of diethylcarbamazine, the standard antifilarial treatment, we conducted a randomized, double-blind trial in 40 South Indian men with lymphatic filariasis caused by Wuchereria bancrofti. Patients were randomly assigned to one of three treatments: a single low dose of ivermectin (mean [+/- SE], 21.3 +/- 0.7 micrograms per kilogram of body weight; n = 13) followed by placebo for 12 days; a single high dose of ivermectin (mean, 126.2 +/- 3.7 micrograms per kilogram; n = 13) followed by placebo for 12 days; or diethylcarbamazine for 13 days (6 mg per kilogram per day for 12 days preceded by 3 mg per kilogram for 1 day; n = 14). Eleven patients were initially assigned to receive placebo and after five days were reassigned to one of the three treatment groups. At day 12 there was complete clearance of microfilariae from the blood in all 26 men who took ivermectin and in 11 of the 14 men who took diethylcarbamazine. At six months the numbers of detectable microfilariae (as a percentage of the pretreatment values) were 18.3 percent after low-dose ivermectin and 19.5 percent after high-dose ivermectin, as compared with 6.0 percent after diethylcarbamazine (P less than 0.05). The side effects were confined to the first five days and were similar in the three treatment groups. We conclude that in lymphatic filariasis, the clinical response to a single dose of ivermectin compares favorably with that after the standard 12-day course of diethylcarbamazine. Given the practical advantages of single-dose administration, ivermectin should become a useful medication for the control of bancroftian filariasis.