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.

Ki-67 Proliferation Index Is Associated With Tumor Grade and Survival in Pleural Epithelioid Mesotheliomas.

Pleural epithelioid mesothelioma (PEM) is divided into low and high grades based on nuclear atypia, mitoses, and necrosis in the tumor. Assessing mitoses and nuclear atypia tend to be labor-intensive with limited reproducibility. Ki-67 proliferation index was shown to be a prognostic factor in PEM, but its performance has not been directly correlated with tumor grade or mitotic score. This study evaluated the potential of Ki-67 index as a surrogate of tumor grade. We also compared the predictability of mitoses and Ki-67 index for overall survival (OS). Ninety-six PEM samples from 85 patients were identified from the surgical pathology file during 2000-2021 at our institution, and all glass slides were reviewed by 2 pulmonary pathologists to confirm the diagnosis and assign the tumor grade. Digital image analysis (DIA) was done for Ki-67 index. The agreement on tumor grading between 2 reviewers was moderate (kappa value = 0.47). The correlation between mitotic count (average count by 2 reviewers) and Ki-67 index was 0.65. The areas under the curve for predicting tumor grade by mitotic score and Ki-67 index were 0.84 and 0.74 (reviewer 1) and 0.85 and 0.81 (reviewer 2), respectively. High Ki-67 index and mitoses were significantly associated with poor OS ( P =0.03 and 0.0005, using 30% and 10/2 mm 2 as cutoffs, respectively). In conclusion, Ki-67 index by DIA was associated with tumor grade as well as mitotic count, and its predictability for OS was comparable to that of mitotic score, thus being a potential surrogate for tumor grade.

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.

A case-control autopsy series of liver pathology associated with novel coronavirus disease (COVID-19).

BACKGROUND: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for coronavirus disease 2019 (COVID-19) is most well-known for causing pulmonary injury, a significant proportion of patients experience hepatic dysfunction. The mechanism by which SARS-CoV2 causes liver injury is not fully understood. The goal of this study was to describe the hepatic pathology in a large cohort of deceased patients with COVID-19 as compared to a control group of deceased patients without COVID-19. METHODS: Consented autopsy cases at two institutions were searched for documentation of COVID-19 as a contributing cause of death. A group of consecutive consented autopsy cases during the same period, negative for SARS-CoV-2 infection, was used as a control group. The autopsy report and electronic medical records were reviewed for relevant clinicopathologic information. H&E-stained liver sections from both groups were examined for pertinent histologic features. Select cases underwent immunohistochemical staining for CD 68 and ACE2 and droplet digital polymerase chain reaction (ddPCR) assay for evaluation of SARS-CoV2 RNA. RESULTS: 48 COVID-19 positive patients (median age 73, M:F 3:1) and 40 COVID-19 negative control patients (median age 67.5, M:F 1.4:1) were included in the study. The COVID-19 positive group was significantly older and had a lower rate of alcoholism and malignancy, but there was no difference in other comorbidities. The COVID-19 positive group was more likely to have received steroids (75.6 % vs. 36.1 %, p < 0.001). Hepatic vascular changes were seen in a minority (10.6 %) of COVID-19 positive cases. When all patients were included, there were no significant histopathologic differences between groups, but when patients with chronic alcoholism were excluded, the COVID-19 positive group was significantly more likely to have steatosis (80.9 % vs. 50.0 %, p = 0.004) and lobular inflammation (45.7 % vs. 20.7 %, p = 0.03). Testing for viral RNA by ddPCR identified 2 of the 18 (11.1 %) COVID-19 positive cases to have SARS-CoV-2 RNA detected within the liver FFPE tissue. CONCLUSIONS: The most significant findings in the liver of COVID-19 positive patients were mild lobular inflammation and steatosis. The high rate of steroid therapy in this population may be a possible source of steatosis. Hepatic vascular alterations were only identified in a minority of patients and did not appear to play a predominant role in COVID-19 mediated hepatic injury. Low incidence of SARS-CoV-2 RNA positivity in liver tissue in our cohort suggests hepatic injury in the setting of COVID-19 may be secondary in nature.

Fatal Infections Differentially Involve Allograft and Native Lungs in Single Lung Transplant Recipients.

CONTEXT.­: Respiratory infections complicate lung transplantation and increase the risk of allograft dysfunction. Allograft lungs may have different susceptibilities to infection than native lungs, potentially leading to different disease severity in lungs of single lung transplant recipients (SLTRs). OBJECTIVE.­: To study whether infections affect allograft and native lungs differently in SLTRs but similarly in double LTRs (DLTRs). DESIGN.­: Using an institutional database of LTRs, medical records were searched, chest computed tomography studies were systematically reviewed, and histopathologic features were recorded per lung lobe and graded semiquantitatively. A multilobar-histopathology score (MLHS) including histopathologic data from each lung and a bilateral ratio (MLHSratio) comparing histopathologies between both lungs were calculated in SLTRs and compared to DLTRs. RESULTS.­: Six SLTRs died of infection involving the lungs. All allografts showed multifocal histopathologic evidence of infection, but at least 1 lobe of the native lung was uninvolved. In all 5 DLTRs except 1, histopathologic evidence of infection was seen in all lung lobes. On computed tomography, multifocal ground-glass and/or nodular opacities were found in a bilateral distribution in all DLTRs but in only 2 of 6 SLTRs. In SLTRs, the MLHSAllograft was higher than MLHSNative (P = .02). The MLHSratio values of SLTR and DLTR were significantly different (P < .001). CONCLUSIONS.­: Allograft and native lungs appear to harbor different susceptibilities to infections. The results are important for the management of LTRs.

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.

Two tumors with combined features of bronchiolar adenoma/ciliated muconodular papillary tumor and sclerosing pneumocytoma.

OBJECTIVES: Bronchiolar adenoma/ciliated muconodular papillary tumor (BA/CMPT) and sclerosing pneumocytoma (SP) are both rare and morphologically unique peripheral lung tumors with indolent behavior. These tumors have not been previously described as showing overlapping morphologic features and are generally genetically distinct. METHODS: Two cases were recently encountered that show hybrid morphologic features between BA/CMPT and SP, and the morphology and immunophenotype are described in detail. RESULTS: Both cases showed interstitial round cells typical of SP (TTF1+, EMA+), as well as areas more typical of BA/CMPT. One case showed BRAFV600E expression in the BA/CMPT areas but not in the SP-like cells. CONCLUSIONS: Although it is possible that these cases represent collision tumors or are examples of unusual metaplastic epithelial changes in SP, they also raise the possibility that these 2 entities could occasionally coexist in true hybrid tumors.

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.

Clinical outcomes of stage-IV non-small-cell lung cancer in young patients and the impact of tumor markers.

INTRODUCTION: Non-Small Cell Lung Cancer (NSCLC) diagnosed at a younger age have patterns of care, responses to treatment, and outcomes not entirely clear. A particular feature includes more advanced stages at diagnosis. Our objective was to characterize these young patients with advanced disease and evaluate the impact of targeted therapies. METHODS: Analyzing our cohort of 18,252 newly diagnosed NSCLC patients, we defined Young-age versus Norm-age based on the age distribution at the time of diagnosis. Stage-IV patients were investigated on their clinical information and outcomes; deaths were considered lung cancer-related. Primary outcome was overall survival (OS). Multivariate Cox models were built to evaluate independent prognostic factors in comparative age groups. RESULTS: We found 4,267 patients with stage-IV NSCLC (359 Young-age; 3,908 Norm-age). Young patients had predominance of females (52.6% vs. 43.3%, P = 0.001), never-smokers (43.2% vs. 14.8%, P < 0.001), and adenocarcinoma (73.5% vs. 62.5%, P < 0.001). Mean OS was 21.1 months in the Young and 15.1 months in Norm, respectively (P < 0.001). Young patients were more often treated with surgery (6.7% vs. 5.0%), chemotherapy (53.2% vs. 44.1%), and targeted therapy (10.6% vs. 5.7%). Molecular studies were assessed in patients when the mutation tests became clinically available (93 Young, 875 Norm) and revealed a critical role of targeted therapy in the improved survival of both age groups. DISCUSSION: Young patients with stage-IV NSCLC have a specific profile and benefit more when treated with surgery and targeted therapy. Molecular testing is critical in this population, where improved survival was identified. A more aggressive approach to this population needs to be considered.

A diagnostic approach to paratesticular lesions with tubulopapillary architecture: a series of 16 serous borderline tumors/low-grade serous carcinoma and 14 well-differentiated papillary mesothelial tumors and mesothelioma.

As there is limited literature on paratesticular tumors of müllerian and mesothelial origin, we reviewed archived cases of serous borderline tumors (n = 15), low-grade serous carcinoma (n = 1), well-differentiated papillary mesothelial tumors (WDPMTs; n = 2), and mesothelioma (n = 12), for relevant clinicopathologic features. Molecular profiling data from the American Association for Cancer Research (AACR) GENIE registry was accessed for 8 additional patients with testicular mesothelioma. For tumors of mesothelial origin, the median age at surgical excision was 62 years, the median size was 4.5 cm, and they consistently exhibited positivity for mesothelial markers (CK5/6, calretinin, WT1, and D2-40). Recurrent alterations of the NF2 gene were identified in 3 of 8 patients (38%), and alterations of BAP1 and CDKN2A were relatively infrequent. While one patient with WDPMT had a recurrence, a second patient with WDPMT progressed to a biphasic mesothelioma 2 years after initial resection. For tumors of müllerian origin, the median age at surgical excision was 45 years, the median size was 2.5 cm, and these exhibited consistent positivity for ER, WT1, and PAX8. Although no recurrences were documented in patients with serous borderline tumors, a single patient with a low-grade serous carcinoma developed widely metastatic disease and died of disease-related complications. Our study emphasizes the need for close clinical follow-up in patients with WDPMT and highlights the prognostic significance of documenting invasive behavior in tumors of müllerian origin as they can have an aggressive clinical course. Finally, our results suggest that NF2 alterations may play an important role in the pathogenesis of testicular mesothelioma.

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.

Immunohistochemical expression of carbonic anhydrase 9, glucose transporter 1, and paired box 8 in von Hippel-Lindau disease-related lesions.

von Hippel-Lindau (VHL) disease occurs secondary to pathogenic alterations of the VHL tumor suppressor gene, manifesting with cysts and tumors in multiple organ systems. VHL protein (pVHL) is a known downregulator of hypoxia inducible factor-1a (HIF1a). Loss of function of pVHL is associated with upregulation of the HIF1a pathway including carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1). Paired box 8 (PAX8) is an important transcription factor regulator of mesonephric development. We investigated the role of immunohistochemistry in the assessment of CA9, GLUT1, and PAX8 expression in VHL disease-related lesions. Clinicopathologic information and archived pathology material from 5 patients with VHL disease were reviewed and evaluated for expression of CA9, GLUT1, and PAX8. The spectrum of VHL disease-related lesions included hemangioblastoma, endolymphatic sac tumor, pulmonary microcysts, pheochromocytoma, pancreatic neuroendocrine tumor and serous cystadenoma, renal cysts, renal cell carcinoma, and epididymal papillary cystadenoma. CA9 was expressed in all lesions and exhibited diffuse positivity (15/15 lesions, 100%; 5/5 patients), while GLUT1 expression was focal/weak or absent in some instances (strong positive: 12/15 lesions, 80%; 5/5 patients). PAX8 was expressed only in renal and epididymal lesions. CA9 and GLUT1 are consistently overexpressed in VHL disease-related lesions, reflecting upregulation of the HIF1a pathway. PAX8 is only expressed in genitourinary lesions, mirroring organ-specific differentiation. A combination of CA9 and GLUT1 immunostains is useful in screening lesions of patients with VHL spectrum manifestations, which may be targeted by the recently Food and Drug Administration-approved HIF-2a inhibitors.

Loss of Methylthioadenosine Phosphorylase by Immunohistochemistry Is Common in Pulmonary Sarcomatoid Carcinoma and Sarcomatoid Mesothelioma.

OBJECTIVES: Differentiating malignant pleural mesothelioma from benign reactive mesothelial processes can be quite challenging. Ancillary tests such as BRCA1-associated protein 1 (BAP1) immunohistochemistry and p16 fluorescence in situ hybridization (FISH) are helpful tools to aid in this distinction. Immunohistochemistry for methylthioadenosine phosphorylase (MTAP) has recently been proposed as an effective surrogate marker for p16 FISH and is an attractive alternative test due to shorter turnaround time. There are little data regarding the specificity of MTAP loss for mesothelioma or whether it may be useful to distinguish mesothelioma from the most common entity in the differential diagnosis, sarcomatoid carcinoma. METHODS: We studied well-characterized cases of sarcomatoid carcinoma (n = 34) and sarcomatoid mesothelioma (n = 62), which were stained for MTAP (clone 2G4) and BAP1 (clone C-4). RESULTS: Loss of MTAP expression was observed in 17 (50%) of 34 pulmonary sarcomatoid carcinomas; BAP1 expression was retained in all of the cases in which it was performed (n = 31). MTAP expression was lost in 38 (61%) of 62 sarcomatoid mesotheliomas; BAP1 was lost in 6 (10%) of 62. In the six cases with BAP1 loss, five also had loss of MTAP, while MTAP expression was retained in one. CONCLUSIONS: Loss of MTAP expression by immunohistochemistry is common in pulmonary sarcomatoid carcinoma, as it is present in half of cases. This rate is similar to what is observed in sarcomatoid mesothelioma (61%). Therefore, this stain is not useful to distinguish between these two malignancies. MTAP loss is more common than BAP1 loss in the setting of sarcomatoid mesothelioma (61% vs 10%, respectively).

Acute Kidney Injury in Severe COVID-19 Has Similarities to Sepsis-Associated Kidney Injury: A Multi-Omics Study.

OBJECTIVE: To compare coronavirus disease 2019 (COVID-19) acute kidney injury (AKI) to sepsis-AKI (S-AKI). The morphology and transcriptomic and proteomic characteristics of autopsy kidneys were analyzed. PATIENTS AND METHODS: Individuals 18 years of age and older who died from COVID-19 and had an autopsy performed at Mayo Clinic between April 2020 to October 2020 were included. Morphological evaluation of the kidneys of 17 individuals with COVID-19 was performed. In a subset of seven COVID-19 cases with postmortem interval of less than or equal to 20 hours, ultrastructural and molecular characteristics (targeted transcriptome and proteomics analyses of tubulointerstitium) were evaluated. Molecular characteristics were compared with archived cases of S-AKI and nonsepsis causes of AKI. RESULTS: The spectrum of COVID-19 renal pathology included macrophage-dominant microvascular inflammation (glomerulitis and peritubular capillaritis), vascular dysfunction (peritubular capillary congestion and endothelial injury), and tubular injury with ultrastructural evidence of mitochondrial damage. Investigation of the spatial architecture using a novel imaging mass cytometry revealed enrichment of CD3+CD4+ T cells in close proximity to antigen-presenting cells, and macrophage-enriched glomerular and interstitial infiltrates, suggesting an innate and adaptive immune tissue response. Coronavirus disease 2019 AKI and S-AKI, as compared to nonseptic AKI, had an enrichment of transcriptional pathways involved in inflammation (apoptosis, autophagy, major histocompatibility complex class I and II, and type 1 T helper cell differentiation). Proteomic pathway analysis showed that COVID-19 AKI and to a lesser extent S-AKI were enriched in necroptosis and sirtuin-signaling pathways, both involved in regulatory response to inflammation. Upregulation of the ceramide-signaling pathway and downregulation of oxidative phosphorylation in COVID-19 AKI were noted. CONCLUSION: This data highlights the similarities between S-AKI and COVID-19 AKI and suggests that mitochondrial dysfunction may play a pivotal role in COVID-19 AKI. This data may allow the development of novel diagnostic and therapeutic targets.

Three-Dimensional Surface Imaging and Printing in Anatomic Pathology.

Three-dimensional (3D) imaging is increasingly being incorporated into a variety of medical specialties: surgery and radiology being but two prominent examples. Image-intensive disciplines, such as anatomic pathology (AP), represent excellent potential candidates for further exploration of this innovative technology. Multiple potential use cases exist within AP, involving patient care, education, and research. These use cases broadly include direct utilization of the 3D digital assets for viewing on a 2D screen, populating 3D extended reality platforms (virtual reality, augmented reality, and mixed reality) as well as generation of 3D printed photorealistic specimen models. Herein, these use cases are explored with specific regard to our experiences and yet unrealized potential. Future directions and considerations are also discussed.

Antifibrotics Modify B-Cell-induced Fibroblast Migration and Activation in Patients with Idiopathic Pulmonary Fibrosis.

B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and ß-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell-secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell-mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

Heterogeneity of PD-L1 expression between invasive and lepidic components of lung adenocarcinomas.

The dynamics of PD-L1 expression are poorly understood over the development of lung adenocarcinomas from pre-invasive lesions to fully invasive carcinomas. Given the importance of PD-L1 expression for the selection of patients to receive immunotherapy in the metastatic setting and possibly in the neoadjuvant setting, we sought to evaluate the agreement of PD-L1 expression in invasive and lepidic components of resected tumor specimens. We stained 86 adenocarcinomas for PD-L1 using the SP263 clone. We assessed the agreement of PD-L1 expression by tumor cells and immune cells between lepidic and invasive components. When both lepidic and invasive components were considered, PD-L1 positive immune cells and tumor cells were observed in 50 (58.1%) and 18 (20.9%) samples, respectively, using a ≥ 1% PD-L1 expression cutoff. Using a ≥ 1% cutoff for PD-L1 expression, positively stained tumor cells were observed in 11 (13%) lepidic and 15 (17%) invasive patterns, with agreement in 76 (88%) specimens and disagreement in 10 (12%) specimens (ĸ = 0.549). At ≥ 1% PD-L1 expression cutoff, PD-L1 positive immune cells were observed in 31 (35%) lepidic and 32 (37%) invasive patterns with an agreement of PD-L1 expression in 49 (57%) specimens and disagreement in 37 (43%) specimens (ĸ = 0.073). In our study of early stage adenocarcinomas of the lung, there was poor agreement in PD-L1 expression between paired invasive and lepidic components of tumors. Our data suggest that the non-invasive tumor components may not be as immunostimulatory as the invasive components, resulting in less adaptive expression of PD-L1.

Utility of Immunohistochemistry for MUC4 and GATA3 to Aid in the Distinction of Pleural Sarcomatoid Mesothelioma From Pulmonary Sarcomatoid Carcinoma.

CONTEXT.­: Distinguishing pulmonary sarcomatoid carcinoma from pleural sarcomatoid mesothelioma is challenging because of overlapping histology, immunophenotype, and clinical features. Reliable immunohistochemical markers to aid in this distinction would be very valuable. Recent studies have proposed that MUC4 expression is common in sarcomatoid carcinoma but not in sarcomatoid mesothelioma, with the converse pattern reported for GATA3. OBJECTIVE.­: To further explore the utility of MUC4 and GATA3 in distinguishing pulmonary sarcomatoid carcinoma from sarcomatoid mesothelioma. DESIGN.­: Well-characterized cases of sarcomatoid carcinoma (n = 32) and sarcomatoid mesothelioma (n = 64) were included. Diagnoses were confirmed by thoracic pathologists with incorporation of immunophenotype, clinical, and radiographic features. Whole-tissue sections were stained for GATA3 and MUC4. RESULTS.­: Patients with sarcomatoid carcinoma and sarcomatoid mesothelioma had similar mean age and male predominance. GATA3 was positive in 63 of 64 sarcomatoid mesotheliomas (98%; 42 diffuse, 16 patchy, 5 focal), and 15 of 32 sarcomatoid carcinomas (47%; 3 diffuse, 8 patchy, 4 focal). MUC4 was positive in 2 of 64 sarcomatoid mesotheliomas (3%; 1 patchy, 1 focal), and in 12 of 32 sarcomatoid carcinomas (38%; 5 diffuse, 6 patchy, 1 focal). CONCLUSIONS.­: Diffuse GATA3 expression favors sarcomatoid mesothelioma over sarcomatoid carcinoma, which rarely shows diffuse expression (sensitivity and specificity of diffuse staining 66% and 94%, respectively). Focal and patchy GATA3 expression is observed in both tumor types, and therefore is not helpful in this distinction. Sensitivity of MUC4 for sarcomatoid carcinoma was low in our cohort, positive in only 38% with frequent patchy staining, but it was quite specific.

Chromosomal Junction Detection from Whole-Genome Sequencing on Formalin-Fixed, Paraffin-Embedded Tumors.

DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (>500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (<2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected >90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.