Best-Practice Biomarker Testing of Oesophago-Gastric Cancer in the UK: Expert Consensus Recommendations Developed Using a Modified Delphi.

AIMS: Oesophago-gastric cancers (OGCs) are amongst the most commonly diagnosed malignancies worldwide and are associated with high disease-related mortality. Predictive biomarkers are molecules that can be objectively measured and used to indicate a likely response to therapeutic intervention, thus facilitating individualised cancer therapy. However, there remains variation in uptake and implementation of biomarker testing across the UK. MATERIALS AND METHODS: We conducted a modified Delphi study to formulate consensus recommendations for best-practice biomarker testing of OGC in the UK. We employed two rounds of online questionnaires followed by a virtual consensus meeting. Biomarkers for discussion included HER2, MSI/MMR, and PD-L1. Topics comprised the overall biomarker pathway, pre-analytical, analytical, and post-analytical considerations, including challenges in current practice. RESULTS: Twenty-six and eighteen participants completed the first and second round Delphi questionnaire, respectively, with an even split of pathologists and oncologists from across the UK. There was consensus (>80% agreement) across several topics, including the requirements for standardisation of the pathway, which must include coordination throughout the tissue journey, requirements for a quality-assured process to ensure accuracy and validity of testing, plus the need for clear, detailed information on the pathology report to support treatment decisions. There was consensus amongst oncologists regarding reflex testing of all biomarkers depending on histology; however, concerns over capacity in relation to workload and availability of pathologists were evident among the pathologists. Overall, participants were in the opinion that reflex testing improves the speed of treatment decisions and improves patient care. CONCLUSION: The recommendations reflect best-practices and should be implemented to support rapid multidisciplinary team decision-making within oesophago-gastric cancer. Results reflect the need for standardisation and demonstrate the challenges faced in clinical practice by those requesting and testing biomarkers for oesophago-gastric cancer, suggesting significant concerns relating to pathologist capacity.

Benefit of chemotherapy as part of treatment for HPV DNA-positive but p16-negative squamous cell carcinoma of the oropharynx.

BACKGROUND: To determine (a) the cause of an improvement in survival from oropharyngeal squamous cell carcinoma (OSCC) in South East Scotland and (b) whether this improvement was human papillomavirus (HPV) and p16 subtype-dependent. METHODS: Clinicopathological characteristics and outcome data for patients referred with OSCC from 1999 to 2001 (Cohort-1) and 2003 to 2005 (Cohort-2) were obtained. Molecular HPV detection and immunohistochemistry for p16 were performed from paraffin blocks. RESULTS: Cohort-1 and Cohort-2 contained 118 and 136 patients, respectively. Kaplan-Meier analysis revealed significantly improved survival in Cohort-2 (P<0.0001). Sub-classification according to HPV and p16 status revealed no improvement in survival in Class-I (HPV-ve/p16-ve; 47 patients) or Class-III (HPV+ve/p16+ve; 77 patients). However in Class-II (HPV+ve/p16-ve; 56 patients) an increase in 5-year cause-specific survival from 36% in Cohort-1 to 73% in Cohort-2 was detected (P=0.0001).Proportional hazards analysis of 217 patients treated radically demonstrated that significant variables were p16 (P<0.0001), N stage (P=0.0006) and cohort (P=0.0024). Removing cohort from the variables offered to the model showed that, whereas p16 (P<0.0001) and N stage (P=0.0016) remain significant, chemotherapy (P=0.0163) and T stage (P=0.0139) are now significant. This suggests that much of the cohort effect is due to the higher use of chemotherapy in the second cohort. CONCLUSION: These data suggest that HPV+ve/p16-ve patients constitute a separate subclass of OSCC who may particularly benefit from chemotherapy. They imply that p16 status cannot be considered a surrogate for HPV status, and those trials to de-escalate treatment in HPV+ve OSCC should take p16 status into account.

ALK immunohistochemistry is highly sensitive and specific for the detection of ALK translocated lung adenocarcinomas: lessons from an audit of lung cancer molecular testing.

Background The approval of novel targeted treatments for epidermal growth factor receptor (EGFR)-positive and anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer has led to the increased requirement for mutation testing. Results We report our experience of ALK testing with immunohistochemistry (IHC) and fluorescence in-situ hybridisation (FISH) and present the prevalence of EGFR, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) and ALK mutations. From January 2011 to May 2014, we found mutation rates of EGFR, KRAS and ALK to be 10.4% (67/643), 35.8% (86/240) and 2.3% (7/304), respectively. ALK-rearrangements were found to be associated with never smokers (p < 0.001) and younger patients (≤ 50 years old) (p < 0.001). ALK IHC protein expression in tumour cells is 100% sensitive (7 IHC+/7 FISH+) and 96.6% specific (113 IHC-/117 FISH-) for ALK-rearrangements by FISH. ALK-rearranged tumours were wildtype for EGFR and KRAS. Conclusion Our findings support the use of ALK protein expression and KRAS mutation testing as part of the molecular diagnostic algorithm for lung adenocarcinomas.

p73alpha is a candidate effector in the p53 independent apoptosis pathway of cisplatin damaged primary murine colonocytes.

AIMS: Colonocytes were derived from wild-type (wt) and p53 deficient mice to investigate p53 dependent and independent death pathways after cisplatin treatment, and the role of p53 in growth regulation of primary, untransformed epithelial cells. METHODS: Wt and p53 null colonocytes were exposed to cisplatin and DNA synthesis, apoptosis, and p53, p21, and p73 expression were investigated after six, 12, and 24 hours. Major p73 isoforms were identified by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Cisplatin treated wt cells exhibited cell cycle arrest, whereas p53 null cells continued to synthesise DNA, although both cell types died. Apoptosis was significantly higher in cisplatin treated wt and p53 null colonocytes than in controls at all timepoints, although apoptosis was lower in cisplatin treated p53 null colonocytes than in wt cells. p53 expression was upregulated in cisplatin treated wt colonocytes. p21 expression was high and remained unchanged in cisplatin treated wt cells, although it was reduced in the absence of p53. p73 was investigated because it could account for p53 independent p21 expression and p53 independent death. RT-PCR detected full length p73alpha. p73 transcript levels remained unchanged, whereas p73 protein accumulated in the nucleus of cisplatin treated cells, irrespective of genotype. CONCLUSIONS: p53 is essential for cell cycle arrest, but not apoptosis in primary murine colonocytes. Apoptosis is reduced in cisplatin treated p53 null cells. Nuclear accumulation of endogenous p73 after cisplatin treatment suggests a proapoptotic role for p73alpha in the absence of p53 and collaboration with p53 in wt colonocytes.

A cautionary lesson on the use of targeted methods for EGFR mutation analysis: a case report.

Epidermal growth factor receptor (EGFR) mutation analysis is recommended for lung cancer patients prior to the prescription of first-line EGFR tyrosine kinase inhibitors in order to predict response to treatment. There are many methods available to identify mutations in the EGFR gene; a large number of clinical laboratories use the therascreen EGFR RGQ PCR kit (Qiagen). We report a case where this kit detected an exon 19 deletion, predicting sensitivity to tyrosine kinase inhibitors (TKIs), which on further analysis was found to be a 2 bp indel (c.2239_2240delinsCC, p.(Leu747Pro)). Two of four published cases with this mutation were found to be associated with resistance to EGFR TKI. The sample was also tested using two other commercial kits, one of which indicated a deletion. This is a rare mutation making the erroneous detection of a deletion unlikely; however, it is important that clinical laboratories are aware of the potential failings of two commercial kits for EGFR mutation analysis.