The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF-mutant colorectal cancer cell lines.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor Vemurafenib (PLX4032) has shown significant increases in response rates and overall survival, only minor responses to Vemurafenib treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MEK inhibition in BRAFMT CRC. Methods: Paired BRAFMT/WT RKO and VACO432 CRC cells and non-isogenic BRAFMT LIM2405, WiDR, HT-29 and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interactions between MEK1/2 and JAK1/2 or c-MET inhibition were assessed using the MTT cell viability assays and Flow Cytometry. Apoptosis was measured using Western Blotting for PARP, cleaved caspase 3, 8 and 9, and caspase 3/7 and 8 activity assays. Results: Treatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in synergy and significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or trametinib in BRAFMT CRC cells. The RTK c-MET is activated upstream of STAT3 following MEK1/2 inhibition. Inhibition of c-MET and MEK1/2, using pharmacological inhibitors (crizotinib and AZD6244), results in synergy and increased cell death in BRAFMT CRC cells. Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition in vitro. Combinations of JAK/MEKi or MET/MEKi can be a potential novel treatment strategy for poor prognostic BRAFMT advanced CRC patients.

Abstract B64: The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF mutant colorectal cancer.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor PLX4032 has shown significant increases in response rates and overall survival compared to standard Dacarbazine treatment, only minor responses to PLX4032 treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumors has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MAPK inhibition in BRAFMT CRC.

Methods: Paired BRAFMT/WT RKO and VACO432 CRC cell line models and non-isogenic BRAFMT LIM2405, WiDR and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interaction between MEK1/2 and JAK1/2 inhibition was assessed using the MTT cell viability assays and flow cytometry. Apoptosis was measured using Western blotting for PARP, cleaved caspase 3/8 and caspase 8, 3/7 activity assays.

Results: Treatment with MEK1/2 inhibitors AZD6244, GSK1120212, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or GSK1120212 in BRAFMT CRC cells. In addition, combination of MEK1/2 and JAK/STAT3 inhibition resulted in strong synergy with CI values between 0.3 and 0.7 in BRAFMT CRC cells.

Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition. These data provide a strong rationale for further investigation of combination of MEK1/2 and JAK/STAT3 inhibition in BRAFMT in vivo models.

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p

A new generation of companion diagnostics: cobas BRAF, KRAS and EGFR mutation detection tests

The cobas® (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an ‘end-to-end’ solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use.

Non-pharmacological interventions for cognitive impairment due to systemic cancer treatment (Review)

Background

It is estimated that up to 75% of cancer survivors may experience cognitive impairment as a result of cancer treatment and given the increasing size of the cancer survivor population, the number of affected people is set to rise considerably in coming years. There is a need, therefore, to identify effective, non-pharmacological interventions for maintaining cognitive function or ameliorating cognitive impairment among people with a previous cancer diagnosis.
Objectives

To evaluate the cognitive effects, non-cognitive effects, duration and safety of non-pharmacological interventions among cancer patients targeted at maintaining cognitive function or ameliorating cognitive impairment as a result of cancer or receipt of systemic cancer treatment (i.e. chemotherapy or hormonal therapies in isolation or combination with other treatments).
Search methods

We searched the Cochrane Centre Register of Controlled Trials (CENTRAL), MEDLINE, Embase, PUBMED, Cumulative Index of Nursing and Allied Health Literature (CINAHL) and PsycINFO databases. We also searched registries of ongoing trials and grey literature including theses, dissertations and conference proceedings. Searches were conducted for articles published from 1980 to 29 September 2015.
Selection criteria

Randomised controlled trials (RCTs) of non-pharmacological interventions to improve cognitive impairment or to maintain cognitive functioning among survivors of adult-onset cancers who have completed systemic cancer therapy (in isolation or combination with other treatments) were eligible. Studies among individuals continuing to receive hormonal therapy were included. We excluded interventions targeted at cancer survivors with central nervous system (CNS) tumours or metastases, non-melanoma skin cancer or those who had received cranial radiation or, were from nursing or care home settings. Language restrictions were not applied.
Data collection and analysis

Author pairs independently screened, selected, extracted data and rated the risk of bias of studies. We were unable to conduct planned meta-analyses due to heterogeneity in the type of interventions and outcomes, with the exception of compensatory strategy training interventions for which we pooled data for mental and physical well-being outcomes. We report a narrative synthesis of intervention effectiveness for other outcomes.
Main results

Five RCTs describing six interventions (comprising a total of 235 participants) met the eligibility criteria for the review. Two trials of computer-assisted cognitive training interventions (n = 100), two of compensatory strategy training interventions (n = 95), one of meditation (n = 47) and one of physical activity intervention (n = 19) were identified. Each study focused on breast cancer survivors. All five studies were rated as having a high risk of bias. Data for our primary outcome of interest, cognitive function were not amenable to being pooled statistically. Cognitive training demonstrated beneficial effects on objectively assessed cognitive function (including processing speed, executive functions, cognitive flexibility, language, delayed- and immediate- memory), subjectively reported cognitive function and mental well-being. Compensatory strategy training demonstrated improvements on objectively assessed delayed-, immediate- and verbal-memory, self-reported cognitive function and spiritual quality of life (QoL). The meta-analyses of two RCTs (95 participants) did not show a beneficial effect from compensatory strategy training on physical well-being immediately (standardised mean difference (SMD) 0.12, 95% confidence interval (CI) -0.59 to 0.83; I2= 67%) or two months post-intervention (SMD - 0.21, 95% CI -0.89 to 0.47; I2 = 63%) or on mental well-being two months post-intervention (SMD -0.38, 95% CI -1.10 to 0.34; I2 = 67%). Lower mental well-being immediately post-intervention appeared to be observed in patients who received compensatory strategy training compared to wait-list controls (SMD -0.57, 95% CI -0.98 to -0.16; I2 = 0%). We assessed the assembled studies using GRADE for physical and mental health outcomes and this evidence was rated to be low quality and, therefore findings should be interpreted with caution. Evidence for physical activity and meditation interventions on cognitive outcomes is unclear.
Authors' conclusions

Overall, the, albeit low-quality evidence may be interpreted to suggest that non-pharmacological interventions may have the potential to reduce the risk of, or ameliorate, cognitive impairment following systemic cancer treatment. Larger, multi-site studies including an appropriate, active attentional control group, as well as consideration of functional outcomes (e.g. activities of daily living) are required in order to come to firmer conclusions about the benefits or otherwise of this intervention approach. There is also a need to conduct research into cognitive impairment among cancer patient groups other than women with breast cancer.