Danilo Rocco

EGFR and KRAS mutations detection on lung cancer liquid-based cytology: a pilot study

In advanced non-small-cell lung carcinomas epidermal growth factor receptor (EGFR) and KRAS testing is often performed on cytology. Liquid-based cytology (LBC), which eliminates the need for slide preparation by clinicians, may be very useful. In 42 LBC DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after smear preparation and laser capture microdissection (LCM) of Papanicolaou-stained cells. EGFR and KRAS mutational analyses were performed by direct sequencing. On CytoLyt-derived DNA four EGFR (9%) and five KRAS (12%) gene mutations were found. When direct sequencing was performed after LCM, the rate of cases that displayed either EGFR or KRAS mutations increased from 21% to 40%. Although time-consuming, LCM makes direct sequencing highly sensitive even on LBC preparations containing only a few cells.

EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non–small cell lung carcinoma patients

Epidermal growth factor receptor (EGFR) mutations are reliably detected by referral laboratories, even if most lung cancer cytology specimens sent to such laboratories contain very few cells. However, EGFR mutations may be distributed heterogeneously within tumors, thereby raising concerns that mutations detected on cytology are not representative of the entire tumor and, thus, are less reliable in predicting response to tyrosine kinase inhibitor (TKI) treatment than mutations detected on histology. To address this issue, the authors reviewed their clinical practice archives and compared the outcome of TKI treatment among patients who were selected by cytology versus patients who were selected by histology.

Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients

Background:When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.Methods:Our panel (‘SiRe’) covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.Results:SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.Conclusions:SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

EGFR mutations detection on liquid-based cytology: is microscopy still necessary?

Currently, there is a trend towards an increasing use of liquid-based cytology (LBC) to diagnose non–small cell lung cancer. In this study, to detect epidermal growth factor receptor mutations, different molecular techniques were applied to LBC samples with and without laser capture microdissection (LCM). In 58 LBCs, DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after slide preparation and LCM of Papanicolaou-stained cells. The rate of mutant cases obtained by direct sequencing was discordant between CytoLyt-derived (10.3%) and LCM-derived (17.2%) DNA. However, the same mutant rate (17.2%) was achieved on the matched samples by high-resolution melting analysis, fragment and TaqMan assays. Thus, LCM and direct sequencing may be replaced by more sensitive non-sequencing methods directly performed on CytoLyt-derived DNA, an easier and faster approach to improve epidermal growth factor receptor testing standardisation on LBCs.

UbcH10 overexpression in human lung carcinomas and its correlation with EGFR and p53 mutational status

INTRODUCTION UbcH10 codes for the cancer related E2 Ubiquitin Conjugating Enzyme, an enzymatic molecule with a key role in the ubiquitin-proteasome pathway. Current studies have suggested a critical role of UbcH10 in a variety of malignancies, including human thyroid, breast, ovarian and colorectal carcinomas. The aim of this study has been to extend the analysis of UbcH10 expression to lung cancer. This neoplasia represents one of the leading cause of cancer mortality worldwide, and new tools for an accurate diagnosis/prognosis are needed. METHODS The expression levels of UbcH10 were analysed in human non-small cell lung carcinoma (NSCLC) by quantitative RT-PCR and tissue microarray immunohistochemistry, and these values were correlated with the clinicopathological features of the patients affected by NSCLC. RESULTS Our results demonstrate that UbcH10 is overexpressed in NSCLC compared to the normal lung tissue. Moreover, UbcH10 expression is significantly higher in squamous cell and large cell carcinomas than in adenocarcinomas, and directly and inversely correlated with the mutational status of p53 and EGFR, respectively. The suppression of UbcH10 expression by RNAi resulted in a drastic reduction of proliferation and migration abilities of lung carcinoma cell lines. CONCLUSION These results, taken together, indicate that UbcH10 overexpression has a critical role in lung carcinogenesis, and the evaluation of UbcH10 expression levels may be a new tool for the characterisation of NSCLC.

EGFR mutation detection on routine cytological smears of non-small cell lung cancer by digital PCR: a validation study

Highly sensitive genotyping techniques are useful to detect epidermal growth factor receptor (EGFR) mutations on lung cancer cytological samples, when these specimens feature only few neoplastic cells. This study aimed to validate digital PCR (dPCR) methodology on cytological material. In plasmid model system, dPCR allowed for the detection of a minimal percentage (1%) of EGFR mutant alleles. Cytological samples (n=30), with neoplastic cell percentage ranging from 10% to 80% and yielding a quantity of extracted DNA ranging from 1.75 to 60 ng/µL were selected. Results previously generated by fragment length and TaqMan assays (n=8 exon 19 deletions, n=2 L858R mutations and n=20 wild-type DNA) were compared with those obtained by dPCR. Data were highly concordant (96.6%). However, dPCR detected an additional L858R mutation that had been missed by TaqMan assay on a paucicellular smear. This mutation was confirmed by cloning PCR products and sequencing. Thus, dPCR can reliably be used to increase EGFR mutation detection rate on scarcely cellular lung cancer smears.

Restoration of CBX7 expression increases the susceptibility of human lung carcinoma cells to irinotecan treatment.

Lung cancer is one of the most common causes of cancer-related death worldwide in men and women, and, despite the recent remarkable scientific advances, drug treatment is still unsatisfactory. Polycomb protein chromobox homolog 7 (CBX7) is involved in several biological processes, including development and cancer progression, indeed the lack of CBX7 protein correlates with a highly malignant phenotype and a poor prognosis. However, its role in lung cancer still remains unknown. Since CBX7 is drastically downregulated in human lung carcinomas, we investigated whether restoration of CBX7 expression could affect growth property of lung cancer cells and modulate their sensitivity to treatment with irinotecan and etoposide, two chemoterapy drugs most commonly used in lung cancer therapy. Here, we demonstrate that restoration of CBX7 in two human lung carcinoma cell lines (A549 and H1299), in which this protein is not detectable, leads to a decreased proliferation (at least in part through a downregulation of phosphorylated ERK and phosphorylated p38) and an increased apoptotic cell death after drug exposure (at least in part through the downregulation of Bcl-2, phosphorylated Akt, and phosphorylated JNK). Taken together, these results suggest that the retention of CBX7 expression may play a role in the modulation of chemosensitivity of lung cancer patients to the treatment with irinotecan and etoposide.

Targeting immune checkpoints in non small cell lung cancer

HighlightsOncogenic drivers and mechanisms of adaptive immunity are changing the landscape of lung cancer treatment.Cancers can disrupt cancer cell immune recognition by interfering with immune‐check‐point pathways.Immune check point molecules PD‐1, PD‐L1 and CTLA‐4 are relevant targets for cancer therapy.Based on clinical trials, anti PD‐1 and anti PD‐L1 agents have been approved for advanced NSCL. &NA; Cancers have the ability to disrupt immune response by interfering with adaptive immunity. Blocking checkpoint pathways has become a target for pharmacological research in lung cancer with particular focus on peptides PD‐1 and CTLA‐4. A number of immune check‐point inhibitors (ICIs) targeting both PD‐1 and CTLA‐4 pathways are under investigation within clinical trials, of which Nivolumab, Pembrolizumab and Atezolizumab have already been approved for lung cancer treatment by both FDA and EMA. Employed as single‐agents in current practice for the treatment of advanced non‐small cell lung cancer (NSCLC) ICIs have exhibited advantages in terms of overall survival and response rate with some responses being durable. Evaluating combinations of different inhibitors, dosing and sequencing within a multimodal therapy approach, together with better management of toxicity represents a new challenge for future research of therapy targeting immune check‐points.

Cell free DNA analysis by SiRe ® next generation sequencing panel in non small cell lung cancer patients: focus on basal setting

Background Non small cell lung cancer (NSCLC) is diagnosed in most cases on small tissue samples, such as cytological preparations and histological biopsies; these limited tissue specimens may be not always sufficient for testing epidermal growth factor receptor (EGFR) mutations and other relevant predictive biomarkers. Cell-free DNA (cfDNA) can be used as a surrogate for EGFR mutational testing, whenever tissue is unavailable. However, the detection of gene mutations on cfDNA is challenging; in fact, the extremely low concentration of circulating tumor DNA requires the implementation of highly sensitive and validated next generation techniques. Methods Thus, we have recently validated a novel next generation sequencing (NGS) assay, employing the SiRe® gene panel to detect on cfDNA mutations of EGFR and KRAS, NRAS, BRAF, cKIT and PDGFR genes. In this current study, we report on a series of NSCLC patients, without available tissue for EGFR testing, who prospectively underwent SiRe® NGS analysis. Results The results confirm the high clinical performance, in terms of success rate and mutation detection, of NGS based analysis of cfDNA. Conclusions SiRe® NGS panel represent an effective diagnostic tool in cfDNA analysis setting.

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.