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* 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. (Arch Pathol Lab Med. 2024;148:e111-e153; doi: 10.5858/arpa.2023-0483-CP, In 2014, the Pathology and Laboratory Quality Center for Evidence-Based Guidelines ('The Center') of the College of American Pathologists (CAP) published a clinical practice guideline that created recommendations for analytic [...]

Context.--RNA-based next-generation sequencing (NGS) assays are being used with increasing frequency for comprehensive molecular profiling of solid tumors. Objective.--To evaluate factors that might impact clinical assay performance. Design.--A 4-month retrospective review of cases analyzed by a targeted RNA-based NGS assay to detect fusions was performed. RNA extraction was performed from formalin-fixed, paraffin-embedded tissue sections and/or cytology smears of 767 cases, including 493 in-house and 274 outside referral cases. The types of samples included 422 core needle biopsy specimens (55%), 268 resection specimens (35%), and 77 cytology samples (10%). Results.--Successful NGS fusion testing was achieved in 697 specimens (90.9%) and correlated positively with RNA yield (P < .001) and negatively with specimen necrosis (P = .002), decalcification (P < .001), and paraffin block age of more than 2 years (P = .001). Of the 697 cases that were successfully sequenced, 50 (7.2%) had clinically relevant fusions. The testing success rates and fusion detection rates were similar between core needle biopsy and cytology samples. In contrast, RNA fusion testing was often less successful using resection specimens (P = .007). Testing success was independent of the tumor percentage in the specimen, given that at least 20% tumor cellularity was present. Conclusions.--The success of RNA-based NGS testing is multifactorial and is influenced by RNA quality and quantity. Identification of preanalytical factors affecting RNA quality and yield can improve NGS testing success rates. (Arch Pathol Lab Med. 2021;145:1405-1412; doi: 10.5858/arpa.2020-0415-OA), With the increasing use of targeted therapeutic agents in solid tumors, RNA-based next-generation sequencing (NGS) assays are progressively used for comprehensive molecular analysis. (1) Chromosome translocations and/or gene fusions are [...]

Intratumoral heterogeneity (ITH) is a fundamental property of cancer; however, the origins of ITH remain poorly understood. We performed single-cell transcriptome profiling of peritoneal carcinomatosis (PC) from 15 patients with gastric adenocarcinoma (GAC), constructed a map of 45,048 PC cells, profiled the transcriptome states of tumor cell populations, incisively explored ITH of malignant PC cells and identified significant correlates with patient survival. The links between tumor cell lineage/state compositions and ITH were illustrated at transcriptomic, genotypic, molecular and phenotypic levels. We uncovered the diversity in tumor cell lineage/state compositions in PC specimens and defined it as a key contributor to ITH. Single-cell analysis of ITH classified PC specimens into two subtypes that were prognostically independent of clinical variables, and a 12-gene prognostic signature was derived and validated in multiple large-scale GAC cohorts. The prognostic signature appears fundamental to GAC carcinogenesis and progression and could be practical for patient stratification. Single-cell analysis of gastric cancer samples tracks the cell of origin of metastatic lesions and identifies an independent prognostic signature of the clinical outcome., Author(s): Ruiping Wang [sup.1] , Minghao Dang [sup.1] , Kazuto Harada [sup.2] [sup.12] , Guangchun Han [sup.1] , Fang Wang [sup.3] , Melissa Pool Pizzi [sup.2] , Meina Zhao [sup.2] [...]

* Context.--The approval of pembrolizumab for treatment of patients with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) advanced cancers has led to increased requests for MSI and/or MMR immunoperoxidase (IPOX) testing. Diagnoses for patients with advanced-stage cancer are frequently made from cytology specimens. Objective.--To investigate the feasibility of using cell block (CB) preparations of effusions for MMR IPOX evaluation. Design.--Surgical pathology cases of colorectal and endometrial carcinomas with known MMR/MSI status and matched effusions with available CBs were identified. Cell block sections were evaluated for adequacy and stained with MMR IPOX (MSH2, MSH6, MLH1, and PMS2). The CBs were reviewed, the number of tumor cells quantified, and MMR IPOX was interpreted as retained, lost, suboptimal, or noncontributory. Results.--We identified 748 cases with MMR/MSI testing on surgical specimens having matched effusions. Of these, 131 cases (17.5%) had an available CB and 53 were deemed adequate for MMR IPOX staining. MMR IPOX results between effusion CBs and surgical pathology specimens were concordant in 45 of 53 (85%), inconclusive in 6 of 53 (11%), and discordant in 2 of 53 (4%) cases. Conclusions.--There was high concordance of MMR IPOX testing between cytologic and surgical specimens, with no false-positive and 2 false-negative CB results. Limited tumor cells, staining in cells indefinite as tumor, tumor staining heterogeneity, and lack of internal control staining were problematic in some cases. Our findings indicate that cytologic effusion specimens may be suitable substrates for MMR IPOX biomarker testing; however, inconclusive cases need to be interpreted with caution. (Arch Pathol Lab Med. 2021;145:46-54; doi: 10.5858/arpa.2019-0398-OA), NA mismatch repair (MMR) is a highly conserved process involving the 4 key genes mutL homologue 1 (MLH1), mutS homologue 2 (MSH2), mutS homologue 6 (MSH6), and postmeiotic segregation increased [...]

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/m2/dose orally twice a day (dose level 1 [DL1], n = 15) or 350 mg/m2/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/m2/dose orally twice a day. Limited single-agent efficacy was observed in a biomarker-selected cohort of refractory pediatric tumors. Ulixertinib, a novel targeted ERK inhibitor with no previous pediatric data, was successfully evaluated in a national precision medicine basket trial (Pediatric MATCH). [ABSTRACT FROM AUTHOR]

Context.--The need for appropriate specimen use for ancillary testing has become more commonplace in the practice of pathology. This, coupled with improvements in technology, often provides less invasive methods of testing, but presents new challenges to appropriate specimen collection and handling of these small specimens, including thoracic small biopsy and cytology samples. Objective.--To develop a clinical practice guideline including recommendations on how to obtain, handle, and process thoracic small biopsy and cytology tissue specimens for diagnostic testing and ancillary studies. Methods.--The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Core needle biopsy, touch preparation, fine-needle aspiration, and effusion specimens with thoracic diseases including malignancy, granulomatous process/sarcoidosis, and infection (eg, tuberculosis) were deemed within scope. Ancillary studies included immunohistochemistry and immunocytochemistry, fluorescence in situ hybridization, mutational analysis, flow cytometry, cytogenetics, and microbiologic studies routinely performed in the clinical pathology laboratory. The use of rapid on-site evaluation was also covered. Results.--Sixteen guideline statements were developed to assist clinicians and pathologists in collecting and processing thoracic small biopsy and cytology tissue samples. Conclusions.--Based on the systematic review and expert panel consensus, thoracic small specimens can be handled and processed to perform downstream testing (eg, molecular markers, immunohistochemical biomarkers), core needle and fine-needle techniques can provide appropriate cytologic and histologic specimens for ancillary studies, and rapid on-site cytologic evaluation remains helpful in appropriate triage, handling, and processing of specimens. doi: 10.5858/arpa.2020-0119-CP, With the advances in minimally invasive sampling techniques, the ability to provide a diagnosis and perform multiple ancillary studies on limited-volume tissue samples, including small biopsy and cytology specimens, has [...]

Context.--Advanced-stage non-small cell lung carcinoma patients on EGFR-targeted tyrosine kinase inhibitors frequently present with an acquired EGFR T790M resistance mutation. Early detection using a high-sensitivity assay is critical to allow patients to switch to third-generation tyrosine kinase inhibitors. The detection of EGFR T790M mutation is often challenging because of low tumor fraction in posttreatment specimens. Because a large fraction of non-small cell lung carcinoma patients are given a diagnosis by cytology, evaluating a high-sensitivity technique for EGFR T790M detection in these specimens is essential. Objective.--To evaluate a high-sensitivity droplet digital polymerase chain reaction (ddPCR) assay for EGFR T790M using different cytologic specimen preparations. Design.--A total of 42 cytology samples, including smears and cell block preparation, were evaluated for EGFR T790M using ddPCR. The results of the mutation assay were compared to the patient's known EGFR T790M mutation status. Results.--The ddPCR assay successfully determined the EGFR T790M mutation status in 36 of 42 samples (86%), including samples with low tumor fraction ([less than or equal to] 20%). In 4 cases the results of the ddPCR assay could not be compared because the mutation status was unknown at the time of collection of the cytology sample. There was 1 false-positive result, with borderline positivity, and 1 false-negative result. Overall sensitivity and specificity of the ddPCR assay were 93% and 96%, respectively. Conclusions.--Our results indicate that EGFR T790M ddPCR is a highly sensitive and specific mutational assay that can be used reliably in cytologic specimens, including samples with low tumor fraction. doi: 10.5858/arpa.2019-0411-OA, The management of non-small cell lung carcinoma (NSCLC), particularly lung adenocarcinoma, has undergone a paradigm shift, with patients harboring sensitizing somatic mutations in EGFR showing a dramatic response to targeted [...]

* Context.--Social media sites are increasingly used for education, networking, and rapid dissemination of medical information, but their utility for facilitating research has remained largely untapped. Objective.--To describe in detail our experience using a social media platform (Twitter) for the successful initiation, coordination, and completion of an international, multi-institution pathology research study. Design.--Following a tweet describing a hitherto-unreported biopsy-related histologic finding in a mediastinal lymph node following endobronchial ultrasound-guided transbronchial needle aspiration, a tweet was posted to invite pathologists to participate in a validation study. Twitter's direct messaging feature was used to create a group to facilitate communication among participating pathologists. Contributing pathologists reviewed consecutive cases of mediastinal lymph node resection following endobronchial ultrasound-guided transbronchial needle aspiration and examined them specifically for biopsy site changes. Data spreadsheets containing deidentified data and digital photomicrographs of suspected biopsy site changes were submitted via an online file hosting service for central review by 5 pathologists from different institutions. Results.--A total of 24 pathologists from 14 institutions in 5 countries participated in the study within 143 days of study conception, and a total of 297 cases were collected and analyzed. The time interval between study conception and acceptance of the manuscript for publication was 346 days. Conclusions.--To our knowledge, this is the first time that a social media platform has been used to generate a research idea based on a tweet, recruit coinvestigators publicly, communicate with collaborating pathologists, and successfully complete a pathology study. (Arch Pathol Lab Med. 2020;144:878-882; doi: 10.5858/arpa.2019-0297-OA), The utility of social media as an instrument for education, advocacy, and community development in medicine is well documented, and applications of social media in these spheres are expanding exponentially. [...]

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTMBRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction-based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTMBRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation-detecting tests.

BackgroundThe purpose of this study was to determine the prevalence of MET amplification and mutation among GU malignancies and its association with clinical factors and responses to c-MET inhibitors.Patients and methodsPatients with GU malignancies referred to our Phase I Clinical Trials Program were evaluated for MET mutation and amplification and outcomes using protocols with c-MET inhibitors.ResultsMET amplification was found in 7 of 97 (7.2%) patients (4/27 renal [all clear cell], 1/18 urothelial, and 2/12 adrenocortical carcinoma), with MET mutation/variant in 3 of 54 (5.6%) (2/20 renal cell carcinoma [RCC] [1 clear cell and 1 papillary] and 1/16 prostate cancer). No demographic characteristics were associated with specific MET abnormalities, but patients who tested positive for mutation or amplification had more metastatic sites (median, 4 vs. 3 for wild type MET). Median overall survival after phase I consultation was 6.1 and 11.5 months for patients with and without a MET alteration, respectively (hazard ratio, 2.8; 95% confidence interval, 1.1 to 6.9; P = .034). Twenty-nine (25%) patients were treated according to a c-MET inhibitor protocol. Six (21%) had a partial response (prostate and RCC) and 10 (34%) had stable disease as best response. Median time to tumor progression was 2.3 months (range, 0.4-19.7) for all treated patients with no responses in patients with a MET abnormality or single-agent c-MET inhibitor treatment.ConclusionMET genetic abnormalities occur in diverse GU malignancies and are associated with a worse prognosis in a phase I setting. Efficacy of c-MET inhibitors was more pronounced in patients without MET abnormalities and when combined with other targets/drugs.

We sought to investigate the demographics and tumor-associated features in patients with gastroesophageal (GE) malignancies referred to our Phase I Program who had formalin-fixed, paraffin-embedded tissue from archival or new biopsies tested for MET mutation and/or amplification. MET amplification was found in 5 of 76 (6.6%) patients (3/34 [8.8%] esophageal, 2/26 [7.7%] gastric and none in 22 gastroesophageal junction cancers). The only MET mutation detected in 3 of 41 (7.3%) patients was N375S. No demographic and histologic characteristics were associated with specific MET abnormalities. Median overall survival was 3 and 5 months for patients with and without a MET alteration, respectively (hazard ratio [HR] = 2.1; 95% CI, 0.8 to 5.5; P=.14). Sixteen of 81 (20%) patients were enrolled in a c-MET inhibitor trial. Best responses were stable disease in 3 patients (19%), including a patient with esophageal adenocarcinoma that remained on the trial for 9.9 months (wild-type for MET abnormality). All tumors with MET abnormality (n=3) progressed on a c-MET inhibitor in fewer than 2 months. In conclusion, MET abnormalities can be found in a small group of patients with GE adenocarcinoma and further studies are necessary to better characterize the prognostic and predictive impact of MET alterations.

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. [ABSTRACT FROM AUTHOR]

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. This study explores the use of an RNA‐based next‐generation sequencing assay for detecting genomic alterations in non–small cell lung cancer cytology specimens in comparison to fluorescence in situ hybridization testing. The results have important implications for clinical oncology practice, including selection of biomarkers and biomarker testing workflow, in addition to specimen collection and optimizing the use of cytology samples. [ABSTRACT FROM AUTHOR]

Simple Summary: Epidermal growth factor receptor variant III (EGFRvIII) is caused by the deletion of six exons and the fusion of exons 1 to exon 8. EGFRvIII occurs frequently in glioblastoma, a type of high-grade brain tumor; however, its presence in other solid tumors is not well characterized. Upon reviewing 4331 solid tumor cases tested via the 610-gene sequencing platform, EGFRvIII was identified in 17 cases, including 16 brain tumors and one breast tumor. EGFRvIII-positive brain tumors were all glioblastoma with wild-type IDH1/2 status, most with EGFR amplification and EGFR mutation. The only EGFRvIII-positive breast lesion was in a young female patient. A separate breast case tested outside our institution with reported EGFRvIII was noted in a young female patient. Both EGFRvIII-positive breast tumors showed high-grade sarcomatoid morphology. In summary, EGFRvIII is rare, occurring primarily in glioblastoma and rarely in breast sarcomatoid neoplasm. This select group of patients may benefit from chemotherapy and/or targeted anti-EGFR therapy. 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. [ABSTRACT FROM AUTHOR]