Rare <Emphasis Type="Italic">RAS</Emphasis> Mutations in Metastatic Colorectal Cancer Detected During Routine <Emphasis Type="Italic">RAS</Emphasis> Genotyping Using Next Generation Sequencing

Background

Overall survival of metastatic colorectal cancer (mCRC) patients has been improved with the addition of targeted therapy such as anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs) to standard chemotherapy. Retrospective studies and randomized trials showed that the presence of RAS mutations was linked to the absence of clinical response to anti-EGFR mAbs. Patients harboring KRAS and NRAS mutations on exons 2, 3 or 4 have little or no benefit from anti-EGFR therapies. Polymerase chain reaction (PCR)-based assays are routinely used to assess KRAS and NRAS status, whereas deep sequencing with next generation sequencing (NGS) currently represents an alternative method.

Objective

The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples.

Method

DNA was extracted from 188 consecutive formalin-fixed paraffin embedded samples of histologically proven colorectal cancer tumor tissue from patients with mCRC. Following amplification, DNA was sequenced by ultra-deep pyrosequencing. Non-hotspot mutations identified by NGS (frequency of mutated allele range [1.8–70.6 %]) were confirmed by Sanger direct-sequencing when possible.

Results

NGS procedure was applicable in 94 % of the cases and detected mutations in 62 % of the samples. Nine uncommon mutational profiles were found with a frequency of mutated allele ?>?1 %. Silent mutations were found in 3.6 % of the samples. Mutations at or near functional domains of RAS proteins, other than defined hotspots, were found in 3.6 %. NGS proved to be accurate, sensitive and suitable for routine RAS genotyping.

Conclusion

Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations.

     

The combined roles of ADAMTS13 and VWF in murine models of TTP, endotoxemia, and thrombosis

Ultralarge von Willebrand factor (UL-VWF) multimers are thought to play a central role in pathogenesis of the disease thrombotic thrombocytopenic purpura (TTP); however, experimental evidence in support of this hypothesis has been difficult to establish. Therefore, to examine directly the requirement for VWF in TTP pathogenesis, we generated ADAMTS13-deficient mice on a TTP-susceptible genetic background that were also either haploinsufficient (Vwf+/-) or completely deficient (Vwf-/-) in VWF. Absence of VWF resulted in complete protection from shigatoxin (Stx)-induced thrombocytopenia, demonstrating an absolute requirement for VWF in this model (Stx has been shown previously to trigger TTP in ADAMTS13-deficient mice). We next investigated the requirements for ADAMTS13 and VWF in a murine model of endotoxemia. Unlike Stx-induced TTP findings, LPS-induced thrombocytopenia and mortality were not affected by either VWF or ADAMTS13 deficiency, suggesting divergent mechanisms of thrombocytopenia between these 2 disorders. Finally, we show that VWF deficiency abrogates the ADAMTS13-deficient prothrombotic state, suggesting VWF as the only relevant ADAMTS13 substrate under these conditions. Together, these findings shed new light on the potential roles played by ADAMTS13 and VWF in TTP, endotoxemia, and normal hemostasis.     

Occupational Snake Bites: a Prospective Case Series of Patients Reported to the ToxIC North American Snakebite Registry

Introduction

In the developing world, occupation has been identified as a risk factor for snake bite. Such an association has not been described in the USA. The objective of this study was to describe the epidemiology and clinical manifestations of occupational snake bite in patients reported to the ToxIC North American Snakebite Registry (NASBR).

Methods

This was a prospective case series of patients reported to the ToxIC NASBR between January 1, 2014 and November 5, 2015. Variables collected included snake species, patient demographics, date and location of exposure, occupation, bite location, clinical manifestations, and management.

Results

Of 180 adult snake bites reported, 25 (13.9 %; 95 % CI 9.2–19.8 %) were occupational in nature. Rattlesnake envenomations were common (80 %). Most snake bites (96 %) occurred in men. Occupations most associated with snake bite were landscaping (28 %) and working directly with snakes (24 %). Fifty-six percent of bites occurred in an outdoor work environment. Seventy-six percent of envenomations were to the upper extremities. Intentional interaction occurred in 40 % of cases, all of which sustained finger envenomations. No cases presented with apparent acute ethanol intoxication.

Conclusions

The majority of occupational snake bites occurred in men working outdoors and were unintentional injuries. Bites involving the upper extremity tended to result from intentional interactions. Acute ethanol intoxication did not appear to be involved with occupational envenomations.

     

Utility of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) in patients with end-stage liver disease awaiting liver transplant.

Cognitive impairment is common among patients with end-stage liver disease (ESLD). This study examined cognitive dysfunction in patients with ESLD using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). METHOD: 66 patients with ESLD awaiting liver transplant were recruited. Patients were evaluated with the RBANS, Peabody Picture Vocabulary Test-Revised, and Beck Depression Inventory-II. RESULTS: Patients with ESLD uniformly performed below expectations on all RBANS index scores compared to the healthy normative sample (all p's<.0001) and they also displayed a "subcortical" pattern of cognitive performance (p<.0001). Performances on RBANS attention, language, immediate memory, and total index scores were correlated with education and ethnicity (r's range=|.32-.57|; p's<.01). There was no association between performance on any of the RBANS index scores or subtests and ESLD patient characteristics. In summary, the RBANS appears to adequately characterize known patterns of cognitive dysfunction in ESLD patients.     

VEGF-related protein isolated from Vipera palestinae venom, promotes angiogenesis

Therapeutic angiogenesis is one of the major approaches in designing new therapies for cardiovascular diseases. vpVEGF was purified from Vipera palestinae venom using two steps of reverse-phase HPLC. Structurally, vpVEGF belongs to the VEGF-F1 family of snake venom proteins, and potently stimulated dHMVEC proliferation in a VEGFR-2 dependent manner. This growth factor appeared to be a chemoattractant for migration of these cells and stimulated their radial migration in a collagen gel. The stimulatory effect on dHMVEC was correlated with activation of the MAPK Erk1/2 signaling pathway. In vivo vpVEGF induced angiogenesis in a Japanese quail assay and in a Matrigel plug assay in mice. Although in the quail assay vpVEGF showed lower activity than hrVEGF-A165 in mammalian-related systems there were no significant differences. The experiments with dHMVEC, as well as angiogenesis in vivo suggest that the pro-angiogenic effect of vpVEGF is related to its interaction with VEGFR-2 (flk-1).     

A unique window of opportunity for practical reform of cancer clinical trials

With rapid modifications in cancer clinical trial operations necessitated by the global pandemic over the last year, there is now an unprecedented opportunity to reform clinical research permanently and solidify innovative practices that have clearly been effective. On the basis of the authors' experience and recommendations from other institutions, a set of specific proposals for clinical trial reform are identified that can be implemented immediately by sponsors, regulators, and study sites. Improvements in clinical trial processes should include increased leverage of technology to facilitate remote trial activity and electronic documents, more efficient and effective communication of adverse event information, and better study design to optimize inclusion criteria, required research procedures, and data collection. The authors suggest that such reform will preserve patient safety and study integrity, address unnecessary and inefficient pre-pandemic constraints, improve access to clinical trials for patients, and speed improvements in cancer care.     

B7-H3 and PD-L1 Expression Are Prognostic Biomarkers in a Multi-racial Cohort of Patients with Colorectal Cancer

BackgroundImmunotherapy has emerged as an effective and durable treatment modality for solid cancers. However, its use in colorectal cancer (CRC) is limited to deficient mismatch repair (dMMR) tumors. As such, assessing immune regulatory proteins from the B7-CD28 family, other than PD-1, PD-L1, and CTLA-4, is critical. This study aimed to evaluate the expression of novel protein regulators in a racially diverse population of patients with CRC.MethodsA tumor microarray was created for 214 samples from a multiracial patient population with metastatic CRC, and expression of HHLA2, B7-H3, PD-L1, CK7, CK20, and CDX2 was determined. The expression pattern was scored as 0 to 12, based on tumor tissue prevalence and the intensity. Clinical information was obtained by chart review and vital statistics from the National Death Index. Associations between low and high expression groups for each protein by race/ethnic groups were assessed, and Kaplan–Meier curves were plotted to evaluate association with survival.ResultsThe median age at diagnosis was 61 years, with a female predominance. The majority of the patients were diagnosed with de novo metastatic disease with left-sided, moderately differentiated tumors. There were no racial disparities in the expression of any protein. Overall, a high frequency of tumors had no expression of B7-H3 (62.5%) or PD-L1 (43.5%). Low expression of both PD-L1 and B7-H3 was a significant prognostic biomarker associated with better survival (median overall survival, 43.3 months vs. 24.6 months; P < .01).ConclusionIn this multiracial tumor microarray of CRC samples, low PD-L1 and B7-H3 expression was associated with an improved prognosis. There was no significant variation among races with respect to the relevant CRC protein markers.