Cancers of Unknown Primary Origin: Real-World Clinical Outcomes and Genomic Analysis at the European Institute of Oncology.

BACKGROUND: Cancer of unknown primary origin (CUP) poses a significant challenge due to poor clinical outcomes and limited treatment options. As such, further definition of clinicopathological factors and genomic profile to better adapt treatment strategies is required. METHODS: Medical records were interrogated to retrospectively include CUP with available clinical and genomics data at the European Institute of Oncology. Next-generation sequencing (NGS) included targeted panels. Statistical analyses were conducted with R Software 4.2.2. RESULTS: A total of 44 patients were included. With a median follow-up of 39.46 months (interquartile range [IQR] 35.98-47.41 months), median PFS (mPFS) to first-line regimen was 3.98 months (95% CI 3.22-5.98), with a clinical benefit rate of 26% (95% CI 14%-49%), and disease control rate (DCR) limited to 48.28%. Most patients (26 of 31, 83.87%) received platinum-doublet chemotherapy, with no statistically significant difference between first-line treatment regimens. Median OS (mOS) was 18.8 months (95% CI 12.3-39.9), with a 12-month OS rate of 66% (95% CI 50%-85%). All patients received comprehensive genomic profiling (CGP). For 11 patients, NGS was unsuccessful due to low sample quantity and/or quality. For the remaining, TP53 (n = 16, 48%) and KRAS (n = 10, 30%) represented the most altered (alt) genes. No microsatellite instability was observed (0 of 28), while 6 of 28 (21.43%) tumors carried high TMB (≥10 mutation per megabase). Eight of 33 tumors (24.2%) displayed at least one actionable alteration with potential clinical benefit according to ESCAT. Only 2 of them received targeted therapy matched to genomic alterations, with a combined mPFS of 2.63 months (95% CI 1.84-not evaluable) as third-line regimens. Six patients received anti-PD1/PD-L1 therapy, showing a meaningful mPFS of 13 months (95% CI 2.04-not evaluable). CONCLUSION: CUP exhibits poor prognosis with limited benefits from standard treatment regimens. A significant proportion of CUPs carry actionable alterations, underscoring the importance of genomic profiling to gather additional treatment opportunities. In addition, immunotherapy might represent a valuable treatment option for a subset of CUP. Finally, accurate definition of sequencing methods and platforms is crucial to overcome NGS failures.

Digital counting of tissue cells for molecular analysis: the QuANTUM pipeline.

The estimation of tumor cellular fraction (TCF) is a crucial step in predictive molecular pathology, representing an entry adequacy criterion also in the next-generation sequencing (NGS) era. However, heterogeneity of quantification practices and inter-pathologist variability hamper the robustness of its evaluation, stressing the need for more reliable results. Here, 121 routine histological samples from non-small cell lung cancer (NSCLC) cases with complete NGS profiling were used to evaluate TCF interobserver variability among three different pathologists (pTCF), developing a computational tool (cTCF) and assessing its reliability vs ground truth (GT) tumor cellularity and potential impact on the final molecular results. Inter-pathologist reproducibility was fair to good, with overall Wk ranging between 0.46 and 0.83 (avg. 0.59). The obtained cTCF was comparable to the GT (p = 0.129, 0.502, and 0.130 for surgical, biopsies, and cell block, respectively) and demonstrated good reliability if elaborated by different pathologists (Wk = 0.9). Overall cTCF was lower as compared to pTCF (30 ± 10 vs 52 ± 19, p < 0.001), with more cases < 20% (17, 14%, p = 0.690), but none containing < 100 cells for the algorithm. Similarities were noted between tumor area estimation and pTCF (36 ± 29, p < 0.001), partly explaining variability in the human assessment of tumor cellularity. Finally, the cTCF allowed a reduction of the copy number variations (CNVs) called (27 vs 29, - 6.9%) with an increase of effective CNVs detection (13 vs 7, + 85.7%), some with potential clinical impact previously undetected with pTCF. An automated computational pipeline (Qupath Analysis of Nuclei from Tumor to Uniform Molecular tests, QuANTUM) has been created and is freely available as a QuPath extension. The computational method used in this study has the potential to improve efficacy and reliability of TCF estimation in NSCLC, with demonstrated impact on the final molecular results.

Prospective evaluation of EBUS-TBNA specimens for programmed death-ligand 1 expression in non-small cell lung cancer patients: a pilot study / Avaliação prospectiva de espécimes obtidos por meio de EBUS-TBNA quanto à expressão de programmed death-ligand 1 em pacientes com câncer pulmonar de células não pequenas: um estudo piloto

ABSTRACT Objective: EBUS-TBNA cytological sampling is routinely performed for pathological diagnosis, mediastinal staging, and molecular testing in lung cancer patients. EBUS-TBNA samples are not formally accepted for testing programmed death-ligand 1 (PD-L1) expression. The objective of the study was to compare the feasibility, reproducibility, and accuracy of PD-L1 expression assessment in cytological specimens and histological samples. Methods: We prospectively collected histological (transbronchial forceps biopsy) and cytological (EBUS-TBNA) samples from peribronchial neoplastic lesions during an endoscopic procedure at the same target lesion for the pathological diagnosis and molecular assessment of stage IV non-small cell lung cancer (NSCLC). Results: Fifteen patients underwent the procedure. Adequate cytological samples (at least 100 neoplastic cells) were obtained in 12 cases (92.3%). Assessment of PD-L1 expression was similar between histological and cytological samples (agreement rate = 92%). Sensitivity and diagnostic accuracy of EBUS-TBNA cytological specimens were 88.9% and 100%, respectively. Conclusions: The evaluation of PD-L1 expression in EBUS-TBNA cytological specimens is feasible and presents good reproducibility when compared with routine histological samples. EBUS-TBNA cytological samples could be used for the assessment of PD-L1 expression in patients with NSCLC as a minimally invasive approach in stage IV NSCLC cancer patients.

RESUMO Objetivo: A amostragem citológica por meio de EBUS-TBNA é realizada rotineiramente para diagnóstico anatomopatológico, estadiamento mediastinal e teste molecular em pacientes com câncer de pulmão. As amostras obtidas por meio de EBUS-TBNA não são formalmente aceitas para testar a expressão da proteína programmed death-ligand 1 (PD-L1, ligante de morte celular programada 1). O objetivo do estudo foi comparar a viabilidade, reprodutibilidade e precisão da avaliação da expressão de PD-L1 em espécimes citológicos e amostras histológicas. Métodos: Foram coletadas prospectivamente amostras histológicas (obtidas por meio de biópsia transbrônquica com pinça) e citológicas (obtidas por meio de EBUS-TBNA) de lesões neoplásicas peribrônquicas durante um procedimento endoscópico na mesma lesão-alvo para o diagnóstico anatomopatológico e avaliação molecular de câncer pulmonar de células não pequenas (CPCNP) em estágio IV. Resultados: Quinze pacientes foram submetidos ao procedimento. Amostras citológicas adequadas (pelo menos 100 células neoplásicas) foram obtidas em 12 casos (92,3%). A expressão de PD-L1 nas amostras histológicas e citológicas foi semelhante (taxa de concordância = 92%). A sensibilidade e precisão diagnóstica das amostras citológicas obtidas por meio de EBUS-TBNA foram de 88,9% e 100%, respectivamente. Conclusões: A avaliação da expressão de PD-L1 em espécimes citológicos obtidos por meio de EBUS-TBNA é viável e apresenta boa reprodutibilidade quando comparada com amostras histológicas rotineiras. Amostras citológicas obtidas por meio de EBUS-TBNA podem ser usadas para avaliar a expressão de PD-L1 como uma abordagem minimamente invasiva em pacientes com CPCNP em estágio IV.