Corrigendum: A novel BRCA1 splicing variant detected in an early onset triple-negative breast cancer patient additionally carrying a pathogenic variant in ATM: a case report.

[This corrects the article DOI: 10.3389/fonc.2023.1102184.].

BRAFV600 variant allele frequency predicts outcome in metastatic melanoma patients treated with BRAF and MEK inhibitors.

BACKGROUND: The prognostic impact of variant allele frequency (VAF) on clinical outcome in BRAFV600 mutated metastatic melanoma patients (MMPs) receiving BRAF (BRAFi) and MEK inhibitors (MEKi) is unclear. MATERIALS AND METHODS: A cohort of MMPs receiving first line BRAFi and MEKi was identified by inspecting dedicated databases of three Italian Melanoma Intergroup centres. VAF was determined by next generation sequencing in pre-treatment baseline tissue samples. Correlation between VAF and BRAF copy number variation was analysed in an ancillary study by using a training and a validation cohort of melanoma tissue samples and cell lines. RESULTS: Overall, 107 MMPs were included in the study. The VAF cut-off determined by ROC curve was 41.3%. At multivariate analysis, progression-free survival (PFS) was significantly shorter in patients with M1c/M1d [HR 2.25 (95% CI 1.41-3.6, p < 0.01)], in those with VAF >41.3% [HR 1.62 (95% CI 1.04-2.54, p < 0.05)] and in those with ECOG PS ≥1 [HR 1.82 (95% CI 1.15-2.88, p < 0.05)]. Overall survival (OS) was significantly shorter in patients with M1c/M1d [HR 2.01 (95% CI 1.25-3.25, p < 0.01)]. Furthermore, OS was shorter in patients with VAF >41.3% [HR 1.46 (95% CI 0.93-2.29, p = 0.06)] and in patients with ECOG PS ≥1 [HR 1.52 (95% CI 0.94-2.87, p = 0.14)]. BRAF gene amplification was found in 11% and 7% of samples in the training and validation cohort, respectively. CONCLUSIONS: High VAF is an independent poor prognostic factor in MMP receiving BRAFi and MEKi. High VAF and BRAF amplification coexist in 7%-11% of patients.

A novel BRCA1 splicing variant detected in an early onset triple-negative breast cancer patient additionally carrying a pathogenic variant in ATM: A case report.

The widespread adoption of gene panel testing for cancer predisposition is leading to the identification of an increasing number of individuals with clinically relevant allelic variants in two or more genes. The potential combined effect of these variants on cancer risks is mostly unknown, posing a serious problem for genetic counseling in these individuals and their relatives, in whom the variants may segregate singly or in combination. We report a female patient who developed triple-negative high grade carcinoma in the right breast at the age of 36 years. The patient underwent bilateral mastectomy followed by combined immunotherapy and chemotherapy (IMpassion030 clinical trial). Two years later she developed a skin recurrence on the right anterior chest wall. Despite intensive treatment, the patient died at 40-year-old due to disease progression. Gene panel testing of patient's DNA revealed the presence of a protein truncating variant in ATM [c.1672G>T; p.(Gly558Ter)] and of a not previously reported variant in the BRCA1 exon 22 donor splice site [c.5406+6T>C], whose clinical significance was unknown. The analysis of patient's RNA revealed the up-regulation of two alternative BRCA1 mRNA isoforms derived from skipping of exon 22 and of exons 22-23. The corresponding predicted protein products, p.(Asp1778GlyfsTer27) and p.(Asp1778_His1822del) are both expected to affect the BRCA1 C Terminus (BRCT) domain. The two variants were observed to co-occur also in the proband's brother who, in addition, was heterozygous for a common variant (c.4837A>G) mapped to BRCA1 exon 16. This allowed to ascertain, by transcript-specific amplification, the lack of functional mRNA isoforms expressed by the c.5406+6T>C allele and provided evidence to classify the BRCA1 variant as pathogenic, according to the guidelines of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To our knowledge, excluding two cases detected following the screening of population specific recurrent variants, only one ATM/BRCA1 double heterozygote has been reported in the literature, being the case here described the one with the youngest age at cancer onset. The systematic collection of cases with pathogenic variants in more than one cancer predisposition gene is needed to verify if they deserve ad hoc counseling and clinical management.

RET-MAP: An International Multicenter Study on Clinicobiologic Features and Treatment Response in Patients With Lung Cancer Harboring a RET Fusion.

INTRODUCTION: Nearly 1% to 2% of NSCLCs harbor RET fusions. Characterization of this rare population is still incomplete. METHODS: This retrospective multicenter study included patients with any-stage RET positive (RET+) NSCLC from 31 cancer centers. Molecular profiling included DNA/RNA sequencing or fluorescence in situ hybridization analyses. Clinicobiological features and treatment outcomes (per investigator) with surgery, chemotherapy (CT), immune checkpoint blockers (ICBs), CT-ICB, multityrosine kinase inhibitors, and RET inhibitors (RETis) were evaluated. RESULTS: For 218 patients included between February 2012 and April 2022, median age was 63 years, 56% were females, 93% had adenocarcinoma, and 41% were smokers. The most frequent fusion partner was KIF5B (72%). Median tumor mutational burden was 2.5 (range: 1-4) mutations per megabase, and median programmed death-ligand 1 expression was 10% (range: 0%-55%). The most common metastatic sites were the lung (50%), bone (43%), and pleura (40%). Central nervous system metastases were found at diagnosis of advanced NSCLC in 21% of the patients and at last follow-up or death in 31%. Overall response rate and median progression-free survival were 55% and 8.7 months with platinum doublet, 26% and 3.6 months with single-agent CT, 46% and 9.6 months with CT-ICB, 23% and 3.1 months with ICB, 37% and 3 months with multityrosine kinase inhibitor, and 76% and 16.2 months with RETi, respectively. Median overall survival was longer in patients treated with RETi versus no RETi (50.6 mo [37.7-72.1] versus 16.3 mo [12.7-28.8], p < 0.0001). CONCLUSIONS: Patients with RET+ NSCLC have mainly thoracic and bone disease and low tumor mutational burden and programmed death-ligand 1 expression. RETi markedly improved survival, whereas ICB may be active in selected patients.

Lorlatinib beyond progression plus platinum/pemetrexed for ALK-positive non-small cell lung cancer patients: report of two cases.

Lorlatinib is an active treatment for advanced anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) pretreated with ALK-tyrosine kinase inhibitors (-TKIs). However, there is paucity of data on the activity of platinum/pemetrexed chemotherapy administered at the time of progression on lorlatinib. In addition, it is uncertain whether continuation of lorlatinib beyond progression (LBP) would provide any additional clinical benefit. Here, we describe two cases experiencing an exceptional response to platinum/pemetrexed chemotherapy plus LBP and make an attempt to identify which patients' characteristics and biologic profiles of the tumor could predict benefit from such an approach. In this report, presence of controlled brain metastases, rapidly progressing extracranial disease, and presence of ALK-dependent mechanisms of resistance were associated with benefit from platinum/pemetrexed chemotherapy plus lorlatinib beyond progression.

Prognosis of ALK-rearranged non-small-cell lung cancer patients carrying TP53 mutations.

Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death. Gene rearrangements involving the anaplastic lymphoma kinase (ALK) tyrosine kinase identify a clinical and molecular subset of NSCLC patients, who benefit from the monotherapy with ALK tyrosine kinase inhibitors. Nonetheless, responsiveness to TKIs and prognosis of these patients are influenced by several factors, including resistance mechanisms and mutations affecting genes involved in key molecular pathways of cancer cells. In a cohort of 98 NSCLC patients with ALK gene rearrangements, we investigated the role of Tumor Protein (TP53) gene mutations in predicting patients prognosis. TP53 mutations were evaluated in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS).Results: In patients with available clinical and TP53 mutation information, we found that 13 patients (20.3%) were affected by TP53 mutations. Considered together, even though showing a trend, TP53 mutations were not associated with PFS and OS. Considering the different TP53 mutations by functionality in terms of disruptive and non-disruptive mutations, we observed that TP53 non-disruptive mutations were able to predict worse OS in the overall case series. Moreover, a worse PFS was seen in the subgroup of patients with TP53 non-disruptive mutation, in first-, second-, and third line of treatment. Our results show that mutations affecting TP53 gene, especially non-disruptive mutations, are able to affect prognosis of ALK-rearranged NSCLC patients.

Inflamed Tumor Phenotype as Predictor of Long-Term Response to Pembrolizumab in an EGFR-Mutated Non-Small Cell Lung Cancer (NSCLC) Patient with Acquired Resistance to Afatinib: a Case Report and Review of the Literature.

Treatment with immune checkpoint inhibitors (ICIs) that target the programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) axis is usually ineffective in patients with epidermal growth factor receptor (EGFR)-mutated advanced non-small cell lung cancer (NSCLC), either as first-line treatment or in later lines. By contrast, especially for patients with common EGFR mutations (exon 19 deletion/L858R point mutation), an orally bioavailable EGFR tyrosine kinase inhibitor (EGFR-TKI) is the best upfront therapy, being able to provide response rates well above 50% and a median progression-free survival ranging from 11 to 19 months, depending on whether a second-generation (e.g., afatinib) or a third-generation (i.e., osimertinib) EGFR-TKI is used. Unfortunately, treatment options for these patients at the time of acquired resistance are limited. As for afatinib-pretreated patients, those who develop a T790M mutation may benefit from osimertinib, whereas platinum-based chemotherapy is the preferable therapeutic strategy for T790M-negative patients as well as for patients who progress on osimertinib administered as first-line therapy. Here, we describe the case of an exon-19-deleted patient who experienced a complete response to the anti-PD-1 agent pembrolizumab upon the development of T790M-negative acquired resistance to afatinib. Furthermore, we discuss this case in the context of the existing literature, especially focusing on the importance of evaluating multiple markers of immune response post-EGFR-TKI and prior to ICI treatment in order to select the best treatment strategy in this clinical scenario.

Molecular profiling of advanced non-small cell lung cancer in the era of immunotherapy approach: a multicenter Italian observational prospective study of biomarker screening in daily clinical practice.

AIMS: Heterogeneous implementation of molecular tests in current diagnostic algorithm at a European and international level is emerging as a major issue for efficient lung cancer molecular profiling. METHODS: From May 2017 until October 2017, N=1612 patients referring to 13 Italian institutions were selected, at advanced stage non-small cell lung cancer (NSCLC), and prospectively evaluated. Principal endpoints were: the percentage of diagnoses performed on cytological and histological material, the proportion of requests for epidermal growth factor receptor (EGFR) mutational status, and resistance mutations detected on tissue and/or liquid biopsy samples after first-generation or second-generation tyrosine kinase inhibitors, the proportion of requests for anaplastic lymphoma kinase (ALK) gene rearrangements, ROS proto-oncogene 1 (ROS1) and Kirsten Rat Sarcoma (KRAS) determinations, the proportion of requests for programmed death-ligand1 (PD-L1) evaluation and, finally, the different assays used for the detection of EGFR mutations, ALK and ROS1 gene rearrangements and PD-L1 expression. RESULTS: Of 1325 patients finally included, only 50.8% requests were related to driver mutations with target agents already available in first-line at that preplanned time, while 49.2% were associated with PD-L1, ROS1, KRAS and others. Multiplex genomic assays (such as next-generation sequencing) were considered by all participating centres. CONCLUSIONS: To the best of our knowledge, this is the first study in a 'real-life daily practice' involving both pathologists and oncologists evaluating routinely workflow and trends towards improvements in molecular requests. Collected data aim to describe the applied algorithms and evolution of molecular screening for stage IV NSCLC in clinical practice.

Genome-wide expression of the residual lung reacting to experimental Pneumonectomy.

BACKGROUND: Acute or chronic irreversible respiratory failure may occur in patients undergoing pneumonectomy. Aim of this study was to determine transcriptome expression changes after experimental pneumonectomy in swine model. Experimental left pneumonectomy was performed in five pigs under general anaesthesia. Both the resected and the remaining lung, after 60 post-operative completely uneventful days, underwent genome-wide bulk RNA-Sequencing (RNA-Seq). RESULTS: Histological analysis showed dilation of air spaces and rupture of interalveolar septa. In addition, mild inflammation, no fibrosis, radial stretch of the bronchus, strong enlargement of airspaces and thinning of the blood supply were observed. Bioinformatic analyses of bulk RNA-Seq data identified 553 Differentially Expressed Genes (DEGs) at adjusted P-value below 0.001, between pre- and post-pneumonectomy. The top 10 up-regulated DEGs were Edn1, Areg, Havcr2, Gadd45g, Depp1, Cldn4, Atf3, Myc, Gadd45b, Socs3; the top 10 down-regulated DEGs were Obscn, Cdkn2b, ENSSSCG00000015738, Prrt2, Amer1, Flrt3, Efnb2, Tox3, Znf793, Znf365. Leveraging digital cytometry tools, no difference in cellular abundance was found between the two experimental groups, while the analysis of cell type-specific gene expression patterns highlighted a striking predominance of macrophage-specific genes among the DEGs. DAVID-based gene ontology analysis showed a significant enrichment of "Extrinsic apoptotic signaling pathway" (FDR q = 7.60 × 10- 3) and "Response to insulin" (FDR q = 7.60 × 10- 3) genes, along with an enrichment of genes involved as "Negative regulators of DDX58/IFIH1 signaling" (FDR q = 7.50 × 10- 4) found by querying the REACTOME pathway database. Gene network analyses indicated a general dysregulation of gene inter-connections. CONCLUSION: This translational genomics study highlighted the existence both of individual genes, mostly dysregulated in certain cellular populations (e.g., macrophages), and gene-networks involved in pulmonary reaction after left pneumonectomy. Their involvement in lung homeostasis is largely supported by previous studies, carried out both in humans and in other animal models (under homeostatic or disease-related conditions), that adopted candidate-gene approaches. Overall, the present findings represent a preliminary assessment for future, more focused, studies on compensatory lung adaptation, pulmonary regeneration and functional reload.

PD-L1 quantification across tumor types using the reverse phase protein microarray: implications for precision medicine.

BACKGROUND: Anti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples. METHODS: PD-L1 expression was measured using five antibody clones (22C3, 28-8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment. RESULTS: Median-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: -0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response. CONCLUSIONS: Continuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

Wild-Type KRAS Allele Effects on Druggable Targets in KRAS Mutant Lung Adenocarcinomas.

KRAS mutations are one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC) and in lung adenocarcinomas in particular. Development of therapeutics targeting KRAS has been incredibly challenging, prompting indirect inhibition of downstream targets such as MEK and ERK. Such inhibitors, unfortunately, come with limited clinical efficacy, and therefore the demand for developing novel therapeutic strategies remains an urgent need for these patients. Exploring the influence of wild-type (WT) KRAS on druggable targets can uncover new vulnerabilities for the treatment of KRAS mutant lung adenocarcinomas. Using commercially available KRAS mutant lung adenocarcinoma cell lines, we explored the influence of WT KRAS on signaling networks and druggable targets. Expression and/or activation of 183 signaling proteins, most of which are targets of FDA-approved drugs, were captured by reverse-phase protein microarray (RPPA). Selected findings were validated on a cohort of 23 surgical biospecimens using the RPPA. Kinase-driven signatures associated with the presence of the KRAS WT allele were detected along the MAPK and AKT/mTOR signaling pathway and alterations of cell cycle regulators. FoxM1 emerged as a potential vulnerability of tumors retaining the KRAS WT allele both in cell lines and in the clinical samples. Our findings suggest that loss of WT KRAS impacts on signaling events and druggable targets in KRAS mutant lung adenocarcinomas.

Higher TLR7 Gene Expression Predicts Poor Clinical Outcome in Advanced NSCLC Patients Treated with Immunotherapy.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of lung cancer. However, their clinical benefit is limited to a minority of patients. To unravel immune-related factors that are predictive of sensitivity or resistance to immunotherapy, we performed a gene expression analysis by RNA-Seq using the Oncomine Immuno Response Assay (OIRRA) on a total of 33 advanced NSCLC patients treated with ICI evaluating the expression levels of 365 immune-related genes. We found four genes (CD1C, HLA-DPA1, MMP2, and TLR7) downregulated (p < 0.05) and two genes (IFNB1 and MKI67) upregulated (p < 0.05) in ICI-Responders compared to ICI-Non-Responders. The Bayesian enrichment computational analysis showed a more complex interaction network that involved 10 other genes (IFNA1, TLR4, CD40, TLR2, IL12A, IL12B, TLR9, CD1E, IFNG, and HLA-DPB1) correlated with different functional groups. Five main pathways were identified (FDR < 0.0001). High TLR7 expression levels were significantly associated with a lack of response to immunotherapy (p < 0.0001) and worse outcome in terms of both PFS (p < 0.001) and OS (p = 0.03). The multivariate analysis confirmed TLR7 RNA expression as an independent predictor for both poor PFS (HR = 2.97, 95% CI, 1.16-7.6, p = 0.023) and OS (HR = 2.2, 95% CI, 1-5.08, p = 0.049). In conclusion, a high TLR7 gene expression level was identified as an independent predictor for poor clinical benefits from ICI. These data could have important implications for the development of novel single/combinatorial strategies TLR-mediated for an efficient selection of "individualized" treatments for NSCLC in the era of immunotherapy.

INSL4 as prognostic marker for proliferation and invasiveness in Non-Small-Cell Lung Cancer.

Non-small-cell-lung cancer accounts for 80-85% of all forms of lung cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of lung cancer, no significant improvements have thus far been achieved in terms of patients' prognosis. Here, we investigated the role of INSL4 - a member of the relaxin-family - in NSCLC. We overexpressed INSL4 in NSCLC cells to analyse in vitro the growth rate and the tumourigenic features. We investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. We found an INSL4 cell growth promoting effect in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, INSL4 overexpression has not similar effect, despite huge basal INSL4-mRNA expression respect to H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, revealed highly difference in the INSL4-mRNA amount. Transfection of NSCLC lines with INSL4-Myc showed huge level of INSL4-mRNA with a very low amount of protein expressed. Notably, similar discrepancy has been observed in NSCLC patients. However, in a cohort of NSCLC patients analysing a database, we found a significant inverse correlation between INSL4 expression and Overall Survival. By combining the in vitro and in vivo results, suggest that in patients whose NSCLC adenocarcinoma spontaneously expressed high levels of INSL4 post-transcriptional modifications affecting INSL4 do not allow to assess precision therapy in selected patients without consider protein INSL4 amount.

Sensitivity to Immune Checkpoint Blockade in Advanced Non-Small Cell Lung Cancer Patients with EGFR Exon 20 Insertion Mutations.

Besides platinum-based chemotherapy, no established treatment option exists for advanced non-small-cell lung cancer (NSCLC) patients with EGFR exon 20 (Ex20ins) insertion mutations. We sought to determine the clinical outcome of patients with this EGFR mutation subtype in the immunotherapy era. Thirty NSCLCs with EGFR Ex20ins mutations were identified, of whom 15 had received immune checkpoint blockade (ICB) treatment as monotherapy (N = 12), in combination with chemotherapy (N = 2) or with another immunotherapeutic agent (N = 1). The response rate was observed in 1 out of 15 patients (6.7%), median progression-free survival (PFS) was 2.0 months and median overall survival (OS) was 5.3 months. A trend towards an inferior outcome in terms of PFS and OS was observed for patients receiving ICB treatment in the first versus second line setting (PFS: 1.6 months versus 2.7 months, respectively, p = 0.16-OS: 2.0 months versus 8.1 months, respectively, p = 0.09). Median OS from the time of diagnosis of advanced disease was shorter for patients treated with ICB versus those who did not receive immunotherapy (12.9 months versus 25.2 months, respectively, p = 0.08), which difference remained associated with a worse survival outcome at multivariate analysis (p = 0.04). Treatment with ICB is poorly effective in NSCLCs with EGFR Ex20ins mutations, especially when given in the first-line setting. This information is crucial in order to select the optimal treatment strategy for patients with this subtype of EGFR mutation.

Kynurenine/Tryptophan Ratio as a Potential Blood-Based Biomarker in Non-Small Cell Lung Cancer.

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) degrade tryptophan (Trp) into kynurenine (Kyn) at the initial step of an enzymatic pathway affecting T cell proliferation. IDO1 is highly expressed in various cancer types and associated with poor prognosis. Nevertheless, the serum Kyn/Trp concentration ratio has been suggested as a marker of cancer-associated immune suppression. We measured Kyn and Trp in blood samples of a wide cohort of non-small-cell lung cancer (NSCLC) patients, before they underwent surgery, and analyzed possible correlations of the Kyn/Trp ratio with either IDO1 expression or clinical-pathological parameters. Low Kyn/Trp significantly correlated with low IDO1 expression and never-smoker patients; while high Kyn/Trp was significantly associated with older (≥68 years) patients, advanced tumor stage, and squamous cell carcinoma (Sqcc), rather than the adenocarcinoma (Adc) histotype. Moreover, high Kyn/Trp was associated, among the Adc group, with higher tumor stages (II and III), and, among the Sqcc group, with a high density of tumor-infiltrating lymphocytes. A trend correlating the high Kyn/Trp ratio with the probability of recurrences from NSCLC was also found. In conclusion, high serum Kyn/Trp ratio, associated with clinical and histopathological parameters, may serve as a serum biomarker to optimize risk stratification and therapy of NSCLC patients.

Heterogeneous Off-Target Effects of Ultra-Low Dose Dimethyl Sulfoxide (DMSO) on Targetable Signaling Events in Lung Cancer In Vitro Models.

Targetable alterations in cancer offer novel opportunities to the drug discovery process. However, pre-clinical testing often requires solubilization of these drugs in cosolvents like dimethyl sulfoxide (DMSO). Using a panel of cell lines commonly used for in vitro drug screening and pre-clinical testing, we explored the DMSO off-target effects on functional signaling networks, drug targets, and downstream substrates. Eight Non-Small Cell Lung Cancer (NSCLC) cell lines were incubated with three concentrations of DMSO (0.0008%, 0.002%, and 0.004% v/v) over time. Expression and activation levels of 187 proteins, of which 137 were kinases and downstream substrates, were captured using the Reverse Phase Protein Array (RPPA). The DMSO effect was heterogeneous across cell lines and varied based on concentration, exposure time, and cell line. Of the 187 proteins measured, all were statistically different in at least one comparison at the highest DMSO concentration, followed by 99.5% and 98.9% at lower concentrations. Only 46% of the proteins were found to be statistically different in more than 5 cell lines, indicating heterogeneous response across models. These cell line specific alterations modulate response to in vitro drug screening. Ultra-low DMSO concentrations have broad and heterogeneous effects on targetable signaling proteins. Off-target effects need to be carefully evaluated in pre-clinical drug screening and testing.

High PD-L1/IDO-2 and PD-L2/IDO-1 Co-Expression Levels Are Associated with Worse Overall Survival in Resected Non-Small Cell Lung Cancer Patients.

Programmed death ligand 1 (PD-L1) expression is a predictive biomarker of the success of PD-1/PD-L1 inhibitor therapy for patients with advanced non-small cell lung cancer (NSCLC) but its role as a prognostic marker for early-stage resectable NSCLC remains unclear. We studied gene expression levels of immune-related genes PD-1, PD-L1, PD-L2, IDO-1, IDO-2 and INFγ in tumor tissue of surgically resected NSCLC and correlated the finding with clinicopathological features and patient outcomes. A total of 191 consecutive early-stage NSCLC patients who underwent curative pulmonary resection were studied. The mRNA expression levels of immune-related genes were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RT2 Profiler PCR Arrays (Qiagen). PD-1, PD-L2 and IDO-2 gene expression levels were significantly higher in patients with squamous histology (p = 0.001, p = 0.021 and p < 0.001; respectively). PD-1, PD-L1 and IDO-2 gene expression levels were significantly higher in patients with higher stage (p = 0.005, p = 0.048 and p = 0.002, respectively). The univariate analysis for recurrence-free survival (RFS) and overall survival (OS) showed that patients with higher levels of three-genes (PD-L1/PD-L2/INFγ) (hazard ratio (HR)) 1.90 (95% confidence interval (CI), 1.13-3.21), p = 0.015) were associated with a worse RFS, while patients with higher levels of both genes (PD-L1/IDO-2) or (PD-L2/IDO-1) were associated with a worse OS (HR 1.63 95% CI, 1.06-2.51, p = 0.024; HR 1.54 95% CI, 1.02-2.33, p = 0.04; respectively). The multivariate interaction model adjusted for histology and stage confirmed that higher levels of three genes (PD-L1/PD-L2/INFγ) were significantly associated with worse RFS (HR 1.98, p = 0.031) and higher levels of both genes (PD-L1/IDO-2) and (PD-L2/IDO-1) with worse OS (HR 1.98, p = 0.042, HR 1.92, p = 0.022). PD-L1/IDO-2 and PD-L2/IDO-1 co-expression high levels are independent negative prognostic factors for survival in early NSCLC. These features may have important implications for future immune-checkpoint therapeutic approaches.

A fully automated assay to detect the expression of pan-cytokeratins and of EML4-ALK fusion protein in circulating tumour cells (CTCs) predicts outcome of non-small cell lung cancer (NSCLC) patients.

BACKGROUND: In advanced non-small cell lung cancer (NSCLC) a recent meta-analysis confirms circulating tumour cells (CTCs) as an independent prognostic indicator of progression-free survival (PFS) and overall survival (OS). However, further investigations are necessary to predict and dynamically monitor the therapy in NSCLC patients using CTCs. To this aim, we combined the classical standard assay (SA) with an expanded cytokeratins profile (EA) and quantified the expression of EML4-ALK fusion protein in CTCs. METHODS: The CellSearch (CS) platform-first marked in vitro diagnostic use (IVD) from Food and Drug Administration (FDA), and "gold standard" for quantifying CTCs - detects EpCAM and cytokeratins (CKs) 8, 18, and 19. Since NSCLC shows different CKs profile, we customized the SA, to recognize CK 4, 5, 6, 7, 8, 10, 13, 14, 18, and 19 (EA). Using both assays we designed a prospective, multi-center study, primarily aimed to enumerate CTCs in advanced NSCLC. Secondarily, we developed an integration of the EA to quantify the expression of EML4-ALK fusion protein in CTCs, and correlated them with PFS and OS. RESULTS: EA identified a significantly much more number of CTC-positive patients (115 out of 180) than SA (103 out of 192; Chi-square =4.0179, with 1 degrees of freedom, P=0.04502). Similar to SA, EA levels were still associated with patient' outcomes. Furthermore, the expression of EML4-ALK on CTCs allowed stratifying NSCLC patients according to a statistically significant difference in PFS. CONCLUSIONS: We proposed here two novel automated tests, to characterize the expression of specific molecules on CTCs. We demonstrated that these integrated assays are robust and actionable in prospective clinical studies, and in the future could allow clinicians to improve both choice and length of treatment in individual NSCLC patient.

Detection of EGFR Mutations in Plasma Cell-Free Tumor DNA of TKI-Treated Advanced-NSCLC Patients by Three Methodologies: Scorpion-ARMS, PNAClamp, and Digital PCR.

Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations.

Prevalence and Spectrum of BRCA Germline Variants in Central Italian High Risk or Familial Breast/Ovarian Cancer Patients: A Monocentric Study.

Hereditary breast and ovarian cancers are mainly linked to variants in BRCA1/2 genes. Recently, data has shown that identification of BRCA variants has an immediate impact not only in cancer prevention but also in targeted therapeutic approaches. This prospective observational study characterized the overall germline BRCA variant and variant of uncertain significance (VUS) frequency and spectrum in individuals affected by breast (BC) or ovarian cancer (OC) and in healthy individuals at risk by sequencing the entire BRCA genes. Of the 363 probands analyzed, 50 (13.8%) were BRCA1/2 mutated, 28 (7.7%) at BRCA1 and 23 (6.3%) at BRCA2 gene. The variant c.5266dupC p.(Gln1756Profs) was the most frequent alteration, representing 21.4% of the BRCA1 variants and 12.0% of all variants identified. The variant c.6313delA p.(Ile2105Tyrfs) of BRCA2 was the most frequent alteration observed in 6 patients. Interestingly, two new variants were identified in BRCA2. In addition, 25 different VUS were identified; two were reported for the first time in BRCA1 and two in BRCA2. The number of triple-negative BCs was significantly higher in patients with the pathogenic BRCA1/2-variant (36.4%) than in BRCA1/2 VUS (16.0%) and BRCA1/2 wild-type patients (10.7%) (p < 0.001). Our study reveals that the overall frequency of BRCA germline variants in the selected high-risk Italian population is about 13.8%. We believe that our results could have significant implications for preventive strategies for unaffected BRCA-carriers and effective targeted treatments such as PARP inhibitors for patients with BC or OC.