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Abstract Background KRAS mutations negatively affect outcome after treatment with cetuximab in metastatic colorectal cancer (mCRC) patients. As only 20% of KRAS wild type (WT) patients respond to cetuximab it is possible that other mutations, constitutively activating the EGFR pathway, are present in the non-responding KRAS WT patients. We retrospectively analyzed objective tumor response rate, (ORR) progression-free (PFS) and overall survival (OS) with respect to the mutational status of KRAS, BRAF, PIK3CA and PTEN expression in mCRC patients treated with a cetuximab-based regimen. Methods 67 mCRC patients were enrolled onto the study. DNA was extracted from paraffin-embedded sections derived from primary or metastatic lesions. Exon 2 of KRAS and exon 15 of BRAF were analyzed by direct sequencing, PIK3CA was evaluated by pyrosequencing and PTEN expression by immunohistochemistry. Results BRAF and PIK3CA mutations were independently associated with worse PFS (p = 0.006 and p = 0.028, respectively) and OS (p = 0.008 and p = 0.029, respectively). No differences in clinical outcome were found between patients who were positive or negative for PTEN expression. Conversely, patients negative for KRAS, BRAF and PIK3CA mutations were characterized by significantly better ORR, PFS and OS than patients with at least one of these mutations. Conclusions BRAF and PIK3CA mutations would seem to be independent predictors of anti-EGFR therapy effectiveness and could be taken into consideration during treatment decision making.

The efficacy and cost-effectiveness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remain uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), and BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in five patients (2.8%) and SMO in four patients (2.2%). Additionally, MAP2K1 , CTNNB1 , and MYC were mutated in three patients (2.4%). Two mutations (1.1%) were observed in ERBB3 , RAF1 , MTOR , JAK1 , and FGFR2 . No significant survival differences were observed based on number of mutations. In total, 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

The real-world, retrospective, NEROnE registry investigated the impact of next-generation sequencing (NGS) in advanced non-small-cell lung cancer (NSCLC) patients (pts) at three oncology units in the north of Italy between January 2020 and December 2022. We focused on the clinical characterization and outcomes of NSCLC with rare molecular alterations: EGFR exon 20 insertion, non-activating EGFR mutations, BRAF V600E and non-V600, ROS1 and RET rearrangements, MET , ErbB2, and FGFR mutations. Overall, these represented 6.4% (62/970) of the pts analysed with NGS in the daily practice. The most heavily represented rare alterations were ROS1 rearrangement (15 pts-24%) and MET exon 14 skipping mutation (11 pts-18%). No associations were found with the demographic and clinical features. Forty-nine pts received targeted therapies, of which 38.8% were first- and 9.8% were second-line. The remaining pts received chemotherapy and/or immunotherapy. In terms of the clinical outcomes, although not statistically significant, a tendency toward shorter OS was seen when therapies other than specific targeted therapies were used (HR: 1.84, 95% CI: 0.79-4.33, p = 0.158). The pts with co-mutations (19.4%) seemed to receive an advantage from the front-line chemotherapy-based regimen. Finally, an NLR score (a well-known inflammatory index) ≥ 4 seemed to be related to shorter OS among the pts treated with immunotherapy alone or in combination with chemotherapy (HR: 2.83, 95% CI: 1.08-7.40, p = 0.033). Prospective evaluations need to be performed to clarify whether these indexes may help to identify patients with oncogene-addicted NSCLC who could benefit from immunotherapy.

Introduction: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples., Methods: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated., Results: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation., Conclusions: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice., (© 2024. The Author(s).)

(1) Background: BRAF mutations affect 4-5% of lung adenocarcinomas. This study aimed to analyze the clinicopathological features of lung carcinomas with BRAF mutations, focusing on V600E vs. non-V600E and the presence of co-mutations. (2) Methods: All BRAF-mutated lung carcinomas were retrieved from a molecular diagnostic unit (the reference unit for four different hospitals). The samples were analyzed using next-generation sequencing. Statistical analyses included log-rank tests for overall survival (OS) and progression-free survival (PFS). (3) Results: In total, 60 BRAF-mutated lung carcinomas were retrieved: 24 (40.0%) with V600E and 36 (60.0%) with non-V600E mutations, and 21 (35.0%) with other co-mutations and 39 (65.0%) with only BRAF mutations. Survival data were available for 54/60 (90.0%) cases. Targeted therapy was documented in 11 cases. Patients with V600E mutations exhibited a better prognosis than patients with non-V600E mutations ( p = 0.008 for OS, p = 0.018 for PFS); this was confirmed in PFS ( p = 0.036) when considering only patients who received no targeted therapy. Patients with co-mutations displayed no prognostic difference compared to patients carrying only BRAF mutations ( p = 0.590 for OS, p = 0.938 for PFS). (4) Conclusions: BRAF-mutated lung carcinomas with V600E (40.0%) had a better prognosis than those without V600E. Concomitant co-mutations (35.0%) did not affect the prognosis.

Lung cancer (LC) is the deadliest malignancy worldwide. In an operable stage I-III patient setting, the detection of minimal residual disease (MRD) after curative treatment could identify patients at higher risk of relapse. In this context, the study of circulating tumor DNA (ctDNA) is emerging as a useful tool to identify patients who could benefit from an adjuvant treatment, and patients who could avoid adverse events related to a more aggressive clinical management. On the other hand, ctDNA profiling presents technical, biological and standardization challenges before entering clinical practice as a decisional tool. In this paper, we review the latest advances regarding the role of ctDNA in identifying MRD and in predicting patients' prognosis, with a particular focus on clinical trials investigating the potential of ctDNA, the technical challenges to address and the biological parameters that influence the MRD detection.

Purpose: Plasma circulating tumor DNA (ctDNA) is a valuable resource for tumor characterization and for monitoring of residual disease during treatment; however, it is not yet introduced in metastatic colorectal cancer (mCRC) routine clinical practice. In this retrospective exploratory study, we evaluated the role of ctDNA in patients with mCRC treated with chemotherapy plus bevacizumab., Materials and Methods: Fifty-three patients were characterized for RAS and BRAF status on tumor tissue before the start of treatment. Plasma was collected at baseline, at first clinical evaluation, and at disease progression. ctDNA analysis was performed using Oncomine Colon cfDNA Assay on the Ion S5 XL instrument., Results: At baseline, from a plasma sample, RAS , BRAF , or PIK3CA mutations were detected in 44 patients. A high correspondence was observed between ctDNA and tumor tissue mutations ( KRAS 100%, NRAS 97.9%, BRAF 97.9%, PIK3CA 90%). Low baseline variant allele frequency (VAF) was found to be associated with longer median progression-free survival (PFS) compared with those with high VAF (15.9 v 12.2 months, P = .02). A higher PFS {12.29 months (95% CI, 9.03 to 17.9) v 8.15 months (95% CI, 2.76 to not available [NA]), P = .04} and overall survival (34.1 months [95% CI, 21.68 to NA] v 11.1 months [95% CI, 3.71 to NA], P = .003) were observed in patients with large decline in VAF at first evaluation., Conclusion: ctDNA analysis is useful for molecular characterization and tumor response monitoring in patients with mCRC. Quantitative variations of released ctDNA are associated with clinical outcomes.

Introduction: Neuroendocrine neoplasms (NENs) are a rare group of tumors exceptionally heterogeneous, with clinical presentation ranging from well differentiated more indolent tumors to poorly differentiated very aggressive forms. Both are often diagnosed after the metastatic spread and require appropriate medical treatment. A high priority need in the management of this disease is the identification of effective therapeutic strategies for advanced and metastatic patients. The recent TALENT trial demonstrated the efficacy of lenvatinib, a multi-tyrosine kinase inhibitor, in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with no other treatment indication. Further development of this drug in advanced NETs is warranted., Methods: We investigated potential clinical and molecular determinants of lenvatinib response in human primary cultures derived from patients with GEP-NET of different grades and sites of origin. We correlated response to treatment with patient clinical characteristics, with the mutational status of 161-cancer associated genes and with the expression levels of MKI-related genes., Results: Lenvatinib exerted a significant antitumor activity in primary GEP-NET cells, with median survival inhibitions similar or higher than those of standard frontline treatments. Of the 11 primary cultures analyzed in our case series, 6 were classified as responder showing a significant survival inhibition, and 5 as non-responder. We observed that the overexpression of HRAS in the original tumor tissue compared to the matched healthy tissue significantly correlated with responsiveness of primary cells to lenvatinib (p=.048). All 5 non-responder cultures showed normal HRAS expression, while of the 6 responder cultures, 4 had HRAS overexpression. Overexpression of HRAS was not associated with gene mutation. None of the other evaluated clinical variables (grade, Ki67, site of origin and syndromic disease) or molecular markers correlated with response., Discussion: Lenvatinib appears to be a highly effective drug for the treatment of NETs. The evaluation of HRAS expression in the tumor tissue might improve patient selection and optimize therapeutic outcome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liverani, Spadazzi, Ibrahim, Pieri, Foca, Calabrese, De Vita, Miserocchi, Cocchi, Vanni, Ercolani, Cavaliere, Ranallo, Chiadini, Prisinzano, Severi, Sansovini, Martinelli, Bongiovanni and Mercatali.)

Aims: Next-generation sequencing (NGS) is becoming a new gold standard for determining molecular predictive biomarkers. This study aimed to evaluate the reliability of NGS in detecting gene fusions, focusing on comparing gene fusions with known and unknown partners., Methods: We collected all gene fusions from a consecutive case series using an amplicon-based DNA/RNA NGS platform and subdivided them into two groups: gene fusions with known partners and gene fusions with unknown partners. Gene fusions involving ALK, ROS1 and RET were also examined by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH)., Results: Overall, 1174 malignancies underwent NGS analysis. NGS detected gene fusions in 67 cases (5.7%), further subdivided into 43 (64.2%) with known partners and 24 (35.8%) with unknown partners. Gene fusions were predominantly found in non-small cell lung carcinomas (52/67, 77.6%). Gene fusions with known partners frequently involved ALK (20/43, 46.5%) and MET (9/43, 20.9%), while gene fusions with unknown partners mostly involved RET (18/24, 75.0%). FISH/IHC confirmed rearrangement status in most (89.3%) of the gene fusions with known partners, but in only one (4.8%) of the gene fusions with unknown partners, with a significant difference (p < 0.001). In 17 patients undergoing targeted therapy, the log-rank test revealed that the overall survival was higher in the known partner group than in the unknown partner group (p = 0.002)., Conclusions: NGS is a reliable method for detecting gene fusions with known partners, but it is less accurate in identifying gene fusions with unknown partners, for which further analyses (such as FISH) are required., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Endometriosis is a benign condition characterized by the presence of ectopic endometrial tissue. It is still debated whether endometriosis is a disease that can predispose to the pathogenesis of endometrial cancer outside the uterus. Deficiencies in mismatch repair (MMR) genes are a known risk factor for developing endometrioid cancer. Starting from two cases of patients with abnormal MMR endometrioid carcinoma of the uterus and synchronous endometrioid carcinoma in non-ovarian and ovarian endometriosis, we performed a somatic mutation profile and phylogenetic analysis of the lesions in order to identify if they were metastasis or primary de novo tumors. In the first case, we identified de novo activating mutations in PIK3CA and KRAS in endometrioid cancer lesions but not in endometriosis. Although the acquisition of a de novo mutation in ESR1 and a decrease in mutant allele fraction (MAF) from the endometrial tumor to the localizations in the endometriosis lesions, the clonal relationship was confirmed by the limited number of heteroplasmic mutations in D-loop mitochondrial DNA region. In the other case, the clonal behavior was demonstrated by the overlap of MAF at each site. Our data support the hypothesis of a retrograde dissemination of tumor cells, moving from the primary carcinoma in the endometrium to ectopic sites of endometriosis where localizations of tumor arise., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Farolfi, Altavilla, Morandi, Capelli, Chiadini, Prisinzano, Gurioli, Molari, Calistri, Foschini and De Giorgi.)

Background: Liquid biopsy analysis for EGFR detection in cell-free DNA (cfDNA) from NSCLC patients has become routine. The aim of this study was to explore its applicability in clinical practice., Methods: We collected data of EGFR-mutated NSCLC patients with liquid biopsy analysis. Data included test timing, concomitant tissue re-biopsy, therapy change, histology, stage, smoking habits, gender and age. All analyses were performed via a real-time PCR method to analyze EGFR mutations at exons 18, 19, 20 and 21. Variant allele frequency was performed for patients with available sequential EGFR mutation analysis in cfDNA. Overall survival was analyzed through the Kaplan-Meier method. We designed flow charts to show the real-life application of liquid biopsy., Results: We found that liquid biopsy is used in treatment-naïve patients as an alternative to EGFR detection in tumor tissue, and in patients with positive or negative EGFR from tumor biopsy. The majority of liquid biopsy analyses were performed in NSCLC patients who were disease progressive during TKI therapy. The presence of EGFR mutation in cfDNA was associated with a worse prognosis. In two patients, VAF of EGFR mutations in cfDNA was concordant with tumor volume changes., Conclusion: These findings suggest that liquid biopsy for EGFR detection can continue to be useful.

The mutational status of the epidermal growth factor receptor (EGFR) guides the stratification of non-small cell lung cancer (NSCLC) patients for treatment with tyrosine kinase inhibitors (TKIs). A liquid biopsy test on cell-free DNA is recommended as a clinical decision-supporting tool, although it has limited sensitivity. Here, we comparatively investigated the extracellular vesicle (EV)-RNA as an independent source for multidimensional and longitudinal EGFR profiling in a cohort of 27 NSCLC patients. We introduced and validated a new rapid, highly specific EV-RNA test with wild-type (WT) and mutant-sensitive probes (E746-A750del, L858R, and T790M). We included a cohort of 20 NSCLC patients with EGFR WT tumor tissues and systematically performed molecular EV-RNA and circulating tumor DNA analyses with clinical data statistics and biophysical profiles of EVs. At the single-patient level, we detected variegated tumor heterogeneity dynamics supported by combinations of driver EGFR mutations. EV-RNA-based mutation analysis showed an unprecedented sensitivity of over 90%. The resistance-associated mutation T790M frequently pre-existed at baseline with a gained EV-transcript copy number at progression, while the general mutational burden was mostly decreasing during the intermediate follow-up. The biophysical profile of EVs and the quantitative assessment of T790M revealed an association with tumor size determined by the sum of the longest diameters in target lesions. Vesicular RNA provides a validated tool suitable for use in clinical practice to investigate the dynamics of common driver EGFR mutations in NSCLC patients receiving TKIs., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The aim of our study was to assess the quality of Tanzanian cervical cancer specimens, evaluating telomerase alterations and human papilloma virus (HPV) infection in relation to histopathological characteristics since these biomarkers are not routinely analyzed. Thirty-two Tanzanian women with invasive cervical cancer were included in the study. Histopathological classification and all the analyses on tissue, including TERT immunohistochemistry, were performed at IRST IRCCS (Meldola, Italy). HPV typization was performed by pyrosequencing. FHACT™ was used to identify chromosomal aberrations. Nonparametric ranking statistics were used. The majority (75 %) of the cases analyzed were squamous carcinoma, while 12.5 % were adenocarcinoma. The presence of HPV infection was confirmed in 26/27 (96.3 %) cases. A high percentage of patients (88 %) were infected with HPV16 of whom 12 (44.4 %) with African type 1, and 4 (14.8 %) with African type 2. TERT expression evaluated in the entire case series showed a median H-score of 130 (range 3-270), with only one negative case. 88 % of the FISH-evaluable samples showed an amplification of the chromosomal region 3q26 (TERC) and/or 5p15, and 20q13, associated with a higher median expression of TERT (P = 0.0226). Despite pre-analytical problems in terms of sample fixation, we showed that the search for biomarkers such as HPV and telomerase is feasible in Tanzanian tissue. These markers could be important risk-stratification tools in this population., (Copyright © 2021. Published by Elsevier GmbH.)

Urinary cell-free DNA offers an important noninvasive source of material for genomic testing also for nonurological tumors. Its clinical utility in monitoring tumor evolution and treatment failure is promising. Here we describe a method to detect cancer mutations into urine from patients affected by colorectal cancer.

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.

Background: Non-small cell lung cancer (NSCLC) is the primary cause of cancer-related deaths worldwide. Epidermal Growth Factor Receptor ( EGFR )-mutated patients usually benefit from TKIs treatment, but a significant portion show unresponsiveness due to primary resistance mechanisms. We investigated the role of TP53 mutations in predicting survival and response to EGFR -TKIs in EGFR-mutated NSCLC patients, to confirm, on an independent case series, our previous results., Methods: An independent retrospective cohort study was conducted, on a case series of 136 EGFR -mutated NSCLC patients receiving first or second generation TKIs as a first line therapy, and a smaller fraction of patients who acquired the T790M resistance mutation and were treated with third generation TKIs in the second or further line of treatment. TP53 mutations were evaluated in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) of the patients., Results: Forty-two patients (30.9%) showed a TP53 mutation. Considered together, TP53 mutations had no significant impact on time-to-event endpoints. Considering the different TP53 mutations separately, exon 8 mutations confirmed their negative effect on PFS (HR 3.16, 95% 1.59-6.28, p = 0.001). In patients who developed the T790M resistance mutation, treated with third generation TKIs, the TP53 exon 8 mutations predicted worse PFS (even though not statistically significant), and OS (HR 4.86, 95% CI: 1.25-18.90, p = 0.023)., Conclusions: TP53 exon 8 mutations confirmed their negative prognostic impact in patients treated with first and second generation TKIs and demonstrated a role in affecting clinical outcome in patients treated with third generation TKIs., Competing Interests: The authors declare no conflict of interest.

The identification of the mutations that drive lung cancer have furnished new targets for the treatment of non-small cell lung cancer (NSCLC) and led to the development of targeted therapies such as tyrosine kinase inhibitors that are used to combat the molecular changes promoting cancer progression. Furthermore, biomarkers identified from gene analysis can be used to detect early lung cancer, determine patient prognosis, and monitor response to therapy. In the present study we analyzed the molecular profile of seventy-three Tunisian patients with lung adenocarcinoma (LAD). Mutational analyses for EGFR and KRAS were performed using direct sequencing, immunohistochemistry or MassARRAY. Anaplastic lymphoma kinase (ALK) rearrangement was evaluated by immunohistochemistry using the D5F3 clone, and p53 expression was also assessed. The median age of patients at diagnosis was 61 years (range 23-82 years). Using different methodologies, EGFR mutations were found in 5.47% of patients and only exon 19 deletions "E746-A750 del" were detected. KRAS mutations were present in 9.58% of cases, while only one patient was ALK-positive. Moreover, abnormal immunostaining of p53 was detected in 56.16% of patients. In conclusion, the detected rates of EGFR and KRAS mutation and ALK rearrangement were lower than those found in European and Asian countries, whereas, abnormal p53 expression was slightly more frequent. Furthermore, given the small sample size of this study, a more comprehensive analysis of this patient set is warranted.

Background: Molecular diagnostics for non-small cell lung cancer (NSCLC) has become the standard of care for personalized treatment. Epidermal growth factor receptor ( EGFR ) mutation and EML4-ALK translocation represent the two most important alterations in first-line treatment decision-making. However, other potentially targetable alterations are also present., Methods: One thousand consecutive NSCLC patients with EGFR wild type (wt) tumors diagnosed by routine molecular analysis were considered. KRAS , BRAF , ERBB2 , PIK3CA , NRAS , ALK , MAP2K1 , RET and DDR2 gene mutations were analyzed using the multiparametric Sequenom MassARRAY ® platform. EML4-ALK and ROS1 rearrangements were also assessed by fluorescent in situ hybridization. HER4 status was determined by direct sequencing., Results: Three hundred and forty-eight (34.8%), 31 (3.1%), 39 (4.4%), 14 (1.8%), 6 (0.7%), 16 (1.8%), 5 (0.6%) and 9 (0.9%) patients showed an alteration in KRAS , BRAF , ALK , ROS1 , NRAS , PIK3CA , MAPK1 /2 and HER2 genes, respectively. Of the 657 patients for whom all markers were determined, 318 (48%) patients had at least one alteration. Eight patients showed overlapping mutations, 4 KRAS mutation/ EML4-ALK translocation, one KRAS mutation/ ROS1 rearrangement, 2 KRAS/PIK3CA mutations, and one BRAF/PIK3CA mutations., Conclusions: About 50% of our patients had a potentially targetable alteration, confirming the usefulness of a multiparametric approach for routine molecular diagnostics aimed at identifying potential therapeutic targets., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

There is evidence of a different response to treatment with regard to the primary tumor localization (right-sided or left-sided) in patients with metastatic colorectal cancer (mCRC). We analyzed the different outcomes and biomolecular characteristics in relation to tumor localization in 122 of the 370 patients with metastatic colorectal cancer enrolled onto the phase III prospective multicenter "Italian Trial in Advanced Colorectal Cancer (ITACa)", randomized to receive first-line chemotherapy (CT) or CT plus bevacizumab (CT + B). RAS and BRAF mutations; baseline expression levels of circulating vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX2), ephrin type-B receptor 4 (EPHB4), hypoxia-inducible factor 1-alpha (HIF-1α), lactate dehydrogenase (LDH), and high-sensitivity C reactive protein (hs-CRP); and inflammatory indexes such as the neutrophil-to-lymphocyte ratio, platelet-lymphocyte rate and systemic immune-inflammation index were evaluated. Patients with right-sided tumors showed a longer median progression-free survival in the CT + B arm than in the CT group (12.6 vs. 9.0 months, respectively, p = 0.017). Baseline inflammatory indexes were significantly higher in left-sided tumors, whereas eNOS and EPHB4 expression was significantly higher and BRAF mutation more frequent in right-sided tumors. Our data suggest a greater efficacy of the CT + B combination in right-sided mCRC, which might be attributable to the lower inflammatory status and higher expression of pro-angiogenic factors that appear to characterize these tumors., Competing Interests: The authors declare no conflict of interest.

Purpose: To analyze the impact of TP53 mutations on response to first-line tyrosine kinase inhibitors (TKI) in patients with EGFR -mutated non-small cell lung cancer (NSCLC). Experimental Design: 136 EGFR -mutated NSCLC patients receiving first-line TKIs were analyzed. TP53 mutations were evaluated in 123 patients in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Results: TP53 mutations were observed in 37 (30.1%), 10 (27.0%), 6 (16.2%), 9 (24.3%), and 12 (32.4%) patients in exons 5, 6, 7, and 8, respectively. DCR was 70% in TP53 -mutated patients compared with 88% in TP53 -wild type (wt) patients [relative risk, RR, of disease progression: 3.17 (95% CI, 1.21-8.48), P = 0.019]. In particular, a 42% DCR was observed in patients with TP53 exon 8 mutation versus 87% in exon 8 wt patients [RR of disease progression 9.6 (2.71-36.63), P < 0.001]. Shorter median PFS and OS were observed in patients with TP53 exon 8 mutations compared with others (4.2 vs. 12.5, P = 0.058, and 16.2 vs. 32.3, P = 0.114, respectively); these differences became significant in the subgroup with EGFR exon 19 deletion (4.2 vs. 16.8, P < 0.001, and 7.6 vs. not reached, P = 0.006, respectively), HR 6.99 (95% CI, 2.34-20.87, P < 0.001) and HR 4.75 (95% CI, 1.38-16.29, P = 0.013), respectively. Conclusions: TP53 mutations, especially exon 8 mutations, reduce responsiveness to TKIs and worsen prognosis in EGFR -mutated NSCLC patients, mainly those carrying exon 19 deletions. Clin Cancer Res; 23(9); 2195-202. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Objectives: Amplification of human telomerase is known to be associated with cervical tumorigenesis, although its role in tumor progression of cervical lesions is still unclear. We aimed to evaluate the role of telomerase in predicting the evolution of cervical lesions., Methods: A total of 50 tissue samples taken by biopsy or conization once or repeatedly from 17 patients with cervical lesions over a 14-year follow-up was analyzed using fluorescence in situ hybridization (FISH) for hTERC gene alterations and immunohistochemistry (IHC) for hTERT expression. The accuracy of the biomarkers was measured using the area under the curve., Results: Telomerase gene amplification is highly indicative of cervical lesion evolution and seems to be a more reliable biomarker than the protein expression detected by IHC. In fact, patients with benign lesions or cervical intraepithelial lesions (CINs) showing hTERC amplification relapsed or progressed into CIN 2 and CIN 3 more frequently than those without any gene amplification. FISH and IHC assays had both 86% sensitivity on conized material and 78% and 40% specificity, respectively., Conclusions: We demonstrated that the most accurate method to evaluate telomerase alterations as prognostic markers in cervical lesions was FISH assay on hTERC gene. The best accuracy was obtained using conized materials.

Background: Epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, and echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation are generally considered to be mutually exclusive. However, concomitant mutations are found in a small number of patients and the effect of these on response to targeted therapy is still unknown., Patients and Methods: We considered 380 non-small-cell lung cancer (NSCLC) patients who underwent nonsequential testing for EGFR and EML4-ALK translocation. KRAS mutation analysis was also performed on 282 patients., Results: We found 1.6%, 1.1%, and 2.5% of patients who showed a double mutation comprising EGFR and EML4-ALK, EGFR and KRAS, and EML4-ALK and KRAS, respectively. Twenty-eight patients with EGFR mutation underwent first-line therapy with a tyrosine kinase receptor; a clinical benefit was observed in 81.8% of patients with EGFR mutations only and in 67% of those who also showed an EML4-ALK translocation. Twelve patients with an EML4-ALK translocation received crizotinib and 7 of these had disease progression within 3 months (2 had a concomitant KRAS mutation and 1 had a concomitant EGFR mutation). Two patients showed stable disease, 1 of whom also had a KRAS mutation. Two patients obtained a partial response and 1 had a complete response; all harbored an EML4-ALK translocation only. The median overall survival of patients who carried an EML4-ALK translocation alone or concomitant with a KRAS mutation was 57.1 (range, 10.7-not reached) and 10.7 (range, 4.6-not reached) months, respectively., Conclusion: Concomitant EGFR, EML4-ALK, or KRAS mutations can occur in NSCLC. Concomitant KRAS mutation and EML4-ALK translocation represents the most common double alteration and confers a poor prognosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Sorafenib is a multi-targeted kinase inhibitor with a demonstrated activity in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC), and it is currently used for the treatment of these pathologies. Ongoing clinical trials are studying its activity in other malignancies, such as non-small-cell lung cancer (NSCLC). However, no biological marker is known to define either the sensitivity or resistance to the drug., Case Presentation: Here we report a case of a patient with two synchronous tumors, HCC and NSCLC, with metastases in the contralateral lung and bone. The patient was treated with gemcitabine as first line, with a resulting progressive disease after two months, and then with sorafenib at standard dosage in the second line setting. After 6 months of treatment CT scan showed a partial response in the primary lesion of the lung, complete response of the metastasis in the contralateral lung, and stability of HCC. The patient had progression in the lung, liver and bone after 13 months of therapy. A molecular characterization of NSCLC and HCC lesions was performed, revealing a BRAF exon 11 mutation (G469V) only in NSCLC. We hypothesize that the response observed in NSCLC lesions could be due to the presence of BRAF mutation, and that this alteration could be responsible in determining sorafenib sensitivity., Conclusions: Results observed in this case encourage further research on the activity of sorafenib in both HCC and NSCLC, based on the presence of BRAF mutation. This could lead to a selection of HCC patients to be treated with this drug, and could help identify a novel treatment strategy for BRAF-mutated NSCLC patients.

Targeted therapy of metastatic colorectal cancer (mCRC) with monoclonal antibody anti-epidermal growth factor receptor (EGFR) agents, such as cetuximab (CTX) or panitumumab, is the treatment strategy of choice in patients characterised by a wild-type (wt) RAS gene status. However, despite selection based on RAS status, a high proportion of patients do not respond to therapy. EGFR methylation has been reported to have a role in predicting the response to anti-EGFR agents. The present study aimed to evaluate the role of EGFR methylation in association with the clinical outcome of patients with mCRC treated with CTX. In total, 64 patients with mCRC were assessed in the present study. Genomic DNA was extracted from tumoral tissue and EGFR methylation and mutation of the KRAS , BRAF and PIK3CA genes were analysed by pyrosequencing. EGFR expression was assessed by immunohistochemistry. The various alterations were analysed by assessing the objective response rate (ORR), progression free survival (PFS) and overall survival (OS) rates. In total, 42 cases (66%) exhibited >10% EGFR methylation and there was no correlation with EGFR expression. Mean EGFR methylation of 41 and 9% was observed in KRAS -mutated and -wt patients, respectively (P=0.05). Conversely, a high EGFR methylation was observed in BRAF -wt patients with compared with patients possessing the mutated gene (18 vs. 3%, respectively; P=0.07). EGFR methylation was significantly correlated with the OS rate [hazard ratio, 0.98; 95% confidence interval (CI), 0.96-1.00; P=0.019], but not PFS rate. In patients with a methylation rate <10 and >10%, the median OS rate was 7.5 months (95% CI, 4.4-9.4 months) and 12.0 months (95% CI, 8.7-13.9 months), respectively (P=0.034). In conclusion, the present study revealed a correlation between EGFR methylation and improved OS rate in patients treated with CTX-based chemotherapy. The presence of EGFR methylation is inversely correlated with BRAF and PIK3CA mutations, indicating that the prognostic value of gene methylation may be worth verifying in further studies.

Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity.

Introduction: Tyrosine kinase inhibitors (TKIs) and anti-anaplastic lymphoma kinase (ALK) agents are highly effective for the treatment of non-small cell lung cancer (NSCLC) patients harbouring specific alterations, and molecular characterisation of the tumour is needed even when limited tumour material is available., Methods: 20 patients with a known epidermal growth factor receptor (EGFR) gene status were enrolled: 10 had mutated and 10 had wild type tumours. FISH analysis was performed on one cytological or histological sample to determine EML4-ALK status, after which the same cells scraped off each slide were used to evaluate the EGFR status., Results: In the 10 EGFR mutated patients, molecular analysis showed the same results as those obtained before the FISH test. One patient with an EGFR mutation also showed an EML4-ALK translocation, and both FISH-positive and FISH-negative cells maintained the EGFR mutation., Conclusions: EGFR mutation analysis can be performed on the same sample previously submitted to the EML4-ALK FISH procedure.

The characterization of circulating tumor cells (CTCs) could substantially improve the management of cancer patients. However, their study is still a matter of debate, often due to lymphocyte contamination. In the present paper, an investigation of CTCs was carried out for the first time using DEPArray, a dielectrophoresis-based platform able to detect and sort pure CTCs. Analyses were conducted on peripheral blood (PB) samples from patients with metastatic colon cancer. After 100% pure cell recovery and whole genome amplification, KRAS gene mutation of CTCs was screened and compared to gene status in the primary tumor tissue. CTCs were found in 21 colon cancer patients (52.5%), with more than three tumor cells per 7.5 ml. KRAS gene mutation analysis, showed a mutational concordance between CTCs and primary tumor in 50% of matched cases. The present study demonstrates for the first time the feasibility of analyzing at the molecular level pure CTCs avoiding lymphocyte contamination using an innovative instrumentation, and a KRAS discordance between CTCs and primary tissue. Our results present dielectrophoresis-based procedures as a new standard in single cell analysis and recovery and invite careful reflection on the value of CTCs characterization., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

In non-small cell lung cancer (NSCLC) patients, somatic EGFR and K-ras mutations predict therapeutic effectiveness and resistance, respectively, to EGFR tyrosine kinase inhibitors (TKIs). Transesophageal ultrasound-guided fine needle aspiration (EUS-FNA) is a validated technique for diagnosis and staging of NSCLC. In the present study, we compared the feasibility and reliability of EGFR and K-ras gene mutation analysis in fixed and fresh mediastinal lymph nodes and extra-lymph nodal samples obtained by EUS-FNA in patients suspicious for NSCLC. Thirty-six patients were enrolled into the study. For each patient, DNA was extracted from both fresh samples and fixed cytological smears. Exons 18-21 of EGFR and exon 2 of K-ras were amplified by PCR and mutation status was determined by direct sequencing and pyrosequencing. All cases were eligible for analysis. NSCLC was diagnosed in 32 patients (25 adenocarcinomas and 7 squamous cell carcinomas) and 4 patients were free of malignancy. Of the 25 patients with adenocarcinoma, EGFR mutations were detected in 2 (8%) fresh tumor samples and in 3 (12%) fixed cytological smears. K-ras mutations were detected in 8 (32%) fresh samples, and in 9 (36%) fixed cytological smears. Fixed and stained cytological samples seem to be more reliable than fresh material for molecular analysis.