CONTEXT.—: The immune microenvironment is involved in fundamental aspects of tumorigenesis, and immune scores are now being developed for clinical diagnostics. OBJECTIVE.—: To evaluate how well small diagnostic biopsies and tissue microarrays (TMAs) reflect immune cell infiltration compared to the whole tumor slide, in tissue from patients with non-small cell lung cancer. DESIGN.—: A TMA was constructed comprising tissue from surgical resection specimens of 58 patients with non-small cell lung cancer, with available preoperative biopsy material. Whole sections, biopsies, and TMA were stained for the pan-T lymphocyte marker CD3 to determine densities of tumor-infiltrating lymphocytes. Immune cell infiltration was assessed semiquantitatively as well as objectively with a microscopic grid count. For 19 of the cases, RNA sequencing data were available. RESULTS.—: The semiquantitative comparison of immune cell infiltration between the whole section and the biopsy displayed fair agreement (intraclass correlation coefficient [ICC], 0.29; P = .01; CI, 0.03-0.51). In contrast, the TMA showed substantial agreement compared with the whole slide (ICC, 0.64; P < .001; CI, 0.39-0.79). The grid-based method did not enhance the agreement between the different tissue materials. The comparison of CD3 RNA sequencing data with CD3 cell annotations confirmed the poor representativity of biopsies as well as the stronger correlation for the TMA cores. CONCLUSIONS.—: Although overall lymphocyte infiltration is relatively well represented on TMAs, the representativity in diagnostic lung cancer biopsies is poor. This finding challenges the concept of using biopsies to establish immune scores as prognostic or predictive biomarkers for diagnostic applications.

CONTEXT.—: Breast cancer with low (1%-10%) estrogen receptor (ER) expression (ER-low positive) constitutes a small portion of invasive breast cancers, and the treatment strategy for these tumors remains debatable. OBJECTIVE.—: To characterize the features and outcomes of ER-low positive patients, and clarify the clinical significance of FOXC1 and SOX10 expression in ER-low positive/HER2-negative tumors. DESIGN.—: Among 9082 patients diagnosed with primary invasive breast cancer, the clinicopathologic features of those with ER-low positive breast cancer were characterized. FOXC1 and SOX10 mRNA levels were analyzed in ER-low positive/HER2-negative cases from public data sets. The expression of FOXC1 and SOX10 in ER-low positive/HER2-negative tumors was evaluated by immunohistochemistry. RESULTS.—: The clinicopathologic study of ER-low positive tumors indicated more aggressive characteristics compared with those tumors with ER >10%, while they had more overlapping features with ER-negative tumors irrespective of the HER2 status. The intrinsic molecular subtype of ER-low positive cases with high FOXC1 and SOX10 mRNA expression was more likely to be nonluminal. Among the ER-low positive/HER2-negative tumors, 56.67% (51 of 90) and 36.67% (33 of 90) were positive for FOXC1 and SOX10, respectively, which was significantly positively correlated with CK5/6 expression. In addition, the survival analysis demonstrated no significant difference between patients who received and who did not receive endocrine therapy. CONCLUSIONS.—: ER-low positive breast cancers biologically overlap more with ER-negative tumors. ER-low positive/HER2-negative cases demonstrate a high rate of FOXC1 or SOX10 expression, and these cases might be better categorized as a basal-like phenotype/subtype. FOXC1 and SOX10 testing may be used for the intrinsic phenotype prediction for ER-low positive/HER2-negative patients.

CONTEXT.—: Ophthalmic pathology is a discipline that relies heavily on a knowledge of clinical ophthalmology. The diagnosis of ocular and periocular lesions can be challenging because some lesions and diseases are unique to this region, whereas others may demonstrate site-specific differences from nonocular counterparts. Because of these challenges, ocular and periocular biopsies are frequently referred to specialized ophthalmic pathology centers for second opinion diagnoses. OBJECTIVE.—: To analyze the referral patterns, diagnostic challenges, and diagnostic discrepancies for second opinion referrals at a dedicated ophthalmic pathology laboratory with an emphasis on lesions of special interest in ophthalmic pathology. DATA SOURCES.—: Data sources included the pathology records of all slides and blocks received in consultation at the referral eye pathology center between December 1, 2015, and December 1, 2022, the personal experience of senior authors, and published peer-reviewed literature. CONCLUSIONS.—: Corneal, intraocular, and conjunctival biopsies are the most common types of cases received in consultation without the referring pathologist's diagnosis, likely reflecting diagnostic challenges. Degenerative intraocular processes occasionally raise concern for a neoplasm. Conjunctival melanocytic lesions are the most common conjunctival biopsies referred for second opinion diagnosis and require careful tissue sampling and clinical-pathologic correlation. Careful clinical-pathologic correlation, a high level of suspicion, and adequate sampling also are required for the accurate diagnosis of periocular sebaceous carcinoma. The diagnostic discrepancies involving uveal, retinal, conjunctival, eyelid, and temporal artery biopsies are most likely to adversely influence patient management and possible outcome. Such specimens may benefit from referral to specialized ophthalmic pathology laboratories.

CONTEXT: Disease courses in COVID-19 patients vary widely. Prediction of disease severity on initial diagnosis would aid appropriate therapy; but few studies include data from initial diagnosis. OBJECTIVE: To develop predictive models of COVID-19 severity based on demographic, clinical, and laboratory data collected at initial patient contact after diagnosis of COVID-19. DESIGN: We studied demographic, clinical laboratory biomarkers at time of diagnosis, using backward logistic regression modeling to determine severe and mild outcomes. Using deidentified data from 14,147 patients who were diagnosed with COVID-19 by polymerase chain reaction SARS-CoV-2 testing at Montefiore Health System, from March 2020 to September 2021. We generated models predicting severe disease (death or more than 90 hospital days) vs mild disease (alive and fewer than 2 hospital days), starting with 58 variables, by backward stepwise logistic regression. RESULTS: Of the 14,147 patients, including whites, blacks and Hispanics, 2,546 (18%) of patients had severe outcomes and 3,395 (24%) had mild outcomes. The final number of patients per model varied from 445 to 755, because not all patients had all available variables. Four models (Inclusive, Receiver Operating Characteristics, Specific, and Sensitive) were identified as proficient in predicting patient outcomes. The parameters that remained in all models were age, albumin, diastolic blood pressure, ferritin, lactic dehydrogenase, socioeconomic status, procalcitonin, B-type natriuretic peptide, and platelet count. CONCLUSIONS: These findings suggest that the biomarkers found within the specific and sensitive models would be most useful to health care providers on their initial severity evaluation of COVID-19.

CONTEXT.—: Hypertrophic lichen planus (HLP) is a variant of lichen planus that can be difficult to diagnose based on histopathologic features alone. Thus, patient clinical history and clinicopathologic correlation are essential considerations to make the correct diagnosis. OBJECTIVE.—: To discuss the clinical and histologic presentation of HLP and provide a thorough review of commonly encountered mimickers in the differential diagnosis. DATA SOURCES.—: Data were derived from a literature review, personal clinical and research experiences, and a review of cases in the archives of a tertiary care referral center. CONCLUSIONS.—: In general, HLP involves the lower extremities and is characterized by thickened, scaly nodules and plaques that are often pruritic and chronic in nature. HLP affects both males and females and is most common in adults 50 to 75 years of age. Unlike conventional lichen planus, HLP tends to have eosinophils and classically displays a lymphocytic infiltrate most concentrated around the tips of rete ridges. The differential diagnosis for HLP is broad and encompasses numerous entities in many different categories, including premalignant and malignant neoplasms, reactive squamoproliferative tumors, benign epidermal neoplasms, connective tissue disease, autoimmune bullous disease, infection, and drug-related reactions. Therefore, a high index of suspicion must be maintained to avoid a misdiagnosis and potential inappropriate treatments.

CONTEXT.—: Cholangiocarcinoma (CCA) is a heterogeneous cancer of the bile duct, and its diagnosis is often challenging. OBJECTIVE.—: To provide insights into state-of-the-art approaches for the diagnosis of CCA. DATA SOURCES.—: Literature review via PubMed search and authors' experiences. CONCLUSIONS.—: CCA can be categorized as intrahepatic or extrahepatic. Intrahepatic CCA is further classified into small-duct-type and large-duct-type, whereas extrahepatic CCA is classified into distal and perihilar according to site of origin within the extrahepatic biliary tree. Tumor growth patterns include mass forming, periductal infiltrating, and intraductal tumors. The clinical diagnosis of CCA is challenging and usually occurs at an advanced tumor stage. Pathologic diagnosis is made difficult by tumor inaccessibility and challenges in distinguishing CCA from metastatic adenocarcinoma to the liver. Immunohistochemical stains can assist in differentiating CCA from other malignancies, such as hepatocellular carcinoma, but no distinctive CCA-specific immunohistochemical profile has been identified. Recent advances in next-generation sequencing-based high-throughput assays have identified distinct genomic profiles of CCA subtypes, including genomic alterations that are susceptible to targeted therapies or immune checkpoint inhibitors. Detailed histopathologic and molecular evaluations of CCA by pathologists are critical for correct diagnosis, subclassification, therapeutic decision-making, and prognostication. The first step toward achieving these goals is to acquire a detailed understanding of the histologic and genetic subtypes of this heterogeneous tumor group. Here, we review state-of-the-art approaches that should be applied to establish a diagnosis of CCA, including clinical presentation, histopathology, staging, and the practical use of genetic testing methodologies.

CONTEXT.—: Rapid onsite evaluation (ROSE) is critical in determining sample adequacy and triaging cytology samples. Although fine-needle aspiration biopsy (FNAB) is the primary method of initial tissue sampling in Tanzania, ROSE is not practiced. OBJECTIVE.—: To investigate the performance of ROSE in determining cellular adequacy and providing preliminary diagnoses in breast FNAB in a low-resource setting. DESIGN.—: Patients with breast masses were recruited prospectively from the FNAB clinic at Muhimbili National Hospital. Each FNAB was evaluated by ROSE for overall specimen adequacy, cellularity, and preliminary diagnosis. The preliminary interpretation was compared to the final cytologic diagnosis and histologic diagnosis, when available. RESULTS.—: Fifty FNAB cases were evaluated, and all were adequate for diagnosis on ROSE and final interpretation. Overall percentage of agreement (OPA) between preliminary and final cytologic diagnosis was 86%, positive percentage of agreement (PPA) was 36%, and negative percentage of agreement (NPA) was 100% (κ = 0.5, P < .001). Twenty-one cases had correlating surgical resections. OPA between preliminary cytologic and histologic diagnoses was 67%, PPA was 22%, and NPA was 100% (κ = 0.2, P = .09). OPA between final cytologic and histologic diagnoses was 95%, PPA of 89%, and NPA of 100% (κ = 0.9, P = <.001). CONCLUSIONS.—: False positive rates of ROSE diagnoses for breast FNAB are low. While preliminary cytologic diagnoses had a high false negative rate, final cytologic diagnoses had overall high concordance with histologic diagnoses. Therefore, the role of ROSE for preliminary diagnosis should be considered carefully in low-resource settings and may need to be paired with additional interventions to improve pathologic diagnosis.

CONTEXT.—: Genetic profiling data of prostatic adenocarcinoma are derived from predominantly White patients. In African Americans, prostatic adenocarcinoma has a poorer prognosis, raising the possibility of distinct genetic alterations. OBJECTIVE.—: To investigate the genomic alterations of prostatic adenocarcinoma metastatic to regional lymph nodes in African American patients, with an emphasis on SPOP mutation. DESIGN.—: We retrospectively reviewed African American patients with pN1 prostatic adenocarcinoma managed with radical prostatectomy and lymph node dissection. Comprehensive molecular profiling was performed, and androgen receptor signaling scores were calculated. RESULTS.—: Nineteen patients were included. The most frequent genetic alteration was SPOP mutations (5 of 17; 29.4% [95% CI: 10.3-56.0]). While most alterations were associated with a high androgen receptor signaling score, mutant SPOP was exclusively associated with a low median and interquartile range (IQR) androgen receptor signaling score (0.788 [IQR 0.765-0.791] versus 0.835 [IQR 0.828-0.842], P = .003). In mutant SPOP, mRNA expression of SPOP inhibitor G3BP1 and SPOP substrates showed a significant decreased expression of AR (33.40 [IQR 28.45-36.30] versus 59.53 [IQR 53.10-72.83], P = .01), TRIM24 (3.95 [IQR 3.28-5.03] versus 9.80 [IQR 7.39-11.70], P = .008), and NCOA3 (15.19 [IQR 10.59-15.93] versus 21.88 [IQR 18.41-28.33], P = .046). CONCLUSIONS.—: African American patients with metastatic prostate adenocarcinoma might have a higher prevalence of mutant SPOP (30%), compared to ∼10% in unselected cohorts with lower expressions of SPOP substrates. In our study, in patients with mutant SPOP, the mutation was associated with decreased SPOP substrate expression and androgen receptor signaling, raising concern for suboptimal efficacy of androgen deprivation therapy in this subset of patients.

CONTEXT.—: In their 2014 article "New Immunohistochemistry for B-cell Lymphoma and Hodgkin Lymphoma," Zhang and Aguilera reviewed new immunohistochemical markers for B-cell lymphoma and Hodgkin lymphoma and described how to use these markers for correct lymphoma diagnoses, using the 2008 World Health Organization classifications. Recently, the World Health Organization's WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues published 2022 updates, and, in quick sequence, a second group published an alternative International Consensus Classification of myeloid neoplasms, acute leukemias, and mature lymphoid neoplasms. Regardless of the system a hematopathologist chooses to follow, updates in the immunohistochemical diagnosis of disease are described in both publications as well as in the primary literature. In addition to updated classifications, the increasing use of small biopsy samples for the evaluation of lymphadenopathy continues to challenge hematopathology diagnosis and increase the utilization of immunohistochemistry. OBJECTIVE.—: To review new immunohistochemical markers or new uses of previously known immunohistochemical markers in the evaluation of hematolymphoid neoplasia for the practicing hematopathologist. DATA SOURCES.—: Data were obtained from a literature review and personal practice experience. CONCLUSIONS.—: The practicing hematopathologist requires knowledge of the ever-expanding repertoire of immunohistochemistry for the diagnosis and treatment of hematolymphoid neoplasia. New markers presented in this article help to complete our understanding of disease, diagnosis, and management.

CONTEXT.—: Breast pathology (BP) is considered to be subject to interobserver variability among pathologists, emphasizing the need for adequate training. However, specifics of BP residency training have not been elucidated. OBJECTIVE.—: To assess the characteristics of BP residency training in the United States. DESIGN.—: A Qualtrics-managed online survey was emailed to program directors of all US pathology residency programs, requesting them to forward the survey link to their pathology residents. RESULTS.—: One hundred seventeen residents' survey responses were evaluable. Most responses (92; 79%) came from residents in university hospital-based programs. Thirty-five respondents (30%) had a dedicated BP rotation in their program. Most respondents believed that BP was an important part of training (96 of 100; 96%) and pathology practice (95 of 100; 95%). Seventy-one respondents believed that their BP training was adequate overall (71 of 100; 71%). Forty-one percent of respondents indicated that they would not like BP to be a significant part of their future practice. The main reasons given were that they had a different preferred area of interest, that they lacked interest in BP, or that breast cases were time-consuming to sign out. CONCLUSIONS.—: Our results show that in the United States, most programs do not offer a dedicated BP rotation, but breast cases are signed out by subspecialized or experienced breast pathologists. In addition, most respondents believed that they received adequate training and would be competent to independently sign out BP in the future. Additional studies addressing new-in-practice pathologists' proficiency in BP will further help elucidate the quality of BP training in the United States.

CONTEXT.—: The neurotrophic tropomyosin receptor kinase (NTRK) family gene rearrangements have been recently incorporated as predictive biomarkers in a "tumor-agnostic" manner. However, the identification of these patients is extremely challenging because the overall frequency of NTRK fusions is below 1%. Academic groups and professional organizations have released recommendations on the algorithms to detect NTRK fusions. The European Society of Medical Oncology proposal encourages the use of next-generation sequencing (NGS) if available, or alternatively immunohistochemistry (IHC) could be used for screening with NGS confirmation of all positive IHC results. Other academic groups have included histologic and genomic information in the testing algorithm. OBJECTIVE.—: To apply some of these triaging strategies for a more efficient identification of NTRK fusions within a single institution, so pathologists can gain practical insight on how to start looking for NTRK fusions. DESIGN.—: A multiparametric strategy combining histologic (secretory carcinomas of the breast and salivary gland; papillary thyroid carcinomas; infantile fibrosarcoma) and genomic (driver-negative non-small cell lung carcinomas, microsatellite instability-high colorectal adenocarcinomas, and wild-type gastrointestinal stromal tumors) triaging was put forward. RESULTS.—: Samples from 323 tumors were stained with the VENTANA pan-TRK EPR17341 Assay as a screening method. All positive IHC cases were simultaneously studied by 2 NGS tests, Oncomine Comprehensive Assay v3 and FoundationOne CDx. With this approach, the detection rate of NTRK fusions was 20 times higher (5.57%) by only screening 323 patients than the largest cohort in the literature (0.30%) comprising several hundred thousand patients. CONCLUSIONS.—: Based on our findings, we propose a multiparametric strategy (ie, "supervised tumor-agnostic approach") when pathologists start searching for NTRK fusions.

CONTEXT.—: Current approaches for characterizing retained lung dust using pathologists' qualitative assessment or scanning electron microscopy with energy-dispersive spectroscopy (SEM/EDS) have limitations. OBJECTIVE.—: To explore polarized light microscopy coupled with image-processing software, termed quantitative microscopy-particulate matter (QM-PM), as a tool to characterize in situ dust in lung tissue of US coal miners with progressive massive fibrosis. DESIGN.—: We developed a standardized protocol using microscopy images to characterize the in situ burden of birefringent crystalline silica/silicate particles (mineral density) and carbonaceous particles (pigment fraction). Mineral density and pigment fraction were compared with pathologists' qualitative assessments and SEM/EDS analyses. Particle features were compared between historical (born before 1930) and contemporary coal miners, who likely had different exposures following changes in mining technology. RESULTS.—: Lung tissue samples from 85 coal miners (62 historical and 23 contemporary) and 10 healthy controls were analyzed using QM-PM. Mineral density and pigment fraction measurements with QM-PM were comparable to consensus pathologists' scoring and SEM/EDS analyses. Contemporary miners had greater mineral density than historical miners (186 456 versus 63 727/mm3; P = .02) and controls (4542/mm3), consistent with higher amounts of silica/silicate dust. Contemporary and historical miners had similar particle sizes (median area, 1.00 versus 1.14 μm2; P = .46) and birefringence under polarized light (median grayscale brightness: 80.9 versus 87.6; P = .29). CONCLUSIONS.—: QM-PM reliably characterizes in situ silica/silicate and carbonaceous particles in a reproducible, automated, accessible, and time/cost/labor-efficient manner, and shows promise as a tool for understanding occupational lung pathology and targeting exposure controls.

CONTEXT.—: Thalassemia is the most widely distributed monogenic autosomal recessive disorder in the world. Accurate genetic analysis of thalassemia is crucial for thalassemia prevention. OBJECTIVE.—: To compare the clinical utility of a third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles with routine polymerase chain reaction (PCR) in genetic analysis of thalassemia and explore the molecular spectrum of thalassemia in Hunan Province. DESIGN.—: Subjects in Hunan Province were recruited and hematologic testing was performed. Five hundred four subjects positive for hemoglobin testing were then used as the cohort and third-generation sequencing and routine PCR were used for genetic analysis. RESULTS.—: Of the 504 subjects, 462 (91.67%) had the same results whereas 42 (8.33%) exhibited discordant results between the 2 methods. Sanger sequencing and PCR testing confirmed the results of third-generation sequencing. In total, third-generation sequencing correctly detected 247 subjects with variants whereas PCR identified 205, which showed an increase in detection of 20.49%. Moreover, α triplications were identified in 1.98% (10 of 504) hemoglobin testing-positive subjects in Hunan Province. Seven hemoglobin variants with potential pathogenicity were detected in 9 hemoglobin testing-positive subjects. CONCLUSIONS.—: Third-generation sequencing is a more comprehensive, reliable, and efficient approach for genetic analysis of thalassemia than PCR, and allowed for a characterization of the thalassemia spectrum in Hunan Province.

CONTEXT.—: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and lethal tumor, characterized by hypercalcemia and early onset and associated with germ-line and somatic SMARCA4 variants. OBJECTIVE.—: To identify all known cases of SCCOHT in the Slovenian population from 1991 to 2021 and present genetic testing results, histopathologic findings, and clinical data for these patients. We also estimate the incidence of SCCOHT. DESIGN.—: We conducted a retrospective analysis of hospital medical records and data from the Slovenian Cancer Registry in order to identify cases of SCCOHT and obtain relevant clinical data. Histopathologic review of tumor samples with assessment of immunohistochemical staining for SMARCA4/BRG1 was undertaken to confirm the diagnosis of SCCOHT. Germ-line and somatic genetic analyses were performed using targeted next-generation sequencing. RESULTS.—: Between 1991 and 2021 we identified 7 cases of SCCOHT in a population of 2 million. Genetic causes were determined in all cases. Two novel germ-line loss-of-function variants in SMARCA4 LRG_878t1:c.1423_1429delTACCTCA p.(Tyr475Ilefs*24) and LRG_878t1:c.3216-1G>T were identified. At diagnosis, patients were ages 21 to 41 and had FIGO stage IA-III disease. Outcomes were poor, with 6 of 7 patients dying of disease-related complications within 27 months from diagnosis. One patient had stable disease for 12 months while receiving immunotherapy. CONCLUSIONS.—: We present genetic, histopathologic, and clinical characteristics for all cases of SCCOHT identified in the Slovenian population during a 30-year period. We report 2 novel germ-line SMARCA4 variants, possibly associated with high penetrance. We estimate the minimal incidence of SCCOHT to be 0.12 per 1 million per year.