Transcriptional subtyping and CD8 immunohistochemistry identifies poor prognosis stage II/III colorectal cancer patients who benefit from adjuvant chemotherapy.

PURPOSE: Transcriptomic profiling of colorectal cancer (CRC) has led to identification of four consensus molecular subtypes (CMS1-4), which have prognostic value in stage II/III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors. However, the clinical value of these classifications in predicting response to standard-of-care adjuvant chemotherapy remains unknown. PATIENTS AND METHODS: Using samples from 4 European sites, we assembled a novel stage II/III CRC patient cohort and performed transcriptomic profiling on 156 samples, targeted sequencing and generated a tissue microarray to enable integrated "multi-omics" analyses. We also accessed data from 2 published stage II/III CRC patient cohorts: GSE39582 and GSE14333 (479 and 185 samples respectively). RESULTS: The epithelial-rich CMS2 subtype of CRC benefitted significantly from adjuvant chemotherapy treatment in both stage II and III disease (p=0.02 and p<0.0001 respectively), while the CMS3 subtype significantly benefitted in stage III only (p=0.00073). Following CRIS sub-stratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from adjuvant chemotherapy in stage II and III disease (p=0.0081 and p<0.0001 respectively), while CRIS-D significantly benefitted in stage III only (p=0.0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk of relapse in both stage II and III disease (p=0.0031). CONCLUSION: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying poor prognostic stage II/III patients who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer.

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer.

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.

Ileal perforation caused by cytomegalovirus infection in an immunocompetent adult.

A 71-year-old woman developed a small bowel perforation due to cytomegalovirus infection. She did not taken any immunosuppressive medication and her cellular immunity was normal. Surgical resection and antiviral therapy with ganciclovir led to complete recovery. As far as we know, this paper reports the first case of small bowel perforation due to cytomegalovirus infection in a non-immunocompromised patient. Nevertheless the patient was known with diabetes mellitus. It should be emphasised that elderly patients have impaired immune defences and may be unsuspected hosts of opportunistic infections.

Ectopic ACTH-syndrome due to a thymic carcinoid tumor.

Investigating a recently developed Cushing Syndrome, we diagnosed in a 47-year-old woman an ectopic ACTH syndrome due to a metastatic carcinoid tumor, most likely a thymic carcinoid tumor. Combined therapy with sandostatin and nizoral and later on with sandostatin, metopirone and orimeten, was not able to suppress the hypercortisolism. A few weeks after surgical adrenalectomy, clinical deterioration ensued, culminating in the patient's death 7 months after diagnosis.