SOX11 expression is restricted to EBV-negative Burkitt lymphoma and associates with molecular genetic features.

SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B-cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV- BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, while in EBV-/SOX11- tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11 expressing BL showed higher frequency of SMARCA4 and ID3 mutations compared to EBV-/SOX11- cases. By RNA-sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or BCR signaling were found between SOX11- and SOX11+ BL cells. However, SOX11+ BL showed higher adhesion to VCAM-1 than SOX11- BL cell lines. Here we demonstrate that EBV- BL comprises two subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

The C250T Mutation of TERTp Might Grant a Better Prognosis to Glioblastoma by Exerting Less Biological Effect on Telomeres and Chromosomes Than the C228T Mutation.

The aim of this study was to determine how TERTp mutations impact glioblastoma prognosis. MATERIALS AND METHODS: TERTp mutations were assessed in a retrospective cohort of 258 uniformly treated glioblastoma patients. RNA-sequencing and whole exome sequencing results were available in a subset of patients. RESULTS: Overall, there were no differences in outcomes between patients with mutated TERTp-wt or TERTp. However, we found significant differences according to the type of TERTp mutation. Progression-free survival (mPFS) was 9.1 months for those with the C250T mutation and 7 months for those with either the C228T mutation or TERTp-wt (p = 0.016). Overall survival (mOS) was 21.9 and 15 months, respectively (p = 0.026). This differential effect was more pronounced in patients with MGMTp methylation (mPFS: p = 0.008; mOS: p = 0.021). Multivariate analysis identified the C250T mutation as an independent prognostic factor for longer mOS (HR 0.69; p = 0.044). We found no differences according to TERTp mutation status in molecular alterations common in glioblastoma, nor in copy number variants in genes related to alternative lengthening of telomeres. Nevertheless, in the gene enrichment analysis adjusted for MGMTp methylation status, some Reactome gene sets were differentially enriched, suggesting that the C250T mutation may exert a lesser effect on telomeres or chromosomes. CONCLUSIONS: In our series, patients exhibiting the C250T mutation had a more favorable prognosis compared to those with either TERPp-wt or TERTp C228T mutations. Additionally, our findings suggest a reduced involvement of the C250T mutation in the underlying biological mechanisms related to telomeres.

Phase II trial of afatinib in patients with advanced urothelial carcinoma with genetic alterations in ERBB1-3 (LUX-Bladder 1).

BACKGROUND: Preclinical and early clinical data suggest that the irreversible ErbB family blocker afatinib may be effective in urothelial cancers harbouring ERBB mutations. METHODS: This open-label, phase II, single-arm trial (LUX-Bladder 1, NCT02780687) assessed the efficacy and safety of second-line afatinib 40 mg/d in patients with metastatic urothelial carcinoma with ERBB1-3 alterations. The primary endpoint was 6-month progression-free survival rate (PFS6) (cohort A); other endpoints included ORR, PFS, OS, DCR and safety (cohorts A and B). Cohort A was planned to have two stages: stage 2 enrolment was based on observed antitumour activity. RESULTS: Thirty-four patients were enroled into cohort A and eight into cohort B. In cohorts A/B, PFS6 was 11.8%/12.5%, ORR was 5.9%/12.5%, DCR was 50.0%/25.0%, median PFS was 9.8/7.8 weeks and median OS was 30.1/29.6 weeks. Three patients (two ERBB2-amplified [cohort A]; one EGFR-amplified [cohort B]) achieved partial responses. Stage 2 for cohort A did not proceed. All patients experienced adverse events (AEs), most commonly (any/grade 3) diarrhoea (76.2%/9.5%). Two patients (4.8%) discontinued due to AEs and one fatal AE was observed (acute coronary syndrome; not considered treatment-related). CONCLUSIONS: An exploratory biomarker analysis suggested that basal-squamous tumours and ERBB2 amplification were associated with superior response to afatinib. CLINICAL TRIAL REGISTRATION: NCT02780687.

MALAT1 expression is associated with aggressive behavior in indolent B-cell neoplasms.

MALAT1 long non-coding RNA has oncogenic roles but has been poorly studied in indolent B-cell neoplasms. Here, MALAT1 expression was analyzed using RNA-seq, microarrays or qRT-PCR in primary samples from clinico-biological subtypes of chronic lymphocytic leukemia (CLL, n = 266), paired Richter transformation (RT, n = 6) and follicular lymphoma (FL, n = 61). In peripheral blood (PB) CLL samples, high MALAT1 expression was associated with a significantly shorter time to treatment independently from other known prognostic factors. Coding genes expressed in association with MALAT1 in CLL were predominantly related to oncogenic pathways stimulated in the lymph node (LN) microenvironment. In RT paired samples, MALAT1 levels were lower, concordant with their acquired increased independency of external signals. Moreover, MALAT1 levels in paired PB/LN CLLs were similar, suggesting that the prognostic value of MALAT1 expression in PB is mirroring expression differences already present in LN. Similarly, high MALAT1 expression in FL predicted for a shorter progression-free survival, in association with expression pathways promoting FL pathogenesis. In summary, MALAT1 expression is related to pathophysiology and more aggressive clinical behavior of indolent B-cell neoplasms. Particularly in CLL, its levels could be a surrogate marker of the microenvironment stimulation and may contribute to refine the clinical management of these patients.

P53 in Penile Squamous Cell Carcinoma: A Pattern-Based Immunohistochemical Framework with Molecular Correlation.

p53 immunohistochemistry (IHC) has been proposed as a surrogate for TP53 mutations in penile squamous cell carcinomas (PSCC). We aimed to evaluate the performance of a pattern-based evaluation of p53 IHC in PSCC. Human papilloma virus (HPV) DNA testing, p16 and p53 IHC, and whole exome sequencing were performed in a series of 40 PSCC. p53 IHC was evaluated following a pattern-based framework and conventional p53 IHC evaluation. Out of 40 PSCC, 12 (30.0%) were HPV-associated, and 28 (70.0%) were HPV-independent. The agreement between the p53 IHC pattern-based evaluation and TP53 mutational status was almost perfect (k = 0.85). The sensitivity and accuracy of the pattern-based framework for identifying TP53 mutations were 95.5% and 92.5%, respectively, which were higher than the values of conventional p53 IHC interpretation (54.5% and 70.0%, respectively), whereas the specificity was the same (88.9%). In conclusions, the pattern-based framework improves the accuracy of detecting TP53 mutations in PSCC compared to the classical p53 IHC evaluation.

Tumorigenic role of Musashi-2 in aggressive mantle cell lymphoma.

SOX11 overexpression has been associated with aggressive behavior of mantle cell lymphomas (MCL). SOX11 is overexpressed in embryonic and cancer stem cells (CSC) of some tumors. Although CSC have been isolated from primary MCL, their relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown. Here, we observed enrichment in leukemic and hematopoietic stem cells gene signatures in SOX11+ compared to SOX11- MCL primary cases. Musashi-2 (MSI2) emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 is directly bound to the MSI2 promoter upregulating its expression in vitro. MSI2 intronic enhancers were strongly activated in SOX11+ MCL cell lines and primary cases. MSI2 upregulation was significantly associated with poor overall survival independently of other high-risk features of MCL. MSI2 knockdown decreased the expression of genes related to apoptosis and stem cell features and significantly reduced clonogenic growth, tumor cell survival and chemoresistance in MCL cells. MSI2-knockdown cells had reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cells in xenotransplanted mouse models. Our results suggest that MSI2 might play a key role in sustaining stemness and tumor cell survival, representing a possible novel target for therapeutic interventions in MCL.

Mutational Status of SMAD4 and FBXW7 Affects Clinical Outcome in TP53-Mutated Metastatic Colorectal Cancer.

Next-generation sequencing (NGS) provides a molecular rationale to inform prognostic stratification and to guide personalized treatment in cancer patients. Here, we determined the prognostic and predictive value of actionable mutated genes in metastatic colorectal cancer (mCRC). Among a total of 294 mCRC tumors examined by targeted NGS, 200 of them derived from patients treated with first-line chemotherapy plus/minus monoclonal antibodies were included in prognostic analyses. Discriminative performance was assessed by time-dependent estimates of the area under the curve (AUC). The most recurrently mutated genes were TP53 (64%), KRAS or NRAS (49%), PIK3CA (15%), SMAD4 (14%), BRAF (13%), and FBXW7 (9.5%). Mutations in FBXW7 correlated with worse OS rates (p = 0.036; HR, 2.24) independently of clinical factors. Concurrent mutations in TP53 and FBXW7 were associated with increased risk of death (p = 0.02; HR, 3.31) as well as double-mutated TP53 and SMAD4 (p = 0.03; HR, 2.91). Analysis of the MSK-IMPACT mCRC cohort (N = 1095 patients) confirmed the same prognostic trend for the previously identified mutated genes. Addition of the mutational status of these genes upon clinical factors resulted in a time-dependent AUC of 87%. Gene set enrichment analysis revealed specific molecular pathways associated with SMAD4 and FBXW7 mutations in TP53-defficient tumors. Conclusively, SMAD4 and FBXW7 mutations in TP53-altered tumors were predictive of a negative prognostic outcome in mCRC patients treated with first-line regimens.

Intrinsic subtypes and therapeutic decision-making in hormone receptor-positive/HER2-negative metastatic breast cancer with visceral crisis: A case report.

Background: CDK4/6 inhibitors (CDKi), namely, palbociclib, ribociclib, and abemaciclib, combined with either an aromatase inhibitor (AI) or fulvestrant are the standard first/second line for hormone receptor-positive(HR+)/HER2-negative(neg) metastatic breast cancer (MBC). However, the choice of one specific CDKi is arbitrary and based on the physician's experience with the drug, toxicity profile, and patient's preferences, whereas biomarkers for optimal patient selection have not been established so far. Moreover, upfront chemotherapy is still recommended in case of clinical presentation with visceral crisis, despite no evidence of superior benefit for chemotherapy regimens against CDKi-based regimens. Recent correlative biomarker analyses from pivotal trials of palbociclib and ribociclib showed that HR+/HER2-neg MBC might respond differently according to the molecular intrinsic subtype, with Luminal A and B tumors being sensitive to both CDKi, Basal-like being insensitive to endocrine therapy, irrespective of CDKi, and HER2-enriched tumors showing a benefit only with ribociclib-based therapy. Clinical case: We hereby present a paradigmatic clinical case of a woman affected by a relapsed HR+/HER2-neg MBC with bone and nodal lesions, presenting with a visceral crisis in the form of lymphangitis carcinomatosis and diagnosed with a molecularly HER2-enriched tumor, successfully treated with upfront ribociclib + fulvestrant. The patient experienced a complete symptomatic and radiologic remission of the lymphangitis with a partial response as best response, according to RECIST 1.1 criteria. The progression-free survival (PFS) was of 20 months, in line with the median PFS observed in the ribociclib + fulvestrant pivotal trial, where, however, patients with visceral crisis had been excluded. Conclusions: This clinical case confirms in the real-world setting that non-luminal subtypes can be found in HR+/HER2-neg disease and may have potential therapeutic implications in the metastatic setting. It also questions the recommendation of upfront chemotherapy in the case of a visceral crisis in the era of CDKi-based regimens. These issues merit further evaluation in prospective and larger studies.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

Comparison of the Idylla™ MSI assay with the Promega™ MSI Analysis System and immunohistochemistry on formalin-fixed paraffin-embedded tissue of endometrial carcinoma: results from an international, multicenter study.

Determination of microsatellite instability (MSI) and mismatch repair deficiency (MMRD), respectively, in endometrial carcinomas (ECs) is important for diagnostic and prognostic purposes, identification of Lynch syndrome carriers, and selection of patients for immunotherapy. The Idylla™ MSI assay is fully automated, does not require non-tumoral tissue, and can be performed in about 150 min. Two hundred forty-two formalin-fixed paraffin-embedded (FFPE) EC samples from 7 international centers were tested by the Idylla™ MSI assay and compared to the Promega™ MSI Analysis System and immunohistochemistry (IHC) for MMR proteins. The cases were selected with an enrichment of MSI EC to around 40%. Concordance was 87.5% between the Idylla™ MSI assay and IHC and 88.58% between IHC and Promega™ MSI assay. Concordance between Idylla™ and Promega™ MSI assays was 89.91%. Discordant results occurred more frequently in cases with MSH6 or PMS2 deficiency. Invalid cases occurred with the three techniques (IHC, 7.00%; Promega™ MSI assay, 5.37%; and Idylla™ MSI assay, 2.47%). The concordance rate between Idylla™ MSI assay and the other 2 methods increased to 88.83% for IHC and to 91.22% for the Promega™ MSI assay when the cutoff of instability in the scoring system was moved from 0.5 to 0.3. The Idylla™ MSI assay is a rapid and highly concordant test for MSI in EC. Modification of the Idylla™ scoring system could increase the sensitivity and specificity of the MSI assay for EC analysis.

Development and validation of the new HER2DX assay for predicting pathological response and survival outcome in early-stage HER2-positive breast cancer.

BACKGROUND: Both clinical and genomic data independently predict survival and treatment response in early-stage HER2-positive breast cancer. Here we present the development and validation of a new HER2DX risk score, and a new HER2DX pathological complete response (pCR) score, both based on a 27-gene expression plus clinical feature-based classifier. METHODS: HER2DX is a supervised learning algorithm incorporating tumour size, nodal staging, and 4 gene expression signatures tracking immune infiltration, tumour cell proliferation, luminal differentiation, and the expression of the HER2 amplicon, into a single score. 434 HER2-positive tumours from the Short-HER trial were used to train a prognostic risk model; 268 cases from an independent cohort were used to verify the accuracy of the HER2DX risk score. In addition, 116 cases treated with neoadjuvant anti-HER2-based chemotherapy were used to train a predictive model of pathological complete response (pCR); two independent cohorts of 91 and 67 cases were used to verify the accuracy of the HER2DX pCR likelihood score. Five publicly available independent datasets with >1,000 patients with early-stage HER2-positive disease were also analysed. FINDINGS: In Short-HER, HER2DX variables were associated with good risk outcomes (i.e., immune, and luminal) and poor risk outcomes (i.e., proliferation, and tumour and nodal staging). In an independent cohort, continuous HER2DX risk score was significantly associated with disease-free survival (DFS) (p=0·002); the 5-year DFS in the low-risk group was 97·4% (94·4-100·0%). For the neoadjuvant pCR predictor training cohort, HER2DX variables were associated with pCR (i.e., immune, proliferation and HER2 amplicon) and non-pCR (i.e., luminal, and tumour and nodal staging). In both independent test set cohorts, continuous HER2DX pCR likelihood score was significantly associated with pCR (p<0·0001). A weak negative correlation was found between the HER2DX risk score versus the pCR score (correlation coefficient -0·19). INTERPRETATION: The two HER2DX tests provide accurate estimates of the risk of recurrence, and the likelihood to achieve a pCR, in early-stage HER2-positive breast cancer. FUNDING: This study received funding from Reveal Genomics, IDIBAPS and the University of Padova.

Revised International Prognostic Index and genetic alterations are associated with early failure to R-CHOP in patients with diffuse large B-cell lymphoma.

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) cases have a poor outcome. Here we analysed clinico-biological features in 373 DLBCL patients homogeneously treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), in order to identify variables associated with early failure to treatment (EF), defined as primary refractoriness or relapse within 12 months from diagnosis. In addition to clinical features, mutational status of 106 genes was studied by targeted next-generation sequencing in 111 cases, copy number alterations in 87, and gene expression profile (GEP) in 39. Ninety-seven cases (26%) were identified as EF and showed significantly shorter overall survival (OS). Patients with B symptoms, advanced stage, high levels of serum lactate dehydrogenase (LDH) or ß2-microglobulin, low lymphocyte/monocyte ratio and higher Revised International Prognostic Index (R-IPI) scores, as well as those with BCL2 rearrangements more frequently showed EF, with R-IPI being the most important in logistic regression. Mutations in NOTCH2, gains in 5p15·33 (TERT), 12q13 (CDK2), 12q14·1 (CDK4) and 12q15 (MDM2) showed predictive importance for EF independently from R-IPI. GEP studies showed that EF cases were significantly enriched in sets related to cell cycle regulation and inflammatory response, while cases in response showed over-representation of gene sets related to extra-cellular matrix and tumour microenvironment.

Case Report: A Case Study Documenting the Activity of Atezolizumab in a PD-L1-Negative Triple-Negative Breast Cancer.

The immune checkpoint inhibitor atezolizumab is approved for PD-L1-positive triple-negative breast cancer (TNBC). However, no activity of atezolizumab in PD-L1-negative TNBC has been reported to date. Here, we present the case study of a woman with TNBC with low tumor infiltrating lymphocytes and PD-L1-negative disease, which achieved a significant response to atezolizumab monotherapy and durable response after the combination of atezolizumab and nab-paclitaxel. The comprehensive genomic analysis that we performed in her tumor and plasma samples revealed high tumor mutational burden (TMB), presence of the APOBEC genetic signatures, high expression of the tumor inflammation signature, and a HER2-enriched subtype by the PAM50 assay. Some of these biomarkers have been shown to independently predict response to immunotherapy in other tumors and may explain the durable response in our patient. Our work warrants further translational studies to identify biomarkers of response to immune checkpoint inhibitors in TNBC beyond PD-L1 expression and to better select patients that will benefit from immunotherapy.

Technical Evaluation of the COBAS EGFR Semiquantitative Index (SQI) for Plasma cfDNA Testing in NSCLC Patients with EGFR Exon 19 Deletions.

The cobas® EGFR Test provides a semiquantitative index (SQI) that reflects the proportion of mutated versus wild-type copies of the EGFR gene in plasma. The significance of SQI as an indirect measure of the variant allele frequency (VAF) or mutated copies/mL remains unclear. The aim of this study was to evaluate the correlation of SQI with the VAF and the number of mutated copies/mL obtained by a digital droplet PCR (ddPCR) test in NSCLC samples. The study included 118 plasma samples from a retrospective cohort of 25 stage IV adenocarcinoma patients with EGFR exon 19 deletions (Ex19Del), obtained before and during tyrosine kinase inhibitor (TKI) treatment. Both SQI and VAF and SQI and mutated copies/mL showed the same significant correlation (r2 = 0.79, p < 0.00001) across the whole study cohort. We found better correlation in samples collected at the baseline between SQI and VAF (r2 = 0.94, p < 0.00001) and SQI and mutated copies/mL (r2 = 0.97, p < 0.00001) compared to samples collected during TKI treatment: r2 = 0.76; p < 0.00001 for SQI and VAF and r2 = 0.75; p < 0.00001 for SQI and mutated copies/mL. The study indicates that SQI is a robust quantitative indirect measure of VAF and the number of mutated copies/mL in plasma from patients with an EGFR Ex19Del mutation. Further studies are desirable to assess the SQI cut-off values related to the clinical status of the patient.

Molecular Landscape of Vulvar Squamous Cell Carcinoma.

Vulvar squamous cell carcinoma (VSCC) is a rare malignancy with dual pathogenesis, Human papillomavirus (HPV)-associated and HPV-independent, with a poorly explored molecular landscape. We aimed to summarize the findings of the series analyzing molecular hallmarks of this neoplasm. In January 2021, we conducted a comprehensive literature search using Pubmed Medline and Scopus to identify publications focused on genomic profiling of VSCC. Observational studies, including both prospective and retrospective designs, evaluating molecular alterations in VSCC were deemed eligible. A total of 14 studies analyzing 749 VSCC were identified. The study series were heterogeneous in HPV testing and sequencing strategies, included small sets of tumors and cancer genes, and commonly lacked survival analysis. Only one extensive targeted next-generation sequencing-based study comprised a large cohort of 280 VSCC. The mutated genes, their number, and frequencies were highly variable between the series. Overall, TP53 and CDKN2A, followed by PIK3CA, HRAS, and PTEN, were the most frequently studied and mutated genes. Mutations involved in the PI3K/AKT/mTOR pathway, including TP53, HRAS, KRAS, and PIK3CA, have been consistently reported across the studies. However, the role of individual mutations or pathways in the development of VSCC remains unclear. In conclusion, heterogeneity and the small sample size of available molecular series contribute to a limited view of the molecular landscape of VSCC. Large-scale genome- or exome-wide studies with robust HPV testing are necessary to improve the molecular characterization of VSCC.

Molecular characterization of advanced non-small cell lung cancer patients by cfDNA analysis: experience from routine laboratory practice.

BACKGROUND: Analysis of circulating free DNA (cfDNA) by the real-time PCR cobas® EGFR Mutation Test v2 (cobas® EGFR Test) is a diagnostic approach used in clinical practice for the characterization of advanced non-small cell lung cancer (NSCLC) patients. The test additionally outputs a semiquantitative index (SQI) which reflects the proportion of mutated versus wild-type copies of the EGFR gene in cfDNA with potential use as a biomarker. CfDNA concentration and cfDNA fragmentation pattern have also shown potential utility as biomarkers for cancer patients. We evaluated the implementation of EGFR testing and cfDNA related parameters in NSCLC patients in routine clinical setting as biomarkers for disease stage and diagnosis. METHODS: A prospective cohort of 173 locally advanced or metastatic NSCLC TKI-naïve patients analyzed by the cobas® EGFR Test were included in the study. Reproducibility of the test was assessed in 56 patients. The concentration of cfDNA and fragment size pattern was measured using fluorometry and microchip electrophoresis respectively. RESULTS: The test showed high diagnostic accuracy when compared to the gold standard of biopsy tumor tissue testing. The SQI value showed a moderate reproducibility (r2=0.70) and did not correlate with cfDNA concentration (r2=0.17, P=0.28) or disease stage (stage III patients SQI =9.1±3.1 and stage IV patients SQI =11.5±4.8, P=0.41). We found differences in SQI values according to the type of EGFR mutation (Ex19Del mutations, SQI =13.6; p.L858R, SQI =8.88; P=0.001). Stage IV patients had higher concentrations of cfDNA (P<0.0001) and higher fractions of cfDNA 100-250 base pairs (bp) fragments (P=0.01) compared to stage III patients. From the ROC curve analysis, cfDNA concentration showed higher AUC compared to cfDNA 100-250 bp fragments (0.86 vs. 0.71). We obtained a cut-off value for cfDNA concentration of 20.3 ng/mL with 72.3% sensitivity and 95% specificity for predicting disease stage in TKI-naïve advanced NSCLC patients. CONCLUSIONS: The study indicates that cfDNA analysis in plasma for EGFR testing by RT-PCR is an accurate and fast method to initially stratify NSCLC patients in a real-world clinical setting. However, the SQI has limited clinical value. The cfDNA concentration and fragmentation pattern have clear potential clinical utility for tumor staging in NSCLC patients.

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non-Small Cell Lung Cancer.

Clinical guidelines promote the identification of several targetable biomarkers to drive treatment decisions in advanced non-small cell lung cancer (NSCLC), but half of all patients do not have a viable biopsy. Specimens from endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) are an alternative source of material for the initial diagnosis of NSCLC, however their usefulness for a complete molecular characterization remains controversial. EBUS-TBNA samples were prospectively tested for several biomarkers by next-generation sequencing (NGS), nCounter, and immunohistochemistry (PD-L1). The primary objectives were to assess the sensitivity of EBUS-TBNA samples for a comprehensive molecular characterization and to compare its performance to the reference standard of biopsy samples. Seventy-two EBUS-TBNA procedures were performed, and 42 NSCLC patients were diagnosed. Among all cytological samples, 92.9% were successfully genotyped by NGS, 95.2% by nCounter, and 100% by immunohistochemistry. There were 29 paired biopsy samples; 79.3% samples had enough tumor material for genomic genotyping, and 96.6% for PD-L1 immunohistochemistry. A good concordance was found between both sources of material: 88.9% for PD-L1, 100% for NGS and nCounter. EBUS-TBNA is a feasible alternative source of material for NSCLC genotyping and allows the identification of patient candidates for personalized therapies with high concordance when compared with biopsy.