Utility and Micro-Costing Framework for TERT Promoter Mutation Analysis in Melanocytic Lesions of Uncertain Malignant Potential: A Retrospective Study in Dutch Local Clinical Practice.

The 2018 WHO edition on the classification of cutaneous melanocytic tumors recognizes eight evolutionary pathways of melanoma and describes tumors of uncertain malignant potential for each. When histology and immunohistochemistry do not support a confident conclusion about its malignant potential, a window of diagnostic uncertainty is created. Mutations in the telomerase reverse transcriptase gene promoter (TERTp) are highly specific for melanoma and can be used as an ancillary technique to acquire a higher level of confidence in the diagnosis. However, little is known about the cost-effectiveness of testing for TERTp mutations. The aims of this study were to determine how often knowledge of the TERTp mutation status contributed to the final diagnosis and to develop a micro-costing framework to calculate cost-effectiveness. A retrospective analysis of all cutaneous melanocytic lesions that were discussed in the Noord-Nederland Melanoma Panel from January 2021 to October 2022 was performed to identify the cases in which the preliminary histopathological diagnosis was uncertain regarding malignancy (ambiguous, likely benign, or likely malignant). For cases in which a TERTp mutation analysis was performed, the final diagnoses were collected, and it was determined whether this impacted the overall conclusion. A micro-costing framework was established to model the financial impact of introducing TERTp mutation analyses and subsequent clinical procedures. The study included 367 cases, of which 175 diagnoses of uncertain malignant potential were initially reported. TERTp mutation analysis was performed for 151/175 (86%). In 38% of these cases, a higher level of confidence regarding malignant potential was obtained. The implementation of TERTp mutation analyses for cutaneous melanocytic proliferations with uncertain malignant potential can narrow the window of diagnostic uncertainty. For the patient group with an initial uncertain diagnosis, the increased cost for molecular testing (86.145 €) was compensated by a reduced overall treatment cost (-122.304 €). A microsimulation model to determine the cost-effectiveness of TERTp mutation analysis projected an overall saving for the healthcare system.

Transcriptomic analyses of lung tissues reveal key genes associated with progression of systemic sclerosis-interstitial lung disease (SSc-ILD).

OBJECTIVE: Systemic sclerosis-interstitial lung disease (SSc-ILD) is the leading cause of death in SSc, affecting around 50 % of the patients. Lung tissue of patients with early-stage SSc-ILD is characterized by a predominant inflammatory response with inconspicuous fibrosis, which may progress to honeycombing fibrosis. Hence, a better understanding of the molecular mechanisms underpinning SSc-ILD pathogenesis is needed to improve treatment options and progression prediction. This transcriptomic study aims to reveal the differential gene expression between control (ctrl) lung tissue and inflammatory, prefibrotic and fibrotic lung tissue to capture progression of early to late phase SSc-ILD. METHODS: Twelve explanted lungs from patients with SSc-ILD were used to analyze gene expression from formalin-fixed paraffin-embedded lung tissues with varying stages of ILD (n = 18) and control lung tissue (n = 6). The SSc-ILD tissues were stratified into three ROIs: inflammatory, prefibrotic, and fibrotic using histological assessments to define a longitudinal simulation of early to late phases of SSc-ILD. The nanoString (nS) nCounter Human Fibrosis Panel was used to profile the transcriptome in the regions of interest. Validation of potential targetswas performed with immunohistochemistry in the same tissues that were used for transcriptome analysis. RESULTS: To validate our simulation model, we performed subgroup analysis that showed an incremental increase in pathway scores related to the severity of fibrosis. Ctrl vs SSc-ILD comparison demonstrated 24 differentially expressed genes, two of which had the most pronounced p-values. Cyclin-dependent kinase inhibitor (cdkn2c) was overexpressed (P = 0.00052) in SSc-ILD compared to ctrl, while expression of Pellino E3 ubiquitin-protein ligase 1 (peli1) showed lower expression (P = 0.0012). Additionally, in all four groups, cdkn2c and peli1 gene expression showed an incremental increase and decrease, respectively. Immunohistochemistry of cdkn2c showed consistent results with the nS analysis. CONCLUSION: More cdkn2c and less peli1 expression were associated with more advanced stages of SSc-ILD on histologic assessment. We report the potential of the cell cycle inhibitor and senescence marker, cdkn2c (p18) to be associated with fibrosis progression.

Unraveling the MicroRNA tapestry: exploring the molecular dynamics of locoregional recurrent rectal cancer.

Introduction: Colorectal cancer (CRC) ranks as the third most prevalent malignancy globally, with a concerning rise in incidence among young adults. Despite progress in understanding genetic predispositions and lifestyle risk factors, the intricate molecular mechanisms of CRC demand exploration. MicroRNAs (miRNAs) emerge as key regulators of gene expression and their deregulation in tumor cells play pivotal roles in cancer progression. Methods: NanoString's nCounter technology was utilized to measure the expression of 827 cancer-related miRNAs in tumor tissue and adjacent non-involved normal colon tissue from five patients with locoregional CRC progression. These expression profiles were then compared to those from the primary colon adenocarcinoma (COAD) cohort in The Cancer Genome Atlas (TCGA). Results and discussion: Intriguingly, 156 miRNAs showed a contrasting dysregulation pattern in reccurent tumor compared to their expression in the TCGA COAD cohort. This observation implies dynamic alterations in miRNA expression patterns throughout disease progression. Our exploratory study contributes to understanding the regulatory landscape of recurrent CRC, emphasizing the role of miRNAs in disease relapse. Notable findings include the prominence of let-7 miRNA family, dysregulation of key target genes, and dynamic changes in miRNA expression patterns during progression. Univariate Cox proportional hazard models highlighted miRNAs associated with adverse outcomes and potential protective factors. The study underscores the need for more extensive investigations into miRNA dynamics during tumor progression and the value of stage specific biomarkers for prognosis.

Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Developments in predictive biomarker testing and targeted therapy in advanced stage non-small cell lung cancer and their application across European countries.

In the past two decades, the treatment of metastatic non-small cell lung cancer (NSCLC), has undergone significant changes due to the introduction of targeted therapies and immunotherapy. These advancements have led to the need for predictive molecular tests to identify patients eligible for targeted therapy. This review provides an overview of the development and current application of targeted therapies and predictive biomarker testing in European patients with advanced stage NSCLC. Using data from eleven European countries, we conclude that recommendations for predictive testing are incorporated in national guidelines across Europe, although there are differences in their comprehensiveness. Moreover, the availability of recently EMA-approved targeted therapies varies between European countries. Unfortunately, routine assessment of national/regional molecular testing rates is limited. As a result, it remains uncertain which proportion of patients with metastatic NSCLC in Europe receive adequate predictive biomarker testing. Lastly, Molecular Tumor Boards (MTBs) for discussion of molecular test results are widely implemented, but national guidelines for their composition and functioning are lacking. The establishment of MTB guidelines can provide a framework for interpreting rare or complex mutations, facilitating appropriate treatment decision-making, and ensuring quality control.

Future perspective for the application of predictive biomarker testing in advanced stage non-small cell lung cancer.

For patients with advanced stage non-small cell lung cancer (NSCLC), treatment strategies have changed significantly due to the introduction of targeted therapies and immunotherapy. In the last few years, we have seen an explosive growth of newly introduced targeted therapies in oncology and this development is expected to continue in the future. Besides primary targetable aberrations, emerging diagnostic biomarkers also include relevant co-occurring mutations and resistance mechanisms involved in disease progression, that have impact on optimal treatment management. To accommodate testing of pending biomarkers, it is necessary to establish routine large-panel next-generation sequencing (NGS) for all patients with advanced stage NSCLC. For cost-effectiveness and accessibility, it is recommended to implement predictive molecular testing using large-panel NGS in a dedicated, centralized expert laboratory within a regional oncology network. The central molecular testing center should host a regional Molecular Tumor Board and function as a hub for interpretation of rare and complex testing results and clinical decision-making.

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.

Formalin-Fixed, Paraffin-Embedded-Targeted Locus Capture: A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors.

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded-targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.

Clinical, histopathological and molecular features of dedifferentiated melanomas: An EORTC Melanoma Group Retrospective Analysis.

PURPOSE: Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases. PATIENTS AND METHODS: A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out. RESULTS: Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome. CONCLUSIONS: Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications.

A novel PPP2R2A::PRKD1 fusion in a cribriform adenocarcinoma of salivary gland.

Cribriform adenocarcinoma of salivary gland (CASG) is a rare, salivary gland tumor. In this report, we describe a case of CASG harboring a novel PPP2R2A::PRKD1 fusion. A 58-year-old female presented with an intraoral mass adjacent to the lower left third molar region. Morphological features at histological examination, immunohistochemical staining (p63+, p40-), and tumor location were indicative of CASG. However, due to the potential focal presence of a biphasic component within the tumor, RNA sequencing was performed to confirm the diagnosis. The subsequently found novel PPP2R2A::PRKD1 fusion adds to the rapidly evolving molecular landscape of salivary gland tumors. Additionally, we report that CASG may show some entrapment of pre-existent salivary gland ducts, which may be misinterpreted as tumor cells with myoepithelial differentiation.

A Micro-Costing Framework for Circulating Tumor DNA Testing in Dutch Clinical Practice.

Circulating tumor DNA (ctDNA) is a promising new biomarker with multiple potential applications in cancer care. Estimating total cost of ctDNA testing is necessary for reimbursement and implementation, but challenging because of variations in workflow. We aimed to develop a micro-costing framework for consistent cost calculation of ctDNA testing. First, the foundation of the framework was built, based on the complete step-wise diagnostic workflow of ctDNA testing. Second, the costing method was set up, including costs for personnel, materials, equipment, overhead, and failures. Third, the framework was evaluated by experts and applied to six case studies, including PCR-, mass spectrometry-, and next-generation sequencing-based platforms, from three Dutch hospitals. The developed ctDNA micro-costing framework includes the diagnostic workflow from blood sample collection to diagnostic test result. The framework was developed from a Dutch perspective and takes testing volume into account. An open access tool is provided to allow for laboratory-specific calculations to explore the total costs of ctDNA testing specific workflow parameters matching the setting of interest. It also allows to straightforwardly assess the impact of alternative prices or assumptions on the cost per sample by simply varying the input parameters. The case studies showed a wide range of costs, from €168 to €7638 ($199 to $9124) per sample, and generated information. These costs are sensitive to the (coverage of) platform, setting, and testing volume.

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods.

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling.

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

Unmet Needs and Perspectives in Oral Cancer Prevention.

Oral potentially malignant disorders (OPMD) may precede oral squamous cell carcinoma (OSCC). Reported rates of malignant transformation of OPMD range from 3 to 50%. While some clinical, histological, and molecular factors have been associated with a high-risk OPMD, they are, to date, insufficiently accurate for treatment decision-making. Moreover, this range highlights differences in the clinical definition of OPMD, variation in follow-up periods, and molecular and biological heterogeneity of OPMD. Finally, while treatment of OPMD may improve outcome, standard therapy has been shown to be ineffective to prevent OSCC development in patients with OPMD. In this perspective paper, several experts discuss the main challenges in oral cancer prevention, in particular the need to (i) to define an OPMD classification system by integrating new pathological and molecular characteristics, aiming (ii) to better identify OPMD at high risk of malignant transformation, and (iii) to develop treatment strategies to eradicate OPMD or prevent malignant transformation.

Detection of NTRK Fusions and TRK Expression and Performance of pan-TRK Immunohistochemistry in Routine Diagnostics: Results from a Nationwide Community-Based Cohort.

Gene fusions involving NTRK1, NTRK2, and NTRK3 are rare drivers of cancer that can be targeted with histology-agnostic inhibitors. This study aimed to determine the nationwide landscape of NTRK/TRK testing in the Netherlands and the usage of pan-TRK immunohistochemistry (IHC) as a preselection tool to detect NTRK fusions. All pathology reports in 2017-2020 containing the search term 'TRK' were retrieved from the Dutch Pathology Registry (PALGA). Patient characteristics, tumor histology, NTRK/TRK testing methods, and reported results were extracted. NTRK/TRK testing was reported for 7457 tumors. Absolute testing rates increased from 815 (2017) to 3380 (2020). Tumors were tested with DNA/RNA-based molecular assay(s) (48%), IHC (47%), or in combination (5%). A total of 69 fusions involving NTRK1 (n = 22), NTRK2 (n = 6) and NTRK3 (n = 41) were identified in tumors from adult (n = 51) and pediatric (n = 18) patients. In patients tested with both IHC and a molecular assay (n = 327, of which 29 NTRK fusion-positive), pan-TRK IHC had a sensitivity of 77% (95% confidence interval (CI), 56-91) and a specificity of 84% (95% CI, 78-88%). These results showed that pan-TRK IHC has a low sensitivity in current routine practice and warrants the introduction of quality guidelines regarding the implementation and interpretation of pan-TRK IHC.

Prevalence of KRAS p.(G12C) in stage IV NSCLC patients in the Netherlands; a nation-wide retrospective cohort study.

OBJECTIVES: The recent accelerated FDA approval of sotorasib, a highly selective KRAS G12C inhibitor, offers new opportunities for the treatment of KRAS p.(G12C)-mutated non-squamous non-small cell lung cancer (NSCLC). The objective of the current study was to the determine the prevalence of KRAS mutations in stage IV non-squamous NSCLC in The Netherlands to reveal the potential impact of upcoming KRAS targeted therapy. MATERIALS AND METHODS: All patients diagnosed with stage IV non-squamous NSCLC in 2013, 2015 and 2017 in the Netherlands were selected by linking the nation-wide Netherlands Cancer Registry (NCR) and the Dutch Pathology Registry (PALGA). Demographic and pathological variables were retrieved from the pathology reports including sex, age, KRAS mutation status, molecular test method used, and the mutation status of other genes. RESULTS: Prevalence for any KRAS mutations in codon 12/13/61/146 was 39.1%. KRAS p.(G12C) was detected in 15.5% of all non-squamous NSCLC cases representing 39.6% of all KRAS-mutant cases. National testing rate for KRAS mutations increased from 70% in 2013 to 82% in 2017. Testing techniques changed significantly over time with next generation sequencing as the main used method in 2017 (71.6%) but did not affect prevalence of KRAS mutations over time. When KRAS was tested as part of a larger panel, the KRAS p.(G12C) mutation was frequently reported with a concurrent mutation in TP53 (47.7%) or STK11 (10.3%). CONCLUSION: The high prevalence for KRAS p.(G12C) offers a promising new specific treatment option for 15% of all stage IV non-squamous NSCLC patients.

CD4+ T cells in classical Hodgkin lymphoma express exhaustion associated transcription factors TOX and TOX2: Characterizing CD4+ T cells in Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

Actionability of on-target ALK Resistance Mutations in Patients With Non-Small Cell Lung Cancer: Local Experience and Review of the Literature.

INTRODUCTION: Non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors. PATIENTS AND METHODS: Local experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors. RESULTS: Of the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 69% to 89%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented. CONCLUSION: Molecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients.