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We investigated correlations between diagnosis according to the 2015 World Health Organization (WHO) classification of unresected lung tumours, molecular analysis and TTF1 expression in small biopsy and cytology specimens from 344 non-small cell lung carcinoma (NSCLC) patients. One case failed testing for EGFR, KRAS and ALK abnormalities and six had insufficient tumour for ALK testing. Overall mutation rate in 343 cases was 48% for the genes tested, with 19% EGFR, 33% KRAS and 4% BRAF mutations, and 5% ALK rearrangements detected. More EGFR-mutant (78%) and ALK-rearranged (75%) tumours had morphologic adenocarcinoma than KRAS-mutant (56%) tumours. Despite no significant difference in the overall rate of any molecular abnormality between morphologic adenocarcinoma (52%) and NSCLC, favour adenocarcinoma (47%) (p = 0.18), KRAS mutations were detected more frequently in the latter group. No significant difference in the overall rate of any molecular abnormality between TTF1 positive (49%) and TTF1 negative tumours (44%) (p = 0.92) was detected, but more EGFR-mutant (97%) and ALK-rearranged tumours (92%) were TTF1 positive than KRAS-mutant tumours (68%). Rates of EGFR, KRAS and BRAF mutations and ALK rearrangements in this Australian NSCLC patient population are consistent with the published international literature. Our findings suggest that 2015 WHO classification of unresected tumours may assist in identifying molecular subsets of advanced NSCLC.

The advent of massively parallel sequencing has caused a paradigm shift in the ways cancer is treated, as personalised therapy becomes a reality. More and more laboratories are looking to introduce next generation sequencing (NGS) as a tool for mutational analysis, as this technology has many advantages compared to conventional platforms like Sanger sequencing. In Australia all massively parallel sequencing platforms are still considered in-house in vitro diagnostic tools by the National Association of Testing Authorities (NATA) and a comprehensive analytical validation of all assays, and not just mere verification, is a strict requirement before accreditation can be granted for clinical testing on these platforms. Analytical validation of assays on NGS platforms can prove to be extremely challenging for pathology laboratories. Although there are many affordable and easily accessible NGS instruments available, there are no standardised guidelines as yet for clinical validation of NGS assays. We present an accreditation development procedure that was both comprehensive and applicable in a setting of hospital laboratory for NGS services. This approach may also be applied to other NGS applications in service laboratories.

We sought to investigate the frequency of mutations in epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) by each pathological subtype for patients with resected pulmonary adenocarcinoma as defined by the IASLC/ATS/ERS classification. Histological examination determined the predominant subtype according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were determined by high-resolution melting and Sanger sequencing. Clinical data were collected from medical records and clinicians. The 178 consecutive patients consisted of 48% males, median age 68 years (range 20-87) and smoking history 78%. The tumour stage was I in 62%, II in 18% and III in 20%. The mutation rates were: EGFR 30%; KRAS 28%. The rate of EGFR mutations in the acinar predominant reference group (n=76), was 37%. The solid predominant subtype showed significantly fewer EGFR mutations [3/33 (9%), odds ratio 0.17 (0.05-0.61), p=0.007]. No differences in mutation rate were observed in other subtypes. No association was found between KRAS mutations and predominant histological subtype. Advanced stage and solid predominant subtype were negative prognostic factors. EGFR mutations can be present in adenocarcinoma of any predominant subtype, however rarely in solid predominant tumours. No association was found between KRAS mutation and the predominant histological subtype.

We investigated if gene copy number (GCN) alterations of the epidermal growth factor receptor (EGFR), as detected by silver enhanced in situ hybridisation (SISH), could be used to select patients for EGFR mutation testing. Resected lung adenocarcinoma specimens with adequate tumour were identified. EGFR SISH was performed using the Ventana Benchmark Ultra platform. EGFR GCN was classified according to the Colorado Classification System. EGFR mutations were scanned by high resolution melting and confirmed by Sanger sequencing. Thirty-four of 96 tumours were EGFR SISH positive (35%), and 31 of 96 tumours harboured one or more EGFR mutations (32%). Of 31 EGFR-mutant tumours, 18 were EGFR SISH positive (58%). There was a statistically significant relationship between the presence of an EGFR mutation and EGFR GCN (p = 0.003). Thirteen of 31 EGFR-mutant tumours were EGFR SISH negative (42%), and 16 of 65 EGFR-wild type tumours were EGFR SISH positive (24%). The sensitivity, specificity, positive predictive value and negative predictive value were 58%, 75%, 52.9% and 79%, respectively. Despite a significant relationship between EGFR GCN alterations and EGFR mutations, our results indicate that EGFR GCN as detected by SISH is not a suitable way to select patients for EGFR mutation testing.

Sir,Zygomycosis is a rare fungal infection occurring in immunosuppressed patients. The infections are acute, rapidly developing and often fulminant with a high mortality. Diagnosis is often difficu...