Shifting from Immunohistochemistry to Screen for ALK Rearrangements: Real-World Experience in a Large Single-Center Cohort of Patients with Non-Small-Cell Lung Cancer.

The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3-7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status.

c-Met immunohistochemistry as reflex test at diagnosis for non-small cell lung cancer: a real-world experience from a monocentric case series.

AIMS: Recent clinical trials have shown promising results with drugs targeting the hepatocyte growth factor receptor (c-Met) for advanced non-small cell lung cancers overexpressing c-Met. We assessed reflex testing of c-Met immunohistochemistry (IHC) at diagnosis for NSCLC in the real-world. METHODS: We retrospectively collected clinical, pathological and molecular data of cases diagnosed with NSCLC in our institution from January 2021 to June 2023. We performed c-Met IHC (SP44 clone) and scored the expression using a H-score and a three-tier classification. RESULTS: 391 cases with interpretable c-Met IHC staining were included. The median age at diagnosis was 70 years (range 25-89 years) including 234 males (male/female ratio 1:5). 58% of the samples came from surgical resections, 35% from biopsies and 8% from cytological procedures. 52% of cases were classified as c-Met-positive (H-score≥150) and 19% were classified as c-Methigh (≥50%, 3+). 43% of the c-Metneg presented with lymph node and/or visceral metastases at diagnosis vs 55% for c-Methigh (p=0.042). 23% of the adenocarcinomas showed c-Methigh expression vs 3% for squamous cell carcinomas (p=0.004). 27% of the c-Metneg cases had a high PD-L1 expression vs 58% of c-Methigh cases (p<0.001). MET ex14 skipping was present in 8% of the c-Methigh cases. CONCLUSIONS: Systematic c-Met testing in daily routine for NSCLC patients is feasible, highlighting a potential correlation with clinicopathological and molecular features.

Characterisation of the protein expression of the emerging immunotherapy targets VISTA, LAG-3 and PRAME in primary uveal melanoma: insights from a southern French patient cohort.

Uveal melanoma (UM) is the most common intraocular tumour in adults, with dismal prognosis once metastases develop, since therapeutic options for the metastatic disease are ineffective. Over the past decade, novel cancer therapies based on immunotherapy have changed the landscape of treatment of different forms of cancer leading to many hopes of improvement in patient overall survival (OS). VISTA, LAG-3 and PRAME are novel promising targets of immunotherapy that have recently gained attention in different solid tumours, but whose relevance in UM remained to be comprehensively evaluated until now. Here, we studied the protein expression of VISTA, LAG-3 and PRAME using immunohistochemistry in representative whole tissue sections from primary UM cases in a cohort of 30 patients from a single centre (Nice University Hospital, Nice, France). The expression of each of these markers was correlated with different clinical and pathological parameters, including onset of metastases and OS. We demonstrated the protein expression of VISTA and LAG-3 in small lymphocytes infiltrating the tumour, while no expression of the proteins was detected in UM cells. For PRAME, nuclear expression was observed in UM cells, but no expression in tumour infiltrating immune cells was identified. Increased levels of VISTA expression in tumour infiltrating lymphocytes (TILs) were associated with nuclear BAP1 expression and better prognosis. Higher levels of LAG-3 in TILs were associated with higher levels of CD8-positive TILs. PRAME nuclear positivity in melanoma cells was associated with epithelioid cell dominant (>90%) UM histological subtype, higher mitotic numbers and a higher percentage of chromosome 8q gain. This study proposes VISTA as a novel relevant immune checkpoint molecule in primary UM and contributes to confirm LAG-3 and PRAME as potentially important immunotherapy targets in the treatment of UM patients, helping to expand the number of immunotherapy candidate molecules that are relevant to modulate in this aggressive cancer.

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer.

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly increased these last few years. The detection of molecular abnormalities is mandatory for the optimal care of advanced or metastatic NS-NSCLC patients, allowing targeted therapies to be administrated with an improvement in overall survival. Nevertheless, these tumors develop mechanisms of resistance that are potentially targetable using novel therapies. Some molecular alterations can also modulate the treatment response. The molecular characterization of NS-NSCLC has to be performed in a short turnaround time (TAT), in less than 10 working days, as recommended by the international guidelines. In addition, the origin of the tissue biopsies for genomic analysis is diverse, and their size is continuously decreasing with the development of less invasive methods and protocols. Consequently, pathologists are being challenged to perform effective molecular technics while maintaining an efficient and rapid diagnosis strategy. Here, we describe the ultra-fast amplicon-based next-generation sequencing (NGS) workflow used in daily routine practice at diagnosis for NS-NSCLC patients. We showed that this system is able to identify the current molecular targets used in precision medicine in thoracic oncology in an appropriate TAT.

Assessment of Different Circulating Tumor Cell Platforms for Uveal Melanoma: Potential Impact for Future Routine Clinical Practice.

Liquid biopsy and circulating tumor cell (CTC) screening has gained interest over the last two decades for detecting almost all solid malignancies. To date, the major limitation in terms of the applicability of CTC screening in daily clinical practice is the lack of reproducibility due to the high number of platforms available that use various technologies (e.g., label-dependent versus label-free detection). Only a few studies have compared different CTC platforms. The aim of this study was to compare the efficiency of four commercially available CTC platforms (Vortex (VTX-1), ClearCell FX, ISET, and Cellsearch) for the detection and identification of uveal melanoma cells (OMM 2.3 cell line). Tumor cells were seeded in RPMI medium and venous blood from healthy donors, and then processed similarly using these four platforms. Melan-A immunochemistry was performed to identify tumor cells, except when the Cellsearch device was used (automated identification). The mean overall recovery rates (with mean recovered cells) were 39.2% (19.92), 22.2% (11.31), 8.9% (4.85), and 1.1% (0.20) for the ISET, Vortex (VTX-1), ClearCell FX, and CellSearch platforms, respectively. Although paramount, the recovery rate is not sufficient to assess a CTC platform. Other parameters, such as the purpose for using a platform (diagnosis, genetics, drug sensitivity, or patient-derived xenograft models), reproducibility, purity, user-friendliness, cost-effectiveness, and ergonomics, should also be considered before they can be used in daily clinical practice and are discussed in this article.

Need for a Dedicated Ophthalmic Malignancy Clinico-Biological Biobank: The Nice Ocular MAlignancy (NOMA) Biobank.

Ophthalmic malignancies include various rare neoplasms involving the conjunctiva, the uvea, or the periocular area. These tumors are characterized by their scarcity as well as their histological, and sometimes genetic, diversity. Uveal melanoma (UM) is the most common primary intraocular malignancy. UM raises three main challenges highlighting the specificity of ophthalmic malignancies. First, UM is a very rare malignancy with an estimated incidence of 6 cases per million inhabitants. Second, tissue biopsy is not routinely recommended due to the risk of extraocular dissemination. Third, UM is an aggressive cancer because it is estimated that about 50% of patients will experience metastatic spread without any curative treatment available at this stage. These challenges better explain the two main objectives in the creation of a dedicated UM biobank. First, collecting UM samples is essential due to tissue scarcity. Second, large-scale translational research programs based on stored human samples will help to better determine UM pathogenesis with the aim of identifying new biomarkers, allowing for early diagnosis and new targeted treatment modalities. Other periocular malignancies, such as conjunctival melanomas or orbital malignancies, also raise specific concerns. In this context, the number of biobanks worldwide dedicated to ocular malignancies is very limited. The aims of this article were (i) to describe the specific challenges raised by a dedicated ocular malignancy biobank, (ii) to report our experience in setting up such a biobank, and (iii) to discuss future perspectives in this field.

Lack of correlation between MET and PD-L1 expression in non-small cell lung cancer revealed by comparative study of matched biopsies and surgical resection samples.

INTRODUCTION: Both MET expression and the PD-L1 tumor proportion score (TPS) are companion diagnostics for treatment of advanced non-small cell lung carcinoma (aNSCLC) patients. We evaluated the rate of correlation between MET expression and the PD-L1 TPS in matched biopsies and surgically resected specimens from NSCLC patients. PATIENTS AND METHODS: This retrospective analysis assessed the prevalence and correlation between MET expression (SP44 clone) and the PD-L1 TPS (22C3 clone) by immunohistochemistry together with molecular alterations determined by targeted next-generation sequencing in matched lung biopsy and surgically lung resected specimens from 70 patients with NSCLC. RESULTS: The study found a significant correlation between the MET H-score in surgical samples and matched biopsies (P-value < 0.0001), as well as between the PD-L1 TPS in paired biopsies and surgical samples (P-value < 0.0001). However, there was no significant correlation between the MET H-score or expression subgroups and the PD-L1 TPS in both types of paired samples (P-value = 0.47, and P-value = 0.90). The MET H-score was significantly higher in adenocarcinoma compared to squamous cell carcinoma (P-value < 0.0001). A mutational analysis showed that the MET H-score was significantly higher in NSCLC cases with targetable molecular alterations (P-value = 0.0095), while no significant correlation was found for the PD-L1 TPS. CONCLUSIONS: Our study found no significant correlation between PD-L1 and MET expression in samples from NSCLC patients, highlighting the importance of personalized treatment strategies based on individual expression profiles. These findings provide valuable insight into the development of effective immunotherapy and targeted therapy for NSCLC patients.

Setting Up an Ultra-Fast Next-Generation Sequencing Approach as Reflex Testing at Diagnosis of Non-Squamous Non-Small Cell Lung Cancer; Experience of a Single Center (LPCE, Nice, France).

The number of genomic alterations required for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has increased and become more complex these last few years. These molecular abnormalities lead to treatment that provides improvement in overall survival for certain patients. However, these treated tumors inexorably develop mechanisms of resistance, some of which can be targeted with new therapies. The characterization of the genomic alterations needs to be performed in a short turnaround time (TAT), as indicated by the international guidelines. The origin of the tissue biopsies used for the analyses is diverse, but their size is progressively decreasing due to the development of less invasive methods. In this respect, the pathologists are facing a number of different challenges requiring them to set up efficient molecular technologies while maintaining a strategy that allows rapid diagnosis. We report here our experience concerning the development of an optimal workflow for genomic alteration assessment as reflex testing in routine clinical practice at diagnosis for NS-NSCLC patients by using an ultra-fast-next generation sequencing approach (Ion Torrent Genexus Sequencer, Thermo Fisher Scientific). We show that the molecular targets currently available to personalized medicine in thoracic oncology can be identified using this system in an appropriate TAT, notably when only a small amount of nucleic acids is available. We discuss the new challenges and the perspectives of using such an ultra-fast NGS in daily practice.

Comparison of Two Rapid Assays for the Detection of BRAF V600 Mutations in Metastatic Melanoma including Positive Sentinel Lymph Nodes.

Testing for the BRAF mutation is mandatory for the management of patients with locally advanced or metastatic melanoma. Molecular analysis based on DNA sequencing remains the gold-standard method for the screening of the different BRAF mutations. These methods must be rapid, sensitive, and specific enough to allow optimal therapeutic management in daily practice and also to include patients in clinical trials. Here, we compared the Idylla BRAF Mutation Test and the anti-BRAF V600E (clone VE1) immunohistochemistry (IHC) in 90 melanoma samples, with a focus on a challenging cohort of 32 positive sentinel lymph nodes. The BRAF status was assessed with both methods independently of the percentage of tumor cells. The concordance rate was calculated excluding both non-contributory analyses and BRAFV600K/R/M mutants due to the specific V600E-IHC test design. The incidence of the BRAFV600E mutation was 33% with both BRAF Idylla and BRAF IHC. The agreement rate was 91% (72/79). Although the agreement rate was high, we suggest that the use of IHC is more suitable for rapid BRAF testing on sentinel lymph node biopsies when associated with a low percentage and scattered tumor cells, which gave a high risk of non-contributory analysis and/or false negative results with the IdyllaTMBRAF Mutation Test.

Analytical validation of automated multiplex chromogenic immunohistochemistry for diagnostic and predictive purpose in non-small cell lung cancer.

OBJECTIVES: The evaluation of an increasing number of diagnostic and predictive markers is playing a central role in precision thoracic oncology. Multiplex immunohistochemistry (mIHC), alongside next-generation sequencing, is ideally situated for this purpose and maximizes tumor tissue preservation for molecular analyses that use increasingly large panels. However, the standardization and validation of mIHC that supports routine clinical laboratory processes are mandatory. After a previous proof-of-concept study, we now (i) optimized two automated four-plex assays on a commercially available IHC autostainer for use in daily practices worldwide and (ii) evaluated the repeatability and concordance of the assessment of the cell density. PATIENTS AND METHODS: Two four-plex mIHC assays [i) TTF1, p40, PD-L1, CD8; and, ii) ALK, ROS1, BRAFV600E, NTRK] were optimized on the BenchMark ULTRA autostainer (Ventana Medical Systems, Inc.), as determined in comparison to conventional IHC chromogenic assays. Intra-site repeatability was evaluated on serial tumor sections from non-small cell lung carcinomas (NSCLC). The concordance was assessed by linear fit to plots of the percentage staining evaluated on tumor sections from 89 NSCLC patients. RESULTS: Following optimization, an average concordance for a staining rate of 95.4% was achieved between conventional IHC and mIHC across all selected markers. Assessment of intra-site repeatability showed strong concordance for all these markers (average, R2 = 0.96; P-value < 0.001). CONCLUSIONS: Our optimized mIHC assay gave a sensitive and repeatable assessment of two panels of eight diagnostic and predictive biomarkers for NSCLC. The availability of standardized protocols to determine these biomarkers on a widely available IHC platform will expand the number of pathology laboratories able to determine the eligibility of patients with NSCLC for targeted treatment or immunotherapy in a reliable and concordant manner, thus providing a unique sample-sparing tool to characterize limited tissue samples in thoracic oncology.

P2RX7B is a new theranostic marker for lung adenocarcinoma patients.

Rationale: The characterization of new theranostic biomarkers is crucial to improving the clinical outcome of patients with advanced lung cancer. Here, we aimed at characterizing the P2RX7 receptor, a positive modulator of the anti-tumor immune response, in patients with lung adenocarcinoma. Methods: The expression of P2RX7 and its splice variants was analyzed by RT-qPCR using areas of tumor and non-tumor lung adenocarcinoma (LUAD) tissues on both immune and non-immune cells. The biological activity of P2RX7 was studied by flow cytometry using fluorescent dyes. Bi-molecular fluorescence complementation and confocal microscopy were used to assess the oligomerization of P2RX7. Tumor immune infiltrates were characterized by immunohistochemistry. Results: Fifty-three patients with LUAD were evaluated. P2RX7A, and 3 alternative splice variants were expressed in LUAD tissues and expression was down regulated in tumor versus adjacent non-tumor tissues. The protein retained biological activity only in immune cells. The P2RX7B splice variant was differentially upregulated in immune cells (P < 0.001) of the tumor and strong evidence of oligomerization of P2RX7A and B was observed in the HEK expression model, which correlated with a default in the activity of P2RX7. Finally, LUAD patients with a high level of P2RX7B had non-inflamed tumors (P = 0.001). Conclusion: Our findings identified P2RX7B as a new theranostic tool to restore functional P2RX7 activity and open alternative therapeutic opportunities to improve LUAD patient outcome.

Targeted Assessment of the EGFR Status as Reflex Testing in Treatment-Naive Non-Squamous Cell Lung Carcinoma Patients: A Single Laboratory Experience (LPCE, Nice, France).

BACKGROUND: Assessment of actionable EGFR mutations is mandatory for treatment-naïve advanced or metastatic non-squamous lung carcinoma (NSLC), but the results need to be obtained in less than 10 working days. For rapid EGFR testing, an EGFR-specific polymerase chain reaction (PCR) assay is an alternative and simple approach compared to next generation sequencing (NGS). Here, we describe how a rapid EGFR-specific PCR assay can be implemented in a single laboratory center (LPCE, Nice, France) as reflex testing in treatment-naïve NSLC. METHODS: A total of 901 biopsies from NSLC with more than 10% of tumor cells were prospectively and consecutively evaluated for EGFR mutation status between November 2017 and December 2019 using the Idylla system (Biocartis NV, Mechelen, Belgium). NGS was performed for nonsmokers with NSLC wild type for EGFR, ALK, ROS1, and BRAF and with less than 50% PD-L1 positive cells using the Hotspot panel (Thermo Fisher Scientific, Waltham, MA, USA). RESULTS: Results were obtained from 889/901 (97%) biopsies with detection of EGFR mutations in 114/889 (13%) cases using the Idylla system. Among the 562 EGFR wild type tumors identified with Idylla, NGS detected one actionable and one nonactionable EGFR mutation. CONCLUSIONS: Rapid and targeted assessment of EGFR mutations in treatment-naïve NSLC can be implemented in routine clinical practice. However, it is mandatory to integrate this approach into a molecular algorithm that allows evaluation of potentially actionable genomic alterations other than EGFR mutations.

Prospective evaluation of NGS-based liquid biopsy in untreated late stage non-squamous lung carcinoma in a single institution.

BACKGROUND: NGS from plasma samples in non-squamous cell lung carcinoma (NSCC) can aid in the detection of actionable genomic alterations. However, the absolute clinical value of NGS in liquid biopsy (LB) made at baseline is currently uncertain. We assessed the impact of plasma-based NGS using an in-house test and an outsourced test in comparison to a routine molecular pathology workflow. METHODS: Twenty-four advanced/metastatic treatment-naïve NSCC patients were prospectively included. NGS analyses were conducted both in-house using the Oncomine cfTNA Panel and in an external testing center using the Foundation Liquid assay. NGS analysis and/or specific molecular based assays were conducted in parallel on tissue or cytological samples. RESULTS: Both LB tests were well correlated. Tissue NGS results were obtained in 67% of patients and demonstrated good correlation with LB assays. Activating EGFR mutations were detected using LB tests in three patients. PD-L1 expression assessed in tissue sections enabled the initiation of pembrolizumab treatment in five patients. CONCLUSION: NGS from LB is feasible in routine clinical practice using an in-house or an outsourced test at baseline. However, the impact on therapy selection was limited in this small series of patients and LB was not able to replace tissue-based testing in our hands.

In-house Implementation of Tumor Mutational Burden Testing to Predict Durable Clinical Benefit in Non-small Cell Lung Cancer and Melanoma Patients.

Tumor mutational burden (TMB) has emerged as an important potential biomarker for prediction of response to immune-checkpoint inhibitors (ICIs), notably in non-small cell lung cancer (NSCLC). However, its in-house assessment in routine clinical practice is currently challenging and validation is urgently needed. We have analyzed sixty NSCLC and thirty-six melanoma patients with ICI treatment, using the FoundationOne test (FO) in addition to in-house testing using the Oncomine TML (OTML) panel and evaluated the durable clinical benefit (DCB), defined by >6 months without progressive disease. Comparison of TMB values obtained by both tests demonstrated a high correlation in NSCLC (R2 = 0.73) and melanoma (R2 = 0.94). The association of TMB with DCB was comparable between OTML (area-under the curve (AUC) = 0.67) and FO (AUC = 0.71) in NSCLC. Median TMB was higher in the DCB cohort and progression-free survival (PFS) was prolonged in patients with high TMB (OTML HR = 0.35; FO HR = 0.45). In contrast, we detected no differences in PFS and median TMB in our melanoma cohort. Combining TMB with PD-L1 and CD8-expression by immunohistochemistry improved the predictive value. We conclude that in our cohort both approaches are equally able to assess TMB and to predict DCB in NSCLC.

Multicenter Evaluation of a Novel ROS1 Immunohistochemistry Assay (SP384) for Detection of ROS1 Rearrangements in a Large Cohort of Lung Adenocarcinoma Patients.

INTRODUCTION: The detection of a ROS1 rearrangement in advanced and metastatic lung adenocarcinoma (LUAD) led to a targeted treatment with tyrosine kinase inhibitors with favorable progression-free survival and overall survival of the patients. Thus, it is mandatory to screen for the ROS1 rearrangement in all these patients. ROS1 rearrangements can be detected using break-apart fluorescence in situ hybridization (FISH), which is the gold standard; however, ROS1 immunohistochemistry (IHC) can be used as a screening test because it is widely available, easy and rapid to perform, and cost-effective. METHODS: We evaluated the diagnostic accuracy and interpathologist agreement of two anti-ROS1 IHC clones, SP384 (Ventana, Tucson, Arizona) and D4D6 (Cell Signaling, Danvers, Massachusetts), in a training cohort of 51 positive ROS1 FISH LUAD cases, and then in a large validation cohort of 714 consecutive cases of LUAD from six routine molecular pathology platforms. RESULTS: In the two cohorts, the SP384 and D4D6 clones show variable sensitivity and specificity rates on the basis of two cutoff points greater than or equal to 1+ (all % tumor cells) and greater than or equal to 2+ (>30% stained tumor cells). In the validation cohort, the D4D6 yielded the best accuracy for the presence of a ROS1 rearrangement by FISH. Interpathologist agreement was moderate to good (interclass correlation 0.722-0.874) for the D4D6 clone and good to excellent (interclass correlation: 0.830-0.956) for the SP384 clone. CONCLUSIONS: ROS1 IHC is an effective screening tool for the presence of ROS1 rearrangements. However, users must be acutely aware of the variable diagnostic performance of different anti-ROS1 antibodies before implementation into routine clinical practice.

Current views on tumor mutational burden in patients with non-small cell lung cancer treated by immune checkpoint inhibitors.

In the last few years, the treatment of patients with non-small cell lung cancer (NSCLC) has impressively benefitted from immunotherapy, in particular from the inhibition of immune checkpoints such as programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1). However, despite the significant survival benefit for some patients with advanced NSCLC, the objective response rates (ORRs) remain relatively low no more than 20-30% with a large proportion of patients demonstrating primary resistance. Although the selection of NSCLC patients for the first-line treatment is currently guided by the expression of PD-L1 in tumor cells as detected by immunohistochemistry, this is not the case for the second-line setting. Moreover, the sensitivity and specificity of PD-L1 expression is modest which has prompted the search for additional predictive biomarkers. In this context, the assessment of the tumor mutational burden (TMB), defined as the total number of nonsynonymous mutations in the coding regions of genes, has recently emerged as an additional powerful biomarker to select patients for immunotherapy. The purpose of our review is to highlight the recent advances as well as the challenges and perspectives in the field of TMB and immunotherapy for patients with NSCLC.

Use of the Ion PGM and the GeneReader NGS Systems in Daily Routine Practice for Advanced Lung Adenocarcinoma Patients: A Practical Point of View Reporting a Comparative Study and Assessment of 90 Patients.

Background: With the integration of various targeted therapies into the clinical management of patients with advanced lung adenocarcinoma, next-generation sequencing (NGS) has become the technology of choice and has led to an increase in simultaneously interrogated genes. However, the broader adoption of NGS for routine clinical practice is still hampered by sophisticated workflows, complex bioinformatics analysis and medical interpretation. Therefore, the performance of the novel QIAGEN GeneReader NGS system was compared to an in-house ISO-15189 certified Ion PGM NGS platform. Methods: Clinical samples from 90 patients (60 Retrospectively and 30 Prospectively) with lung adenocarcinoma were sequenced with both systems. Mutations were analyzed and EGFR, KRAS, BRAF, NRAS, ALK, PIK3CA and ERBB2 genes were compared and sampling time and suitability for clinical testing were assessed. Results: Both sequencing systems showed perfect concordance for the overlapping genes. Correlation of allele frequency was r² = 0.93 for the retrospective patients and r² = 0.81 for the prospective patients. Hands-on time and total run time were shorter using the PGM system, while the GeneReader platform provided good traceability and up-to-date interpretation of the results. Conclusion: We demonstrated the suitability of the GeneReader NGS system in routine practice in a clinical pathology laboratory setting.

Use of circulating tumor cells in prospective clinical trials for NSCLC patients - standardization of the pre-analytical conditions.

BACKGROUND: Circulating tumor cells (CTCs) hold potential for noninvasive diagnosis, prognosis and prediction testing in non-small cell lung cancer (NSCLC) patients. Minimizing degradation or loss of CTCs is pivotal for detection and profiling of the low abundance and fragile CTCs, particularly in clinical trials. We prospectively investigated (NCT02372448) whether a new blood collection device performed better compared to commonly used K3EDTA tubes, when subjected to long-term sample storage. METHODS: Blood samples were drawn into K3EDTA and blood collection tubes (BCT) (Streck), and filtered by the Isolation by SizE of Tumor/Trophoblastic Cells (ISET® system), for CTC detection in two study populations of NSCLC patients; the training set of 14 patients with stage II/IV NSCLC, and the validation set of 36 patients with stage IV NSCLC). MET expression was evaluated by immunocytochemistry (ICC) and anaplastic lymphoma kinase (ALK) gene rearrangement by break-apart fluorescence in situ hybridization (FISH) on ISET-enriched CTCs. RESULTS: Blood processed after 24 h and 48 h in BCT tubes showed stable CTCs counts and integrity, whereas CTCs in K3EDTA tubes showed an altered morphology in all patients. CTCs recovered in BCT or K3EDTA tubes at 24 and 48 h were evaluable by ICC for MET expression and by FISH for ALK rearrangement. CONCLUSIONS: The BCT tubes gave a high yield and preserved the integrity of CTCs after 24 and 48 h of storage at room temperature, which facilitate their molecular characterization in NSCLC patients entering clinical trials.

MiR-223-3p inhibits angiogenesis and promotes resistance to cetuximab in head and neck squamous cell carcinoma.

MicroRNAs (miRs) participate in tumor growth and dissemination by regulating expression of various target genes. MiR-223-3p is suspected of being involved in head and neck squamous cell carcinoma (HNSCC) growth although its precise role has not been elucidated. In this study, we showed that miR-223-3p is present in biopsies of HNSCC patients and that its presence is correlated with high neutrophil infiltrate. We found that overexpression of miR-223-3p slightly increased proliferation of the CAL27 squamous carcinoma cell line both in vitro and in vivo. Moreover, miR-223-3p induced CAL27 apoptosis in an orthotopic xenograft mouse model, counteracting the proliferative effect and resulting in no impact on overall tumor growth. We analyzed the effect of miR-223-3p overexpression on signaling pathways and showed that it induced pERK2, pAKT and AKT, consistent with an increase in cell proliferation. In addition, we found that miR-223-3p reduced the STAT3 level correlating with increased cell apoptosis and inhibited vasculature formation. In HNSCC tissues, miR-223-3p expression was inversely correlated to CD31, highlighting the relationship between miR-223 and vessel formation. Finally, we studied the effect of miR-223-3p on response to selected anticancer agents and showed that cells expressing miR-223-3p are more resistant to drugs, notably cetuximab. In conclusion, our study is the first to show the antiangiogenic properties of miR-223-3p in HNSCC patients and to question whether expression levels of miR-223-3p can be evaluated as an indicator of eligibility for non-treatment of HNSCC patients with cetuximab.

Expression of MET in circulating tumor cells correlates with expression in tumor tissue from advanced-stage lung cancer patients.

Given the difficulty in obtaining adequate tissue in NSCLC, we investigated the utility of circulating tumor cells (CTCs) for MET status assessment in NSCLC patients. We used two platforms for CTC capture, and assessed MET expression in CTCs and matched-bronchial biopsies in patients with advanced-stage III/IV lung adenocarcinoma. Baseline peripheral blood was collected from 256 advanced-stage III/IV NSCLC patients from Genentech clinical trials, and from 106 patients with advanced-stage III/IV lung adenocarcinoma treated at the Department of Pneumology, Pasteur Hospital, Nice. CTCs were enriched using CellSearch (Genentech), or ISET technologies (Pasteur Hospital). MET expression was evaluated by immunofluorescence on CellSearch, and by immunocytochemistry on ISET-enriched CTCs and on matched FFPE tissue sections (Pasteur Hospital). CTCs were detected in 83 of 256 (32%) patients evaluated on CellSearch, with 30 samples (12%) exhibiting ≥ 5 CTCs/7.5 ml blood. On ISET, CTC were observed in 80 of 106 patients (75%), and 79 patients (75%) exhibited ≥ 5 CTCs/4 ml blood. MET expression on ISET CTCs was positive in 72% of cases, and the MET expression on matched-patient tissue was positive in 65% patients using the Onartuzumab IHC scoring algorithm (93% concordance). Quantification of MET expression using H-scores showed strong correlation between MET expression in tissue and CTCs (Spearman correlation, 0.93). MET status in CTCs isolated on ISET filters from blood samples of advanced-stage NSCLC patients correlated strongly with MET status in tumor tissue, illustrating the potential for using CTCs as a non-invasive, real-time biopsy to determine MET status of patients entering clinical trials.