Prognostic Immunohistochemistry for Ki-67 and OTP on Small Biopsies of Pulmonary Carcinoid Tumors: Ki-67 Index Predicts Progression-free Survival and Atypical Histology.

Prognostic stratification of pulmonary carcinoids into "typical" and "atypical" categories requires examination of large tissue volume. However, there is a need for tools that provide similar prognostic information on small biopsy samples. Ki-67 and OTP immunohistochemistry have shown promising prognostic value in studies of resected pulmonary carcinoids, but prognostic value when using biopsy/cytology specimens is unclear. Ki-67 immunohistochemistry was performed on small biopsy/cytology specimens from pulmonary carcinoid tumors (n=139), and labeling index was scored via automated image analysis of at least 500 cells. OTP immunohistochemistry was performed on 70 cases with sufficient tissue and scored as positive or negative (<20% tumor nuclei staining). Higher Ki-67 index was associated with worse disease-specific progression-free survival (ds-PFS), with 3% and 4% thresholds having similarly strong associations with ds-PFS (P<0.001, hazard ratio ≥11). Three-year ds-PFS was 98% for patients with Ki-67 <3% and 89% for patients with Ki-67≥3% (P=0.0006). The optimal Ki-67 threshold for prediction of typical versus atypical carcinoid histology on subsequent resection was 3.21 (AUC 0.68). Negative OTP staining approached significance with atypical carcinoid histology (P=0.06) but not with ds-PFS (P=0.24, hazard ratio=3.45), although sample size was limited. We propose that Ki-67 immunohistochemistry may contribute to risk stratification for carcinoid tumor patients based on small biopsy samples. Identification of a 3% hot-spot Ki-67 threshold as optimal for prediction of ds-PFS is notable as a 3% Ki-67 threshold is currently used for gastrointestinal neuroendocrine tumor stratification, allowing consideration of a unified classification system across organ systems.

Primary Pulmonary Myxoid Sarcoma and Thoracic Angiomatoid Fibrous Histiocytoma: Two Sides of the Same Coin?

Primary pulmonary myxoid sarcoma (PPMS) and thoracic angiomatoid fibrous histiocytoma (AFH) are rare neoplasms with EWSR1 fusions and overlapping morphology. Both tumor types often show epithelial membrane antigen expression, but AFH characteristically co-expresses desmin. We encountered a case of PPMS with the unexpected finding of patchy, strong anaplastic lymphoma kinase (ALK) (previously reported in AFH) and synaptophysin expression. We evaluated a cohort of PPMS and thoracic AFH with systematic morphologic comparison and surveyed for aberrant expression of ALK and synaptophysin. Medical records and slides were reviewed for 16 molecularly confirmed cases of PPMS (n=5) and thoracic AFH (n=11). Each case was scored for morphologic characteristics typical of PPMS and/or AFH. ALK, synaptophysin, chromogranin, desmin, and epithelial membrane antigen immunostains were performed on cases with available tissue. AFH and PPMS cases showed similar age at presentation and long-term tumor behavior. Almost all cases of PPMS and AFH had a fibrous pseudocapsule and lymphoid rim. All PPMS had myxoid stroma and reticular growth pattern, but these features were also present in a subset of AFH. Synaptophysin expression was present in 6 of 11 AFH and 1 of 5 PPMS; all tested cases were negative for chromogranin (n=15). One case of AFH and 1 case of PPMS showed focally strong coexpression of synaptophysin and ALK. AFH and PPMS show considerable clinicopathologic overlap. When supportive, the immunohistochemical findings described may aid in diagnosis before molecular confirmation. PPMS and AFH may be morphologic variants of the same clinicopathologic entity, which can show more immunophenotypic variability than previously reported.

CSF1 expression in xanthogranulomatous epithelial tumor/keratin-positive giant cell-rich tumor.

"Xanthogranulomatous epithelial tumor" (XGET) and "keratin-positive giant cell-rich soft tissue tumor" (KPGCT), two recently described mesenchymal neoplasms, likely represent different aspects of a single entity. Both tumors are composed of only a small minority of tumor cells surrounded by large numbers of non-neoplastic inflammatory cells and histiocytes, suggesting production of a paracrine factor with resulting "landscape effect," as seen in tenosynovial giant cell tumor. Recent evidence suggests that the paracrine factor in XGET/KPGCT may be CSF1, as in tenosynovial giant cell tumor. We hypothesized that CSF1 is overexpressed in XGET/KPGCT. To test our hypothesis, we performed quantitative real time PCR (qPCR) for CSF1 expression and CSF1 RNAscope chromogenic in situ hybridization (CISH) on 6 cases of XGET/KPGCT. All cases were positive with CSF1 CISH and showed increased expression of CSF1 by qPCR. Our findings provide additional evidence that the CSF1/CSF1R pathway is involved in the pathogenesis of XGET/KPGCT. These findings suggest a possible role for CSF1R inhibition in the treatment of unresectable or metastatic XGET/KPGCT.

Nuclear Protein in Testis (NUT) Carcinoma: A Comprehensive Immunohistochemical Analysis of 57 Cases With Consideration of Interpretation and Pitfall Recognition.

CONTEXT.­: Nuclear protein in testis (NUT) carcinoma is an aggressive carcinoma defined by NUTM1 gene rearrangement. Diagnostic challenges include morphologic overlap with poorly differentiated squamous cell carcinoma, small cell carcinoma, thoracic SMARCA4-deficient undifferentiated tumor, and other small round blue cell tumors. OBJECTIVE.­: To comprehensively study the immunohistochemistry (IHC) features of a large cohort of NUT carcinomas. DESIGN.­: Fifty-seven NUT carcinoma cases were identified from 2012-2022, including 38 thoracic/mediastinal, 13 head and neck/sinonasal, and 6 from other sites. Pathology reports and available slides were reviewed. Comprehensive IHC studies were performed on available cases. RESULTS.­: Keratin stains showed variable positivity and were entirely negative in 15% (8 of 55) of cases. p40 was only positive in 65% (24 of 37) of cases, implying inferior sensitivity when compared to p63 (87% sensitivity, 20 of 23 cases) and other squamous cell markers. Neuroendocrine markers were focally/weakly positive in few cases; however, INSM1 was positive in 54% (7 of 13) of cases, indicating a possible diagnostic pitfall. TTF-1 was mostly negative with focal positivity in 26% (10 of 38) of cases. Occasional CD34 (15%, 3 of 20 cases) and CD99 (21%, 3 of 14 cases) positivity could also cause potential diagnostic confusion. S100, desmin, CD45, and SALL4 were rarely positive. BRG1 and INI1 were retained in all cases. Ki-67 proliferative index was high (median, 60%). PD-L1 was negative in all tested cases. CONCLUSIONS.­: This comprehensive IHC study demonstrates the immunohistochemical spectrum of NUT carcinoma. The findings can help narrow the differential diagnosis and recognize potential pitfalls.

Subtype of SCLC Is an Intrinsic and Persistent Feature Through Systemic Treatment.

Introduction: SCLC is an aggressive malignancy with poor outcome. Most patients have disease recurrence despite treatments with multiple modalities. Subtyping of SCLC has been proposed recently, and novel agents targeting specific subtypes are actively being investigated. In this study, we evaluated the plasticity of subtypes in paired pre- and post-treatment samples. The aim was to understand possible subtype evolution after chemotherapy resistance that could lead to alternate targeted therapy strategies. Methods: A total of 68 samples from 32 patients with sufficient paired specimens were identified from 1998 to 2022. ASCL1, NEUROD1, and POU2F3 immunohistochemistry studies were performed on all cases, and subtyping by predominant expression was determined. Subtype comparison in each patient was performed, and expression analysis was performed on the basis of subtypes. Results: Of 32 cases, 28 (88%) had the same subtype in pre- and first post-treatment specimens. Protein expression level of subtype-specific transcription factor remained stable after chemotherapy. Two of five (40%) NEUROD1-predominant SCLC switched to ASCL1-predominant phenotype after treatment. One case had a pitfall of scoring ASCL1 on specimen with marked crushing artifacts. One case revealed the challenge of proper subtyping for samples with borderline POU2F3 expression. Conclusions: Subtype of SCLC generally remains the same after acquiring chemotherapy resistance. Plasticity was observed with rare cases switching from NEUROD1-predominant to ASC1-predominant SCLC. Resubtyping is unnecessary for the consideration of novel subtype-specific targeted agents, except cases with NEUROD1-predominant subtype.

Age-related histopathological findings in temporal arteries.

AIMS: Giant cell arteritis (GCA) is a systemic vasculitis affecting medium and large arteries in patients aged over 50 years. Involvement of temporal arteries (TA) can lead to complications such as blindness and stroke. While the diagnostic gold standard is temporal artery biopsy (TAB), comorbidities and age-related changes can make interpretation of such specimens difficult. This study aims to establish a baseline of TA changes in subjects without GCA to facilitate the interpretation of TAB. METHODS AND RESULTS: Bilateral TA specimens were collected from 100 consecutive eligible postmortem examinations. Subjects were divided into four age groups and specimens semiquantitatively evaluated for eccentric intimal fibroplasia, disruption and calcification of the internal elastic lamina (IEL), medial attenuation and degree of lymphocytic inflammation of the peri-adventitia, adventitia, media and intima. The individual scores of intimal fibroplasia, IEL disruption and medial attenuation were added to yield a 'combined score (CS)'. Seventy-eight 78 decedents were included in the final analysis following exclusion of 22 individuals for either lack of clinical information or inability to collect TA tissue. A total of 128 temporal artery specimens (50 bilateral from individual decedents, 28 unilateral) were available for examination. Intimal proliferation, IEL loss, IEL calcification and CS increased with age in a statistically significant fashion. Comparison of the oldest age group with the others showed statistically significant differences, although this was not uniformly preserved in comparison between the three youngest groups. CONCLUSION: Senescent arterial changes and healed GCA exhibit histological similarity and such changes increase proportionally with age. The CS demonstrates significant association with age overall and represents a potential avenue for development to 'normalise' TA biopsies from older individuals.

Raphal Cord Rupture: A Rare Mechanism of Aortic Regurgitation in Bicuspid Aortic Valve.

A 70-year-old male with chronic aortic regurgitation was referred with abrupt worsening heart failure. Late referral markers were pulmonary hypertension, mitral regurgitation, and tricuspid regurgitation. Evaluation revealed rupture of a raphal cord or fenestrated raphe from the conjoined cusp of a congenitally bicuspid aortic valve, a rare mechanism of aortic regurgitation. (Level of Difficulty: Intermediate.).

Integrating Molecular Sequencing Into the Pathological Diagnosis of Clinically Suspected Non-Small Cell Lung Carcinomas.

Cancer panel sequencing has been rapidly adopted into clinical practice for the detection of actionable genetic variants in advanced non-small cell lung carcinomas. Unexpected molecular results may lead to reexamination of the clinical diagnosis; however, this scenario has not been systematically evaluated. We reviewed cancer panel next-generation sequencing results from 1007 consecutive patients performed for the clinical indication of non-small cell lung carcinoma along with the corresponding clinical history and anatomical pathology findings. The final integrative diagnosis was a cancer of extrapulmonary origin in 12 of 1007 patients (1.2%). Molecular evidence supporting the final diagnoses included the detection of an UV radiation-associated mutational signature (n = 6), gene fusions (n = 2), and mutations (n = 4). The integrative diagnoses included undifferentiated melanoma, cutaneous squamous cell, and basal cell carcinomas, thyroid carcinoma, urothelial carcinoma, hepatocellular carcinoma, pancreatic adenocarcinoma, intrahepatic cholangiocarcinoma, and synovial sarcoma. A small but nonnegligible proportion of clinically suspected non-small cell lung carcinomas had a final diagnosis of cancer of extrapulmonary origin after clinical next-generation sequencing. The integration of clinical, microscopic, and molecular evidence can aid diagnosis and guide personalized oncology care.

Updates in pathology and molecular diagnostics to inform the evolving landscape of thoracic surgery and oncology.

The pathologic assessment of lung cancers provides essential guidance to the surgeon and oncologist who are considering the best treatment strategies for patients with both early and advanced-stage disease. The management of patients with lung cancer is predicated first and foremost on access to an accurate diagnosis, even when the sample size is limited, as is often the case with use of modern, minimally invasive sampling techniques. Once the diagnosis and disease stage are established, predictive biomarker testing may be essential, particularly for those patients with nonsmall cell lung carcinoma (NSCLC) being considered for immunotherapy or genomic biomarker-driven targeted therapy. This review will discuss the best practices for the diagnosis of NSCLC using morphology and immunohistochemistry, thus providing the surgeon with needed information to understand and critically evaluate pathology reports. Controversial and evolving topics including tumor spread through airspaces, evaluation of multiple tumors, and staging based on invasive tumor size will be addressed. Clinical genomic profiling in NSCLC is driven by published guidelines and reflects evidence based on clinical trials and regulatory approvals. In this fast-moving space, surgeons should be aware of the critical immunohistochemical and genomic biomarkers that drive systemic therapy decisions and anticipate when such testing will be required, both to ensure adequate sampling and to advise the pathologist when tumor material will be required for biomarker analysis. The basic approaches to and sample requirements for molecular biomarker testing will be addressed. As biomarker testing moves exclusively from advanced-stage patients into earlier stage disease, the surgeon should be aware of the relevant markers and work with the pathologist and oncologist to ensure that this information is available to facilitate timely access to therapies not just in the advanced setting, but in consideration of neoadjuvant and adjuvant care.

Anatomic Approach to Common and Uncommon Manifestations of Thoracic Leukemias with Radiologic-Pathologic Correlation.

Leukemias are hematopoietic malignancies characterized by the production of abnormal leukocytes in the bone marrow. Clinical manifestations arise from either bone marrow suppression or leukemic organ infiltration. Lymphadenopathy is the most common direct manifestation of intrathoracic leukemia. However, leukemic cells may also infiltrate the lungs, pleura, heart, bones, and soft tissues. Pulmonary complications in patients with leukemia typically include pneumonia, hemorrhage, pulmonary edema, and sequelae of leukemia treatment. However, pulmonary abnormalities can also be related directly to leukemia, including leukemic pulmonary infiltration. The direct, non-treatment-related effects of leukemia on intrathoracic structures will be the focus of this imaging essay. Given the typical anatomic approach for image interpretation, an organ-based depiction of common and less common intrathoracic findings directly caused by leukemic involvement is presented, emphasizing imaging findings with pathologic correlations. Keywords: Leukemia, Pulmonary, Thorax, Soft Tissues/Skin, Hematologic, Bone Marrow © RSNA, 2023.

Clinical Utility Validation of an Automated Ultrarapid Gene Fusion Assay for NSCLC.

Introduction: Gene rearrangements are frequent oncologic drivers in NSCLC, and many are suitable for treatment with Food and Drug Administration-approved or experimental targeted therapies. We evaluated the accuracy, specimen acceptance profile, and limits of detection of a rapid fusion assay (Idylla GeneFusion Assay), a commercially available ultrarapid molecular assay, for its clinical utility. Methods: A collection of 97 specimens which had previously undergone next-generation sequencing testing were analyzed using the rapid fusion assay. Accuracy was evaluated by sensitivity and specificity compared with the next-generation sequencing results. The performance characteristics were tested by using a variety of different clinically relevant specimen types. Limits of detection were assessed by evaluating different input of tumor percentage and material amount. Results: The rapid fusion assay was found to have 100% sensitivity in detecting fusions of ALK, ROS1, RET, NTRK1, and MET exon 14 skipping and 83% sensitivity for NTRK2/3 fusions. There were 100% specificity in detecting fusions of ROS1, RET, NTRK2/3, and MET exon 14 skipping and 98% specificity for ALK. Testing was successful with formalin-fixed paraffin-embedded biopsy and surgical tissues, cell blocks from fine-needle aspiration and pleural fluid (down to 5% tumor content, 18 mm2 tissue scraped), cytology smears (≥300 cells), and previously extracted RNA (minimal 20 ng). Conclusions: The rapid fusion assay is quick, accurate, and versatile, allowing reliable detection of ALK, ROS1, RET fusions, and MET exon 14 skipping in NSCLC, and NTRK fusions. Rapid molecular testing may expedite treatment with appropriate targeted therapies.

Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

Late Complications of COVID-19.

CONTEXT.­: Studies of lungs in patients with COVID-19 have focused on early findings. OBJECTIVE.­: To systematically study histopathologic and imaging features and presence of SARS-CoV-2 RNA in lung tissue from patients in later stages of COVID-19. DESIGN.­: Autopsies, explants, surgical lung biopsies, transbronchial biopsies, cryobiopsies, and needle biopsies from patients with COVID-19 whose onset of symptoms/confirmed diagnosis was more than 28 days before the procedure were studied. Available images were reviewed. Reverse transcription droplet digital polymerase chain reaction for SARS-CoV-2 RNA was performed on lung tissue. RESULTS.­: Of 44 specimens (43 patients; median age, 59.3 years; 26 [60.5%] male) features of acute lung injury (ALI) were seen in 39 (88.6%), predominantly organizing pneumonia and diffuse alveolar damage, up to 298 days after onset of COVID-19. Fibrotic changes were found in 33 specimens (75%), most commonly fibrotic diffuse alveolar damage (n = 22) and cicatricial organizing pneumonia (n = 12). Time between acquiring COVID-19 and specimen was shorter in patients with diffuse ALI (median, 61.5 days) compared with patients with focal (140 days) or no ALI (130 days) (P = .009). Sixteen (of 20; 80%) SARS-CoV-2 reverse transcription droplet digital polymerase chain reaction tests were positive, up to 174 days after COVID-19 onset. Time between COVID-19 onset and most recent computed tomography in patients with consolidation on imaging was shorter (median, 43.0 days) versus in patients without consolidation (87.5 days; P = .02). Reticulations were associated with longer time to computed tomography after COVID-19 onset (median, 82 versus 23.5 days; P = .006). CONCLUSIONS.­: ALI and SARS-CoV-2 RNA can be detected in patients with COVID-19 for many months. ALI may evolve into fibrotic interstitial lung disease.

Two In Cis Variants-Two Worlds Apart.

Precision oncology emphasizes genotyping as one of the mainstays of oncological decision-making. The core information element exchanged between the laboratory and the oncologist is the precise mutation. Specifically, it is the written representation typically in the form of a variant description at the DNA or protein level. These annotations can be confusing, and many commercial laboratories have abandoned DNA-level annotations. Here we present a complex double-point mutation to illustrate a situation where a formally "correct" reporting nomenclature can obscure clinically relevant and potentially clinically actionable information. KEY POINTS: The Human Genome Variation Society (HGVS) currently recommends that "two variants separated by one or more nucleotides should be described individually and not as a combined 'delins' (deletion-inserion)." There remains confusion about the appropriate nomenclature to report variants and the significance of these variants among clinicians. It is the clinically integrated molecular-genetic interpretation that will help clinicians make informed decisions to improve patient care.

Using deep learning to identify bladder cancers with FGFR-activating mutations from histology images.

BACKGROUND: In recent years, the fibroblast growth factor receptor (FGFR) pathway has been proven to be an important therapeutic target in bladder cancer. FGFR-targeted therapies are effective for patients with FGFR mutation, which can be discovered through genetic sequencing. However, genetic sequencing is not commonly performed at diagnosis, whereas a histologic assessment of the tumor is. We aim to computationally extract imaging biomarkers from existing tumor diagnostic slides in order to predict FGFR alterations in bladder cancer. METHODS: This study analyzed genomic profiles and H&E-stained tumor diagnostic slides of bladder cancer cases from The Cancer Genome Atlas (n = 418 cases). A convolutional neural network (CNN) identified tumor-infiltrating lymphocytes (TIL). The percentage of the tissue containing TIL ("TIL percentage") was then used to predict FGFR activation status with a logistic regression model. RESULTS: This predictive model could proficiently identify patients with any type of FGFR gene aberration using the CNN-based TIL percentage (sensitivity = 0.89, specificity = 0.42, AUROC = 0.76). A similar model which focused on predicting patients with only FGFR2/FGFR3 mutation was also found to be highly sensitive, but also specific (sensitivity = 0.82, specificity = 0.85, AUROC = 0.86). CONCLUSION: TIL percentage is a computationally derived image biomarker from routine tumor histology that can predict whether a tumor has FGFR mutations. CNNs and other digital pathology methods may complement genome sequencing and provide earlier screening options for candidates of targeted therapies.

Checkpoint Inhibitor Colitis Shows Drug-Specific Differences in Immune Cell Reaction That Overlap With Inflammatory Bowel Disease and Predict Response to Colitis Therapy.

OBJECTIVES: Checkpoint inhibitor (CPI)-associated colitis can limit therapy and has resemblance to inflammatory bowel disease (IBD). Studies exploring mechanistic similarities between these colitides are limited, yet therapeutic targets for either disorder could emerge from shared pathophysiology. METHODS: The morphology and inflammatory content of colonic biopsy specimens from anti-CTLA-4 and anti-PD-1/PD-L1 antibody-treated patients with CPI colitis were compared with initial biopsy specimens from patients with IBD. Predictors of the need for infliximab were sought in CPI patients. RESULTS: Biopsy specimens from CPI patients showed significantly lower chronicity scores and similar activity scores compared with patients with IBD. Anti-CTLA-4 and IBD groups showed equivalent CD8, CD4, PD-1, and PD-L1 expression, while FoxP3 scores were lower and CD68 scores were higher in anti-CTLA-4 compared with IBD biopsy specimens. Anti-PD-1/PD-L1 group had lower scores for CD8, CD4, and PD-1 and equivalent scores for FoxP3, PD-L1, and CD68 compared with IBD. Anti-CTLA-4 biopsy specimens had higher scores for CD8, PD-1, PD-L1, and CD68 than anti-PD-1/PD-L1 biopsy specimens. CD8/FoxP3 ratios and CD68 scores were higher among CPI patients requiring infliximab therapy for colitis compared with those responding to steroids. CONCLUSIONS: The proinflammatory immune phenotype of anti-CTLA-4-associated colitis has significant overlap with IBD. CD8/FoxP3 ratios may predict therapeutic response in CPI-associated colitis.