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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.

Background and Aim Ductal carcinoma in situ (DCIS) accounts for ~25% of breast cancer. Clinical and histopathological parameters cannot identify cases which will recur or progress. DNA methylation was assessed as a potential biomarker in DCIS. Methods DNA meťhylation was assessed by methylation sensitive high resolution melting on DNAs isolated from formalin fixed, paraffin embedded (FFPE) samples from 82 DCIS and 18 normal samples. Nuclear grade, architectural pattern, and comedo necrosis were evaluated and phenotype determined by immunohistochemical staining for ER, PgR, CK5, Ki-67, and HER2 SISH. Results The median age was 54 years (29-82 years); 47.6% of samples were high, 45.1%, intermediate and 7.3% low grade. There were significant associations between APC, CDH13 , and FOXC1 methylation and grade, and between RASSF1A methylation and comedo necrosis. There was a significant negative relationship between ER and APC , and RARβ methylation, and between PgR status and CDH1, CDH13 , and RARβ methylation. HER2 amplification and CDH13 methylation were significantly associated. There was no significant relationship between methylation and architectural pattern or presence of invasion. Conclusions DNA methylation status is associated with DCIS phenotypic features of known prognostic significance. This, combined with the ability to assess DNA methylation in FFPE material, indicates potential diagnostic utility in clinical risk stratification.

Mast cells from over 80% of adult systemic mastocytosis (SM) patients carry an activating mutation at codon 816 of the KIT gene. In fact, a KIT mutation constitutes one of the minor WHO 2008 classification criteria for SM. The most common is the c.2447A>T: p.Arg816Val (D816V) mutation. The KIT gene encodes a type III transmembrane receptor tyrosine kinase whose ligand is stem cell factor. Activation of KIT mediates proliferation and maturation signalling. D816 V causes constitutive activation of the KIT tyrosine kinase activity, which also results in resistance to the tyrosine kinase inhibitor imatinib mesylate. Therefore knowing the mutational status of KIT codon 816 is important for diagnosis and treatment strategy. The population of mutant mast cells is often low in relation to normal cells and so a sensitive molecular detection technique is required. Several have been reported in the literature, the most sensitive include RT-PCR + RFLP, PNA-mediated PCR and allele-specific PCR. Within the diagnostic setting the Peter MacCallum uses the rapid and highly sensitive allele-specific competitive blocker PCR (ACB-PCR) assay with a sensitivity level of between 1 and 0.1%. Between Jan 2008 and Oct 2010, 338 samples were tested, of which 58% were positive.