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Comprehensive biomarker testing for patients with non-small cell lung cancer is critical for selecting appropriate targeted therapy or immunotherapy. Ensuring timely ordering, processing, and reporting is key to optimizing patient outcomes. However, various factors can prevent or delay patients from being offered the option of treatment selection based on comprehensive biomarker testing. These factors include problems with access to testing, tissue adequacy, turnaround time, and health insurance coverage and billing practices. Turnaround time depends on several logistical and tissue handling factors, which involve institutional policies, processes, resources, testing methodology, and testing algorithms that vary across different practices. In this article, the authors identify key factors that prolong biomarker testing turnaround time, propose strategies to reduce it, and present a process map to aid physicians and key organizational stakeholders in improving testing efficiency., (© 2024 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Purpose: The National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice trial assigned patients age 1-21 years with relapsed or refractory solid tumors, lymphomas, and histiocytic disorders to phase II treatment arms of molecularly targeted therapies on the basis of genetic alterations detected in their tumor. Patients with tumors that harbored prespecified genomic alterations in the cyclinD-CDK4/6-INK4a-Rb pathway with intact Rb expression were assigned and treated with the cdk4/6 inhibitor palbociclib., Methods: Patients received palbociclib orally once daily for 21 days of 28-day cycles until disease progression, intolerable toxicity, or up to 2 years. The primary end point was objective response rate; secondary end points included safety/tolerability and progression-free survival., Results: Twenty-three patients (median age, 15 years; range, 8-21) were enrolled; 20 received protocol therapy and were evaluable for toxicity and response. Of the evaluable patients, the most common diagnoses were osteosarcoma (n = 9) and rhabdomyosarcoma (n = 6). A single actionable gene amplification was found in 19 tumors ( CDK4 , n = 11, CDK6 , n = 2, CCND3 , n = 6), with one tumor harboring two amplifications ( CDK4 and CCND2 ). Hematologic toxicities were the most common treatment-related events. No objective responses were seen. Two patients with tumors harboring CDK4 amplifications (neuroblastoma and sarcoma) had best response of stable disease for six and three cycles. Six-month progression was 10% (95% CI, 1.7 to 27.2)., Conclusion: The CDK4/6 inhibitor palbociclib at 75 mg/m 2 orally daily was tolerable in this heavily pretreated cohort. No objective responses were observed in this histology-agnostic biomarker-selected population with treatment-refractory solid tumors, demonstrating that pathway alteration alone is insufficient in pediatric cancers to generate a response to palbociclib monotherapy.

Purpose: Patients age 1-21 years with relapsed or refractory solid and CNS tumors were assigned to phase II studies of molecularly targeted therapies on the National Cancer Institute-Children's Oncology Group (NCI-COG) Pediatric Molecular Analysis for Therapy Choice (MATCH) trial. Patients whose tumors harbored predefined genetic alterations in the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway and lacked mitogen-activated protein kinase pathway activating alterations were treated with the PI3K/mTOR inhibitor samotolisib., Methods: Patients received samotolisib twice daily in 28-day cycles until disease progression or unacceptable toxicity. A rolling 6 limited dose escalation was performed as, to our knowledge, this was the first pediatric study of samotolisib. The primary end point was the objective response rate; secondary end points included progression-free survival (PFS) and the recommended phase II dose and toxicity of samotolisib in children., Results: A total of 3.4% (41/1,206) of centrally tested patients were matched to this arm. Seventeen patients were treated. Among treated patients, the most common diagnoses included osteosarcoma (n = 6) and high-grade glioma (n = 5) harboring alterations in phosphatase and tensin homolog (n = 6), PIK3CA (n = 5), and tuberous sclerosis complex 2 (n = 3). No objective responses or prolonged stable disease were observed. Three-month PFS was 12% (95% CI, 2 to 31). Two patients experienced dose-limiting toxicities (mucositis and pneumonitis). Dose level 2 (115 mg/m 2 /dose twice daily) was determined to be the recommended phase II dose of samotolisib in children., Conclusion: This nationwide study was successful at identifying patients and evaluating the efficacy of molecularly targeted therapy for rare molecular subgroups of patients in a histology-agnostic fashion. Unfortunately, there was no activity of samotolisib against tumors with PI3K/mTOR pathway alterations. Prospective trials such as the NCI-COG Pediatric MATCH are necessary to evaluate the efficacy of molecularly targeted therapies given their increasing use in clinical practice.

Background: This is a phase II subprotocol of the NCI-COG Pediatric MATCH study evaluating vemurafenib, a selective oral inhibitor of BRAF V600 mutated kinase, in patients with relapsed or refractory solid tumors harboring BRAF V600 mutations., Methods: Patients received vemurafenib at 550 mg/m2 (maximum 960 mg/dose) orally twice daily for 28-day cycles until progression or intolerable toxicity. The primary aim was to determine the objective response rate and secondary objectives included estimating progression-free survival and assessing the tolerability of vemurafenib., Results: Twenty-two patients matched to the subprotocol and 4 patients (18%) enrolled. Primary reasons for non-enrollment were ineligibility due to exclusions of low-grade glioma (n = 7) and prior BRAF inhibitor therapy (n = 7). Enrolled diagnoses were one each of histiocytosis, ameloblastoma, Ewing sarcoma, and high-grade glioma, all with BRAF V600E mutations. Treatment was overall tolerable with mostly expected grade 1/2 adverse events (AE). Grade 3 or 4 AE on treatment were acute kidney injury, hyperglycemia, and maculopapular rash. One patient came off therapy due to AE. One patient (glioma) had an objective partial response and remained on protocol therapy for 15 cycles., Conclusion: There was a low accrual rate on this MATCH subprotocol, with only 18% of those who matched with BRAFV600 mutations enrolling, resulting in early termination, and limiting study results (ClinicalTrials.gov Identifier: NCT03220035)., (© The Author(s) 2024. Published by Oxford University Press.)

Background: The National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice (MATCH) precision oncology platform trial enrolled children aged 1-21 years with treatment-refractory solid tumors and predefined actionable genetic alterations. Patients with tumors harboring alterations in DNA damage repair (DDR) genes were assigned to receive olaparib., Methods: Tumor and blood samples were submitted for centralized molecular testing. Tumor and germline sequencing were conducted in parallel. Olaparib was given twice daily for 28-day cycles starting at a dose 30% lower than the adult recommended phase 2 dose (RP2D). The primary endpoint was the objective response., Results: Eighteen patients matched (1.5% of those screened) based on the presence of a deleterious gene alteration in BRCA1/2, RAD51C/D, or ATM detected by tumor sequencing without germline subtraction or analysis of loss of heterozygosity (LOH). Eleven (61%) harbored a germline mutation, with only one exhibiting LOH. Six patients enrolled and received the olaparib starting dose of 135 mg/m2/dose. Two participants were fully evaluable; 4 were inevaluable because <85% of the prescribed dose was administered during cycle 1. There were no dose-limiting toxicities or responses. Minimal hematologic toxicity was observed., Conclusion: Most DDR gene alterations detected in Pediatric MATCH were germline, monoallelic, and unlikely to confer homologous recombination deficiency predicting sensitivity to olaparib monotherapy. The study closed due to poor accrual., Clinicaltrials.gov Identifier: NCT03233204. IRB approved: initial July 24, 2017., (© The Author(s) 2024. Published by Oxford University Press.)

The identification of targetable genomic alterations in lung cancer is required as standard of care to guide optimal therapy selection. With a constantly evolving landscape of ancillary molecular and biomarker testing in lung cancer, pathologists need to be aware of what specimens to test, how the testing should be performed, and which targets to test for to provide the clinically relevant genomic information necessary to treat these patients. Several guideline statements on the topic are currently available to help pathologists and laboratory personnel best use the small specimens obtained from patients with lung cancer for ancillary molecular testing., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Plasmacytoid urothelial carcinoma (UC) is a rare histologic subtype of bladder cancer that is associated with an aggressive clinical behavior. We analyzed the clinicopathologic and molecular features of plasmacytoid UC in 52 patients from a single institute. The patients included 44 men and 8 women, with a mean age of 64 years (range, 41-91 years). All bladder cancers were high-grade UC, and plasmacytoid component accounted for a mean of 47% of bladder tumors (range, 5-100%). Distinct gene mutations were found in most plasmacytoid UCs (n = 49); the most common mutations were TP53 (n = 30), followed by TERT (n = 20), and CDH1 (n = 18). Copy number analysis was performed in 34 patients, and 13 of them showed copy number variations. Expression of HER2 was analyzed in 18 patients by immunohistochemistry, and 3 of them showed HER2 overexpression, which was confirmed by fluorescence in situ hybridization analysis. Thirty-two patients died of disease in a median of 15 months (range, 1-45 months). No individual gene mutations were significantly associated with clinical outcome, but mutations in the mammalian target of rapamycin (mTOR) pathway, including PICK3CA and PIK3R1 mutations, were associated with a significantly shorter survival duration (p < 0.05). Plasmacytoid UC is an aggressive histologic subtype that demonstrates frequent somatic gene mutations and CNVs, which may underlie its oncogenesis and progression. Gene mutations of the mTOR pathway are associated with poor outcome in a subset of patients with plasmacytoid UC., Competing Interests: Declaration of competing interest All authors declare they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

ALK-fused Spitz melanocytic neoplasms are a distinct subgroup of melanocytic lesions exhibiting unique histopathologic characteristics. These lesions often manifest as exophytic or polypoid tumors, characterized by fusiform-to-epithelioid melanocytes arranged in a nested, fascicular, or plexiform growth pattern. Several fusion partners of the ALK gene have been identified in spitzoid melanocytic neoplasms, with TPM3 and DCTN1 being the most prevalent. Less common fusion partners include NPM1, TPR, CLIP1, GTF3C2, EEF2, MYO5A, KANK1, and EHBP1. The MLPH gene, which encodes melanophilin (MLPH), playing a crucial role in regulating skin pigmentation by acting as a linker between RAB27A and myosin Va during melanosome transport, has also recently been recognized as a rare fusion partner of ALK in Spitz melanocytic neoplasms. Currently, there exists a sparse documentation within English literature, illustrating a limited number of cases featuring MLPH::ALK fusion in Spitz melanocytic neoplasms. In this report, we present two additional cases, including a previously unreported instance of Spitz melanoma, contributing to the expanding knowledge on ALK-fused Spitz melanocytic neoplasms. In addition, we provide a comprehensive review of the clinical, histopathologic, and molecular features observed in documented cases with this novel fusion., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Context.—: In 2014, the College of American Pathologists developed an evidence-based guideline to address analytic validation of immunohistochemical assays. Fourteen recommendations were offered. Per the National Academy of Medicine standards for developing trustworthy guidelines, guidelines should be updated when new evidence suggests modifications., Objective.—: To assess evidence published since the release of the original guideline and develop updated evidence-based recommendations., Design.—: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and update the original guideline recommendations using the Grading of Recommendations Assessment, Development and Evaluation approach., Results.—: Two strong recommendations, 1 conditional recommendation, and 12 good practice statements are offered in this updated guideline. They address analytic validation or verification of predictive and nonpredictive assays, and recommended revalidation procedures following changes in assay conditions., Conclusions.—: While many of the original guideline statements remain similar, new recommendations address analytic validation of assays with distinct scoring systems, such as programmed death receptor-1 and analytic verification of US Food and Drug Administration approved/cleared assays; more specific guidance is offered for validating immunohistochemistry performed on cytology specimens., Competing Interests: Authors’ disclosures of potential conflicts of interest and author contributions are found in the Appendix at the end of this article., (© 2024 College of American Pathologists.)

Purpose: The National Cancer Institute-Children's Oncology Group (NCI-COG) Pediatric MATCH trial assigns patients age 1-21 years with refractory malignancies to phase II treatment arms of molecularly targeted therapies on the basis of genetic alterations detected in their tumor. Patients with activating alterations in the mitogen-activated protein kinase pathway were treated with ulixertinib, an extracellular signal-regulated kinase (ERK)1/2 inhibitor., Methods: As there were no previous pediatric data, ulixertinib was initially tested in a dose escalation cohort to establish the recommended phase II dose (RP2D) before proceeding to the phase II cohort. Ulixertinib was administered at 260 mg/m 2 /dose orally twice a day (dose level 1 [DL1], n = 15) or 350 mg/m 2 /dose orally twice a day (DL2, n = 5). The primary end point was objective response rate; secondary end points included safety/tolerability and progression-free survival (PFS)., Results: Twenty patients (median 12 years; range, 5-20) were treated, all evaluable for response. CNS tumors comprised 55% (11/20) of diagnoses, with high-grade glioma and low-grade glioma most common (n = 5 each). All CNS tumors except one harbored BRAF fusions or V600E mutations. Rhabdomyosarcoma (n = 5) was the most frequent non-CNS diagnosis. DL1 was declared the RP2D in the dose escalation cohort after dose-limiting toxicities in Cycle 1 occurred in 1/6 patients at DL1 and 2/5 patients at DL2, including fatigue, anorexia, rash, nausea, vomiting, diarrhea, dehydration, hypoalbuminemia, and hypernatremia. No objective responses were observed. Six-month PFS was 37% (95% CI, 17 to 58). Three patients with BRAF -altered CNS tumors achieved stable disease >6 months., Conclusion: Ulixertinib, a novel targeted agent with no previous pediatric data, was successfully evaluated in a national precision medicine basket trial. The pediatric RP2D of ulixertinib is 260 mg/m 2 /dose orally twice a day. Limited single-agent efficacy was observed in a biomarker-selected cohort of refractory pediatric tumors.

Introduction: Malignant pleural effusion (MPE) is a frequent complication of advanced malignancies. In this pilot study, we characterized the immune landscapes of MPEs, compared them to their primary tumor (PT) samples from breast carcinoma (BC) and lung adenocarcinoma (LADC), and tested the utility of multiplexed image technology in cytological samples., Materials and Methods: We evaluated the immune contexture of 6 BC and 5 LADC MPEs and their PTs using 3 multiplex immunofluorescence panels. We explored the associations between sample characteristics and pleural effusion-free survival., Results: No MPE samples had positive programmed death-ligand 1 expression in malignant cells, although 3 of 11 PTs has positive programmed death-ligand 1 expression (more than 1% expression in malignant cells). Overall, in LADC samples, cluster of differentiation 3 (CD3)+ T cells and CD3+CD8+ cytotoxic T cells predominated (median percentages for MPEs versus PTs: 45.6% versus 40.7% and 4.7% versus 6.6%, respectively) compared with BC. CD68+ macrophages predominated in the BC samples (medians for MPEs 61.2% versus PTs for 57.1%) but not in the LADC samples. Generally in PTs, CD3+CD8+ forkhead box P3+ T cells and the median distances from the malignant cells to CD3+CD8+Ki67+ and CD3+ programmed cell death protein 1 + T cells correlated to earlier MPE after PT diagnosis., Conclusions: The immune cell phenotypes in the MPEs and PTs were similar within each cancer type but different between BC versus LADC. An MPE analysis can potentially be used as a substitute for a PT analysis, but an expanded study of this topic is essential., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: During the COVID-19 pandemic, the need for digital pathology tools became more urgent. However, there needs to be more knowledge of the use in cytology. We aimed to evaluate current digital cytology practices and attitudes and compare the results with a pre-COVID-19 American Society of Cytopathology (ASC) survey., Materials and Methods: Fourteen survey questions assessing current attitudes toward digital cytology were developed from a 2016 ASC Digital Pathology Survey. Ten new survey questions were also created to evaluate telecytology use. The survey was e-mailed to ASC members over 6 weeks in 2023., Results: A total of 123 individuals responded (116 in 2016). Attitudes toward digital cytology were unchanged; most participants stated digital cytology is beneficial (87% 2023 versus 90% 2016). The percentage of individuals using digital cytology was unchanged (56% in 2016 and 2023). However, telecytology for rapid onsite assessment (ROSE) is now considered the best application (55% 2023 versus 31% 2016). Forty-three institutions reported using digital and telecytology tools; 40% made implementations after 2020; most did not feel that COVID-19 affected digital cytology (56%). Telecytology for ROSE is the most common application now (78%) compared with education (30%) in 2016. Limitations for implementing digital imaging in cytology included inability to focus (38%) and expense (33%)., Conclusions: General attitudes toward digital tools by the cytology community have essentially remained the same between 2016 and now. However, telecytology for ROSE is increasingly being used, which supports a need for validation and competency guidelines., (Copyright © 2024 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

The integration of cancer biomarkers into oncology has revolutionized cancer treatment, yielding remarkable advancements in cancer therapeutics and the prognosis of cancer patients. The development of personalized medicine represents a turning point and a new paradigm in cancer management, as biomarkers enable oncologists to tailor treatments based on the unique molecular profile of each patient's tumor. In this review, we discuss the scientific milestones of cancer biomarkers and explore future possibilities to improve the management of patients with solid tumors. This progress is primarily attributed to the biological characterization of cancers, advancements in testing methodologies, elucidation of the immune microenvironment, and the ability to profile circulating tumor fractions. Integrating these insights promises to continually advance the precision oncology field, fostering better patient outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Many patients with non-small cell lung cancer do not receive guideline-recommended, biomarker-directed therapy, despite the potential for improved clinical outcomes. Access to timely, accurate, and comprehensive molecular profiling, including targetable protein overexpression, is essential to allow fully informed treatment decisions to be taken. In turn, this requires optimal tissue management to protect and maximize the use of this precious finite resource. Here, a group of leading thoracic pathologists recommend factors to consider for optimal tissue management. Starting from when lung cancer is first suspected, keeping predictive biomarker testing in the front of the mind should drive the development of practices and procedures that conserve tissue appropriately to support molecular characterization and treatment selection., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Purpose: Genomic alterations are fundamental for molecular-guided therapy in patients with breast and lung cancer. However, the turn-around time of standard next-generation sequencing assays is a limiting factor in the timely delivery of genomic information for clinical decision-making., Methods: In this study, we evaluated genomic alterations in 54 cerebrospinal fluid samples from 33 patients with metastatic lung cancer and metastatic breast cancer to the brain using the Oncomine Precision Assay on the Genexus sequencer. There were nine patients with samples collected at multiple time points., Results: Cell-free total nucleic acids (cfTNA) were extracted from CSF (0.1-11.2 ng/μl). Median base coverage was 31,963× with cfDNA input ranging from 2 to 20 ng. Mutations were detected in 30/54 CSF samples. Nineteen (19/24) samples with no mutations detected had suboptimal DNA input (< 20 ng). The EGFR exon-19 deletion and PIK3CA mutations were detected in two patients with increasing mutant allele fraction over time, highlighting the potential of CSF-cfTNA analysis for monitoring patients. Moreover, the EGFR T790M mutation was detected in one patient with prior EGFR inhibitor treatment. Additionally, ESR1 D538G and ESR1::CCDC170 alterations, associated with endocrine therapy resistance, were detected in 2 mBC patients. The average TAT from cfTNA-to-results was < 24 h., Conclusion: In summary, our results indicate that CSF-cfTNA analysis with the Genexus-OPA can provide clinically relevant information in patients with brain metastases with short TAT., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Genomic profiling is needed to identify actionable alterations in non-small cell lung cancer (NSCLC). Panel-based testing such as next-generation sequencing (NGS) is often preferred to interrogate multiple alterations simultaneously. In this study, we evaluate the utility of an RNA-based NGS assay to detect genomic alterations in NSCLC cytology specimens and compare these results to fluorescence in situ hybridization (FISH) testing., Methods: A retrospective review was performed of 264 NSCLC cytology specimens that were concurrently tested for gene fusions by RNA-based NGS and ALK, RET, and/or ROS1 by FISH., Results: Genomic alterations were detected in 29 cases by NGS, including ALK, RET, ROS1, NTRK, NUTM1, and FGFR3 fusions and MET exon 14 skipping alterations. Of the 20 cases with ALK, RET, and ROS1 fusions detected by NGS, 16 (80%) were concordant with the corresponding FISH results. Three cases showed discordance, where EML4::ALK (n = 2) and SLC34A2::ROS1 (n = 1) fusions were not detected by the corresponding FISH assay; one case with EZR::ROS1 was inadequate for FISH. No gene fusions were detected in 181 cases by NGS and 54 cases failed testing. The concordance rates for detecting ALK, RET, and ROS1 fusions using NGS and FISH were 97%, 100%, and 99.5%, respectively., Conclusion: RNA-based NGS can be used to detect gene fusions in NSCLC cytology cases with high concordance with FISH results. However, RNA-based NGS may have high failure rates and therefore a low threshold for reflexing inadequate cases to an orthogonal testing method is essential for comprehensive genomic profiling., (© 2023 American Cancer Society.)

Epidermal growth factor receptor variant III (EGFRvIII, the deletion of exons 2-7) is a recurrent intragenic EGFR::EGFR .E1E8 fusion that occurs in high-grade gliomas. The presence of EGFRvIII in other solid tumors has not been well characterized. We retrospectively reviewed advanced malignant solid tumor cases tested by a custom hybrid capture 610-gene next-generation sequencing platform from 2021 to 2022. EGFRvIII was identified in 17 of 4331 (0.4%) cases, including 16 of 238 (7%) brain tumors and 1/301 (0.3%) breast tumors. EGFRvIII-positive brain tumors were all glioblastoma IDH-wildtype, most with concurrent TERT promoter mutation (14 of 16), EGFR amplification (13 of 16), and EGFR mutation (8 of 16). The only EGFRvIII-positive breast lesion was a sarcomatoid neoplasm in a young female patient. A separate breast case tested outside our institution with reported EGFRvIII was noted in a young female patient with a malignant phyllodes tumor with stromal overgrowth. Microscopically, both EGFRvIII-positive breast tumors showed high-grade sarcomatoid morphology with brisk mitotic activity. In summary, EGFRvIII is rare, occurring primarily in glioblastoma and rarely in breast sarcomatoid neoplasm, with no instances identified in other tumor types in our series. This select group of patients may benefit from chemotherapy and/or targeted anti-EGFR therapy.

Background: National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice assigns patients aged 1-21 years with refractory solid tumors, brain tumors, lymphomas, and histiocytic disorders to phase II trials of molecularly targeted therapies based on detection of predefined genetic alterations. Patients whose tumors harbored EZH2 mutations or loss of SMARCB1 or SMARCA4 by immunohistochemistry were treated with EZH2 inhibitor tazemetostat., Methods: Patients received tazemetostat for 28-day cycles until disease progression or intolerable toxicity (max 26 cycles). The primary endpoint was objective response rate; secondary endpoints included progression-free survival and tolerability of tazemetostat., Results: Twenty patients (median age = 5 years) enrolled, all evaluable for response and toxicities. The most frequent diagnoses were atypical teratoid rhabdoid tumor (n = 8) and malignant rhabdoid tumor (n = 4). Actionable alterations consisted of SMARCB1 loss (n = 16), EZH2 mutation (n = 3), and SMARCA4 loss (n = 1). One objective response was observed in a patient with non-Langerhans cell histiocytosis with SMARCA4 loss (26 cycles, 1200 mg/m2/dose twice daily). Four patients with SMARCB1 loss had a best response of stable disease: epithelioid sarcoma (n = 2), atypical teratoid rhabdoid tumor (n = 1), and renal medullary carcinoma (n = 1). Six-month progression-free survival was 35% (95% confidence interval [CI] = 15.7% to 55.2%) and 6-month overall survival was 45% (95% CI = 23.1% to 64.7%). Treatment-related adverse events were consistent with prior tazemetostat reports., Conclusions: Although tazemetostat did not meet its primary efficacy endpoint in this population of refractory pediatric tumors (objective response rate = 5%, 90% CI = 1% to 20%), 25% of patients with multiple histologic diagnoses experienced prolonged stable disease of 6 months and over (range = 9-26 cycles), suggesting a potential effect of tazemetostat on disease stabilization., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The International Academy of Cytology has joined with the International Agency for Research on Cancer to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians, and improve patient care. The WHO System describes 5 categories for reporting lung cytopathology: 'Insufficient/Inadequate/Nondiagnostic', 'Benign', 'Atypical', 'Suspicious for malignancy', and 'Malignant', each one with a clear descriptive term, a definition, a risk of malignancy, and a suggested management algorithm. The key diagnostic cytopathologic features of each of the lesions within each category have been established by consensus through an Expert Editorial Board, who are also the authors of this review and selected for each reporting system and chosen based on their expertise in the field and/or diversity of geographical representation. Many other co-authors from around the world also contributed. The assignment of writing and editing responsibilities used the same model as that used for the WHO Classification of Tumours (https://whobluebooks.iarc.fr/about/faq/). The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and guides in sampling and processing techniques to optimize the handling and preparation of specimens. The WHO System was created by the authors to be applicable globally and is based on cytomorphology with possibilities for additional diagnostic management of the patient. The authors are aware that local medical and pathology resources would differ, especially in low- and middle-income countries. The WHO Tumour Classification for Thoracic Tumors, Fifth Edition, is directly accessible through the online WHO System., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: MET amplification is a known resistance mechanism to EGFR tyrosine kinase inhibitor (TKI) treatment in EGFR -mutant NSCLC. Dual EGFR-MET inhibition has been reported with success in overcoming such resistance and inducing clinical benefit. Resistance mechanisms to dual EGFR-MET inhibition require further investigation and characterization., Methods: Patients with NSCLC with both MET amplification and EGFR mutation who have received crizotinib, capmatinib, savolitinib, or tepotinib plus osimertinib (OSI) after progression on OSI at MD Anderson Cancer Center were included in this study. Molecular profiling was completed by means of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Radiological response was assessed on the basis of Response Evaluation Criteria in Solid Tumors version 1.1., Results: From March 2016 to March 2022, 23 treatments with dual MET inhibitor and osi were identified with a total of 20 patients included. Three patients received capmatinib plus OSI after progression on crizotinib plus OSI. Median age was 64 (38-89) years old and 75% were female. MET amplification was detected by FISH in 14 patients in the tissue, NGS in 10 patients, and circulating tumor DNA in three patients. Median MET gene copy number was 13.6 (6.4-20). Overall response rate was 34.8% (eight of 23). In assessable patients, tumor shrinkage was observed in 82.4% (14 of 17). Median time on treatment was 27 months. Two of three patients responded to capmatinib plus OSI after progression on crizotinib plus OSI. Dual EGFR-MET inhibition was overall well tolerated. Two patients on crizotinib plus OSI and one pt on capmatinib plus OSI discontinued therapy due to pneumonitis. One pt discontinued crizotinib plus OSI due to gastrointestinal toxicity. Six patients were still on double TKI treatment. At disease progression to dual EGFR-MET inhibition, FISH and NGS on tumor and plasma were completed in six patients. Notable resistance mechanisms observed include acquired MET D1246H (n = 1), acquired EGFR C797S (n = 2), FGFR2 fusion (n = 1, concurrent with C797S), and EGFR G796S (n = 1, concurrent with C797S). Four patients lost MET amplification., Conclusions: Dual EGFR and MET inhibition yielded high clinical response rate after progression on OSI. Resistance mechanisms to EGFR-MET double TKI inhibition include MET secondary mutation, EGFR secondary mutation, or loss of MET amplification., (© 2023 The Authors.)

The role of combination chemotherapy with immune checkpoint inhibitors (ICI) (ICI-chemo) over ICI monotherapy (ICI-mono) in non-small cell lung cancer (NSCLC) remains underexplored. In this retrospective study of 1133 NSCLC patients, treatment with ICI-mono vs ICI-chemo associate with higher rates of early progression, but similar long-term progression-free and overall survival. Sequential vs concurrent ICI and chemotherapy have similar long-term survival, suggesting no synergism from combination therapy. Integrative modeling identified PD-L1, disease burden (Stage IVb; liver metastases), and STK11 and JAK2 alterations as features associate with a higher likelihood of early progression on ICI-mono. CDKN2A alterations associate with worse long-term outcomes in ICI-chemo patients. These results are validated in independent external (n = 89) and internal (n = 393) cohorts. This real-world study suggests that ICI-chemo may protect against early progression but does not influence overall survival, and nominates features that identify those patients at risk for early progression who may maximally benefit from ICI-chemo., (© 2023. The Author(s).)

Background: Non-small cell lung carcinoma (NSCLC) patients with BRAF V600E-mutated tumors respond to targeted therapy. Testing for BRAF V600E is commonly performed with molecular methods; however, a mutation-specific VE1 antibody clone can provide an alternative testing option using immunohistochemistry (IHC) for practices using single-gene testing and in situations when the specimen is inadequate for molecular testing. This study evaluates the usefulness of VE1 IHC in screening for BRAF V600E mutations in NSCLC cytology specimens., Methods: The authors retrospectively identified cytology cases with a diagnosis of NSCLC that had BRAF V600E IHC performed on cell block sections with the monoclonal VE1 antibody clone. The BRAF V600E IHC results were compared with those of molecular testing performed with an amplicon-based next-generation sequencing assay., Results: There were 201 NSCLC cases evaluated. The VE1 IHC was positive in seven of seven BRAF V600E-mutated tumors (100%) and was negative in 158 of 158 nonmutated BRAF V600E tumors (100%). Thirty cases did not undergo molecular testing, primarily because of insufficient tissue or because molecular testing was performed on an alternative specimen. Six cases showed equivocal weak/focal staining: Two cases demonstrated BRAF V600E mutations, and four cases were negative by molecular testing., Conclusions: This study suggests that BRAF V600E IHC can be used reliably to screen NSCLC cytology specimens, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining., (© 2022 American Cancer Society.)

The International Academy of Cytology has joined with the International Agency for Research on Cancer (IARC) to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This WHO System defines five categories for reporting lung cytopathology, that is, "Insufficient"/"Inadequate"/"Non-diagnostic," "Benign," "Atypical," "Suspicious for malignancy," and "Malignant," each with a clear descriptive term for the category, a definition, a risk of malignancy and a suggested management algorithm. The key diagnostic cytopathology features of each of the lesions within each category have been established by consensus and will be presented more fully in a subsequent IARC e-book and published hard cover book.The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and provides a review to guide sampling and processing techniques to optimize the handling and preparation of the cytopathology sample emphasizing the cytomorphological differential diagnosis to aid low-resourced settings. The authors recognize that local medical and pathology resources will vary, particularly in low- and middle-income countries, and have developed the WHO System to make it applicable worldwide based on cytomorphology with options for further diagnostic management of the patient.The online WHO System provides a direct link to the WHO Tumour Classification for Thoracic Tumours 5th Edition. It will raise the profile and use of cytopathology by increasing awareness of its current role and its potential role in the era of personalized medicine based on molecular pathology utilizing "small biopsies." Ultimately, the System will improve patient care and outcomes.This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians and improve patient care. The System is based on the current role of lung cytopathology and synthesizes the existing evidence while highlighting areas requiring further research and the future potential role of lung cytopathology., (© 2022 S. Karger AG, Basel.)

Aims: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated., Methods: Cell lines harbouring EML4 (13) -ALK (20) and SLC34A2 (4) -ROS1 (32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides., Results: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms., Conclusions: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches., Competing Interests: Competing interests: UM has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Eli Lilly, Diaceutics, GSK, Merck and Astra Zeneca, unrelated to the current work. Lukas Bubendorf has a consulting or advisory role with Astra Zeneca, AbbVie, Bayer, Boehringer Ingelheim, Eli Lilly, MSD, Pfizer, Takeda and F. Hoffmann-La Roche and has received research funding (institution) from F. Hoffmann-La Roche, MSD and Sanofi. PH reports personal fees (as advisor) from Roche, Astrazeneca, BMS, MSD, Pfizer, Bayer, Amgen, Illumina, Qiagen, Thermo Fisher Scientific, Biocartis, Ed Lilly, unrelated to the current work. SM-B has received personal fees (as consultant and/or speaker bureau) from Astra Zeneca, Roche, BMS, Novartis, GSK, MSD, Targos, Merck, unrelated to the current work. Spasenija Savic Prince received personal fees from MSD, Astra Zeneca, Boehringer Ingelheim, Roche, Pfizer, Bristol-Myers Squibb and Thermo Fisher Scientific, unrelated to the submitted work. EV has received personal fees (as consultant and/or speaker bureau) from Diaceutics, unrelated to the current work. GTr reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer, Boehringer Ingelheim, Eli Lilly, BMS, GSK, Menarini, AstraZeneca, Amgen and Bayer, unrelated to the current work. The other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Ancillary and molecular testing of cytopathology specimens has emerged as a reliable and useful tool to provide diagnostic information and treatment-related biomarker status for the management of cancer patients. The cytology specimens obtained through minimally invasive means have proven suitable testing substrates for a variety of ancillary tests, including immunohistochemistry, fluorescence in situ hybridization, as well as polymerase chain reaction and next generation sequencing molecular techniques. By focusing specifically on the cytology specimen, this review provides an overview of basic testing considerations and assay selection in addition to updates on the ancillary testing of cytologic tumor specimens from the lung, salivary gland, and thyroid., (Copyright © 2022 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Background: A deficiency in DNA mismatch repair function in neoplasms can be assessed by an immunohistochemical (IHC) analysis of the deficiency/loss of the mismatch repair proteins (dMMR) or by PCR-based methods to assess high microsatellite instability (MSI-H). In some cases, however, there is a discrepancy between the IHC and MSI analyses. Several studies have addressed the issue of discrepancy between IHC and MSI deficiency assessment, but there are limited studies that also incorporate genetic/epigenetic alterations. Methods: In this single-institution retrospective chart-review study, we reviewed 706 neoplasms assessed between 2015 and 2021. All eligible neoplasms were assessed by IHC testing, MSI analysis by PCR-based assay, and tumor-normal paired next-generation sequencing (NGS) analysis. Eighty percent of neoplasms with MLH1 protein loss had a concurrent MLH1 promoter methylation analysis. Mutation data for MMR genes, IHC, MSI analysis, and tumor histology were correlated with each other. Results: Fifty-eight (8.2%) of 706 neoplasms had MSI-H by PCR and/or dMMR by IHC. Of the 706 analyzed neoplasms, 688 neoplasms (98%) had concordant results: MSI-H/dMMR (n = 44), microsatellite-stable (MSS)/proficient MMR (pMMR) (n = 625), and MSI-Low (L)/pMMR (n = 19). Of the remaining 18 neoplasms, 9 had a major discordance: MSS/loss of MSH2 and MSH6 (n = 3), MSS/loss of MSH6 (n = 2), MSS/Loss of MLH1 and PMS2 (n = 1), and MSI-High/pMMR (n = 3). In total, 57% of cases with dMMR and 61% of cases with MSI-H had a null mutation of an MMR gene mutation (or methylation of the MLH1 promoter), whereas this figure was 1% for neoplasms with a normal IHC or MSI pattern (p < 0.001). Among 9 cases with major discordance between MSI and IHC, only 3 cases (33%) had an underlying genetic/epigenetic etiology, whereas 37 (76%) of 49 cases with MSI-H and/or dMMR and without major discordance had an underlying genetic abnormality (p = 0.02). Discussion: For most neoplasms, IHC and PCR-based MSI testing results are concordant. In addition, an underlying genetic abnormality (a null mutation of an MMR gene or MLH1 promoter methylation) was attributable to dMMR and/or MSI-H findings. For neoplasms with major discordance in IHC and MSI testing, the addition and integration of NGS results and MLH1 promoter methylation analyses can be beneficial for resolving borderline cases, thereby facilitating patient management.

Aims: In advanced-stage non-small-cell lung cancer (NSCLC), incomplete genotyping for guideline-recommended genomic biomarkers poses a significant challenge to making informed and timely clinical decisions. We report our institution's experience in assessing the adequacy of small specimens for comprehensive genomic profiling for guideline-recommended lung cancer biomarker testing., Methods: We performed a retrospective evaluation of all image-guided procedures for NSCLC performed in our institution between October 2016 and July 2018, including core needle biopsy (CNB) and fine-needle aspiration (FNA) in patients who had undergone genomic profiling for lung cancer. Lung cancer biomarker adequacy, defined as successful testing of guideline-recommended biomarkers including, epidermal growth factor receptor ( EGFR ); serine/threonine protein kinase B-Raf ( BRAF ); anaplastic lymphoma kinase ( ALK ); proto-oncogene tyrosine protein kinase ROS ( ROS1 ); Rearranged during Transfection ( RET ); Tyrosine protein kinase Met ( MET ); and programmed cell death ligand 1 (PD-L1), was evaluated., Results: A total of 865 cases were evaluated in this study, 785 of which included testing of all lung cancer biomarkers. Lung tissue was adequate for biomarker testing in 84% of cases; this rate increased to 87% when biomarker testing was combined with concurrently acquired FNA or CNB specimens. Biomarker testing success correlated strongly with DNA concentration (p<0.0001) and the use of 22G needles in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) procedures (p=0.0035). Biomarker testing of CNB specimens showed a significantly higher success rate than did biomarker testing of cytology FNA specimens (p=0.0005). The adequacy of EBUS-TBNA samples was not significantly different from that of the transthoracic needle aspiration samples (p=0.40). Variables such as age, gender, lesion size, site, diagnosis and number of needle passes showed no significant correlation with success rates in lung cancer biomarker testing., Conclusion: The growing numbers of therapeutic biomarkers in NSCLC requires judicious triage of limited-volume tissue from small specimens. Our study showed that thoracic small tissue specimens can be used successfully to provide prognostic and predictive information for the current guideline-recommended biomarkers for NSCLC in most cases., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)