PD-L1 expression on routine samples of non-small cell lung cancer: results and critical issues from a 1-year experience of a centralised laboratory.

AIMS: Our laboratory is a centralised centre receiving routine non-small cell lung cancer (NSCLC) samples for programmed death ligand-1 (PD-L1) immunohistochemical (IHC) evaluation. Since literature data are not concordant here we review our clinical records to assess the rate of PD-L1 positive and negative NSCLC cases in real-world practice. METHODS: PD-L1 expression was evaluated by a validated 22C3 IHC laboratory developed test on 211 prospectively collected routine NSCLC samples, received from 10 outside institutions. PD-L1 expression was assessed by the tumour proportion score (TPS) and reported by using a three cut-point system: TPS<1, TPS 1%-49% and TPS≥50%. RESULTS: Overall, 193 out of 211 samples (91.5%) meet the criteria for adequacy (more than 100 viable neoplastic cells). In 62.7% (121/193) of samples TPS was <1%; 17.6% of samples (34/193) expressed PD-L1 with a TPS of 1%-49% and 19.7% (38/193) with a TPS of >50%. There was no significant difference in PD-L1 expression rates between different histotypes and site of sampling. Instead, a statistically significant difference was associated to the type of samples: in fact, cytological samples were more frequently negative for PD-L1 expression (TPS<1%) and less often displayed PD-L1 expression at high levels (TPS>50%) than surgical resections and biopsies. CONCLUSIONS: We present a referral laboratory experience on IHC PD-L1 expression of prospectively collected routine NSCLC samples. Data from the real-world practice can better clarify the percentage of PD-L1 positive and negative cases, to establish benchmarks for good practice standards.

EGFR mutant allelic-specific imbalance assessment in routine samples of non-small cell lung cancer.

In non-small cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) gene may undergo both mutations and copy number gains. EGFR mutant allele-specific imbalance (MASI) occurs when the ratio of mutant-to-wild-type alleles increases significantly. In this study, by using a previously validated microfluidic-chip-based technology, EGFR-MASI occurred in 25/67 mutant cases (37%), being more frequently associated with EGFR exon 19 deletions (p=0.033). In a subset of 49 treated patients, we assessed whether MASI is a modifier of anti-EGFR treatment benefit. The difference in progression-free survival and overall survival between EGFR-MASI-positive and EGFR-MASI-negative groups of patients did not show a statistical significance. In conclusion, EGFR-MASI is a significant event in NSCLC, specifically associated with EGFR exon 19 deletions. However, EGFR-MASI does not seem to play a role in predicting the response to first-generation EGFR small molecules inhibitors.

EGFR mutation detection by microfluidic technology: a validation study.

Advanced non-small cell lung cancer samples are tested for epidermal growth factor receptor (EGFR) gene mutations. Their detection by direct sequencing is time-consuming. Conversely, the length analysis of fluorescently labelled PCR products is easier. To avoid labelled primers and the automated capillary electrophoresis apparatus, we validated a fast and sensitive chip-based microfluidic technology. The limit of detection of fragment length assay on microfluidic device was 5%, more sensitive than direct sequencing (12.5%). The novel methodology showed high accuracy in the analysis of samples whose mutational status was known. The accuracy in quantifying mutated alleles (mA) was evaluated by PCR products subcloning; the mA% provided by direct sequencing of subcloned PCR products showed a close correlation with the mA% provided by the microfluidic technology for both exon 19 (R(2)=0.9) and 21 (R(2)=0.9). Microfluidic-based on-chip electrophoresis makes EGFR testing more rapid, sensitive and cost-effective.