Single-Cell Sequencing Illuminates Thymic Development: An Updated Framework for Understanding Thymic Epithelial Tumors.

Thymic epithelial tumors (TETs) are rare tumors for which treatment options are limited. The ongoing need for improved systemic therapies reflects a limited understanding of tumor biology as well as the normal thymus. The essential role of the thymus in adaptive immunity is largely effected by its epithelial compartment, which directs thymocyte (T-cell) differentiation and immunologic self-tolerance. With aging, the thymus undergoes involution whereby epithelial tissue is replaced by adipose and other connective tissue, decreasing immature T-cell production. Against this natural drive toward involution, a fraction of thymuses will instead undergo oncologic transformation, leading to the formation of TETs, including thymoma and thymic carcinoma. The rarity of these tumors restricts investigation of the mechanisms of tumorigenesis and development of rational treatment options. To this end, the development of technologies which allow deep molecular profiling of individual tumor cells permits a new window through which to view normal thymic development and contrast the malignant changes that result in oncogenic transformation. In this review, we describe the findings of recent illuminating studies on the diversity of cell types within the epithelial compartment through thymic differentiation and aging. We contextualize these findings around important unanswered questions regarding the spectrum of known somatic tumor alterations, cell of origin, and tumor heterogeneity. The perspectives informed by single-cell molecular profiling offer new approaches to clinical and basic investigation of thymic epithelial tumors, with the potential to accelerate development of improved therapeutic strategies to address ongoing unmet needs in these rare tumors.

Acquired Resistance to KRASG12C Inhibition in Cancer.

BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRASG12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).

Does Wound VAC Temporization Offer Patient-Reported Outcomes Similar to Single-Stage Excision Reconstruction After Myxofibrosarcoma Resection?

BACKGROUND: Vacuum-assisted closure (VAC) temporization is a promising technique to achieve local control in aggressive soft tissue sarcomas. Despite its previously reported efficacy, adoption of VAC temporization remains limited, primarily due to the scarce literature on patient-reported outcomes (PROs) supporting its efficacy. This study compared the postoperative PROs after VAC temporization or single-stage (SS) excision and reconstruction for patients undergoing surgical resection for myxofibrosarcoma management. METHODS: A retrospective analysis of myxofibrosarcoma patients who underwent surgical resections at our institution from 2016 to 2022 was performed. Postoperative PROs collected prospectively for those treated with VAC temporization or SS excision/reconstruction were compared using a visual analog scale (VAS) for pain and three Patient-Reported Outcomes Measurement Information System (PROMIS) questionnaires: Global Health Short-Form Mental (SF Mental), Global Health Short-Form Physical (SF Physical), and Physical Function Short-Form 10a (SF 10a). Absolute and differential (postoperative minus preoperative) scores at the 1-month, 3-month, 6-month, 1-year, and 2-year time points were compared. RESULTS: The analysis included 79 patients (47 treated with VAC temporization and 32 treated with SS excision/reconstruction). All outcomes were similar between the groups except for physical function 1 year after surgery, in which the differential PROMIS SF 10a scores were higher in the SS group (p = 0.001). All the remaining absolute and differential PROMIS and VAS pain scores were similar between the groups at all time points. Postoperative complications did not differ between the groups. CONCLUSION: The PROs for physical and mental health, physical function, and pain were similar between the myxofibrosarcoma patients who had VAC temporization and those who had SS excision/reconstruction after surgical resection.

Mesenchymal tumours of the pleura: review and update.

Primary mesenchymal tumours of the pleura are uncommon and can be diagnostically challenging due to their overlapping histopathologic and immunophenotypic features. Herein we discuss selected mesenchymal tumours of the pleura, including solitary fibrous tumour, calcifying fibrous tumour, desmoid fibromatosis, synovial sarcoma, schwannoma, malignant peripheral nerve sheath tumour, inflammatory myofibroblastic tumour, follicular dendritic cell sarcoma, epithelioid hemangioendothelioma, and desmoplastic small round cell tumour. We review their clinicopathologic characteristics, along with an update on the relevant immunohistochemical and molecular features.

Anastomosing haemangioma of the mediastinum: Clinicopathological series with radiological and genetic characterisation.

AIMS: Anastomosing haemangiomas are benign tumours with anastomosing vascular channels that may mimic angiosarcoma. While anastomosing haemangiomas have been described in diverse locations, particularly the abdominal/paraspinal region, data on anastomosing haemangiomas in the mediastinum remain limited. We report the clinicopathological, radiological and molecular characteristics of the largest single-institutional series of mediastinal anastomosing haemangiomas. METHODS AND RESULTS: In our pathology archives in 2011-23, we reviewed all vascular lesions involving the mediastinum and identified seven anastomosing haemangiomas. Clinical information was abstracted from medical charts; available radiological imaging was reviewed. Targeted DNA-based next-generation sequencing (447 genes, including GNAQ and GNA11) was performed on five cases. The seven patients included five women and two men, with an age range of 55-77 (median = 72) years. Of the six tumours with available radiology, two each were in the prevascular, visceral and paravertebral mediastinum, with lobulated peripheral enhancement in all tumours examined with contrast enhancement. Six patients underwent tumour resection; one patient received proton radiotherapy. Microscopically, each tumour was solitary and characterised by anastomosing capillary-sized vessels lined by hobnail endothelial cells. Fibrin microthrombi, hyaline globules and extramedullary haematopoiesis were common. In the five tumours analysed by next-generation sequencing, GNAQ p.Q209P was identified in one tumour; no additional reportable alterations were identified in the remaining cases. No recurrence was noted in the four patients with available follow-up of 3-58 (median = 9.5) months after resection. CONCLUSION: While mediastinal anastomosing haemangiomas can microscopically mimic angiosarcoma, awareness of this entity and radiological correlation may help to circumvent this diagnostic pitfall.

Phosphaturic mesenchymal tumor: management and outcomes of ten patients treated at a single institution.

BACKGROUND: Phosphaturic mesenchymal tumor (PMT) is a rare tumor that causes tumor-induced osteomalacia. Patients present with non-specific symptoms secondary to renal phosphate wasting and decreased bone mineralization. We sought to assess: (1) What are the common presenting features, laboratory and imaging findings, histologic findings of phosphaturic mesenchymal tumors? (2) What are the available treatment strategies for phosphaturic mesenchymal tumors and their long-term outcomes in terms of local recurrence and symptom control after treatment? METHODS: We retrospectively identified patients with a histologic diagnosis of PMT located in the axial or appendicular skeleton, or surrounding soft tissues. A total of 10 patients were finally included in our study. RESULTS: Median tumor size was 1.9 cm (range, 1.1 to 6.1) and median time from symptom onset to diagnosis was 3 years (range, 0.5 to 15 years). All patients but one presented with hypophosphatemia (median 1.9 mg/dL, range 1.2 to 3.2). Pre-operative FGF-23 was elevated in all cases (median 423.5 RU/mL, range 235 to 8950). Six patients underwent surgical resection, three were treated percutaneously (radiofrequency ablation or cryoablation), and one refused treatment. Only one patient developed local recurrence and no patients developed metastatic disease. At last follow-up, nine patients showed no evidence of disease and one was alive with disease. CONCLUSION: Phosphaturic mesenchymal tumor is a rare tumor presenting with non-specific symptoms. Surgery is the standard treatment when negative margins can be achieved without significant morbidity. In patients with small tumors in surgically-inaccessible areas, radiofrequency ablation or cryoablation can be performed successfully.

Clear cell mesotheliomas with inactivating VHL mutations and near-haploid genomic features.

Clear cell mesothelioma is uncommon and shows predominance of clear cells with resemblance to clear cell carcinomas. Clinicopathologic and molecular descriptions of clear cell mesothelioma remained limited. In this study, we identified an index patient with clear cell mesothelioma, confirmed by immunohistochemical and ultrastructural studies. Targeted next-generation sequencing revealed the presence of an inactivating VHL mutation. We then systematically searched for VHL-mutant mesotheliomas in a comprehensive genomic profiling database of 1532 mesotheliomas. Collectively, we identified a cohort of four VHL-mutant clear cell mesotheliomas, including three peritoneal and one pleural tumors from three females and one male, with age range of 47-68 (median 63) years. Histologically, each tumor showed a microcystic to tubulopapillary architecture with prominent clear cells. By next-generation DNA sequencing, each of the four clear cell mesotheliomas harbored inactivating VHL mutations, while lacking other alterations typical of mesotheliomas such as BAP1, NF2, SETD2, CDKN2A, CDKN2B, TP53, and PTEN. By using low-pass whole genome sequencing on the index case and targeted next-generation sequencing on the remaining three cases, we identified extensive loss of heterozygosity throughout the genome but consistently sparing chromosomes 5, 7, and 20, characteristic of genomic near-haploidization. In summary, clear cell mesotheliomas were characterized by inactivating VHL mutations and genomic near-haploidization and appeared to represent a distinct clinicopathologic and molecular category of mesotheliomas. Our findings implicate VHL in the pathogenesis of a subset of mesotheliomas, particularly those with clear cell morphology.

NR4A3 fusions characterize a distinctive peritoneal mesothelial neoplasm of uncertain biological potential with pure adenomatoid/microcystic morphology.

A focal adenomatoid-microcystic pattern is not uncommon in peritoneal mesothelioma, but tumors composed almost exclusively of this pattern are distinctly rare and have not been well characterized. A small subset of mesotheliomas (mostly in children and young adults) are characterized by gene fusions including EWSR1/FUS::ATF1, EWSR1::YY1, and NTRK and ALK rearrangements, and often have epithelioid morphology. Herein, we describe five peritoneal mesothelial neoplasms (identified via molecular screening of seven histologically similar tumors) that are pure adenomatoid/microcystic in morphology and unified by the presence of an NR4A3 fusion. Patients were three males and two females aged 31-70 years (median, 40 years). Three presented with multifocal/diffuse and two with a localized disease. The size of the individual lesions ranged from 1.5 to 8 cm (median, 4.7). The unifocal lesions originated in the small bowel mesentery and the mesosigmoid. Treatment included surgery, either alone (three) or combined with hyperthermic intraperitoneal chemotherapy (two), and neoadjuvant or adjuvant chemotherapy (one case each). At the last follow-up (6-13 months), all five patients were alive and disease-free. All tumors were morphologically similar, characterized by extensive sieve-like microcystic growth with bland-looking flattened cells lining variably sized microcystic spaces and lacked a conventional epithelioid or sarcomatoid component. Immunohistochemistry confirmed mesothelial differentiation, but most cases showed limited expression of D2-40 and calretinin. Targeted RNA sequencing revealed an NR4A3 fusion (fusion partners were EWSR1 in three cases and CITED2 and NIPBL in one case each). The nosology and behavior of this morphomolecularly defined novel peritoneal mesothelial neoplasm of uncertain biological potential and its distinction from adenomatoid variants of conventional mesothelioma merit further delineation as more cases become recognized.

Complete tumor necrosis after neoadjuvant chemotherapy defines good responders in patients with Ewing sarcoma.

BACKGROUND: Survival in patients who have Ewing sarcoma is correlated with postchemotherapy response (tumor necrosis). This treatment response has been categorized as the response rate, similar to what has been used in osteosarcoma. There is controversy regarding whether this is appropriate or whether it should be a dichotomy of complete versus incomplete response, given how important a complete response is for in overall survival of patients with Ewing sarcoma. The purpose of this study was to evaluate the impact that the amount of chemotherapy-induced necrosis has on (1) overall survival, (2) local recurrence-free survival, (3) metastasis-free survival, and (4) event-free survival in patients with Ewing sarcoma. METHODS: In total, 427 patients who had Ewing sarcoma or tumors in the Ewing sarcoma family and received treatment with preoperative chemotherapy and surgery at 10 international institutions were included. Multivariate Cox proportional-hazards analyses were used to assess the associations between tumor necrosis and all four outcomes while controlling for clinical factors identified in bivariate analysis, including age, tumor volume, location, surgical margins, metastatic disease at presentation, and preoperative radiotherapy. RESULTS: Patients who had a complete (100%) tumor response to chemotherapy had increased overall survival (hazard ratio [HR], 0.26; 95% CI, 0.14-0.48; p < .01), recurrence-free survival (HR, 0.40; 95% CI, 0.20-0.82; p = .01), metastasis-free survival (HR, 0.27; 95% CI, 0.15-0.46; p ≤ .01), and event-free survival (HR, 0.26; 95% CI, 0.16-0.41; p ≤ .01) compared with patients who had a partial (0%-99%) response. CONCLUSIONS: Complete tumor necrosis should be the index parameter to grade response to treatment as satisfactory in patients with Ewing sarcoma. Any viable tumor in these patients after neoadjuvant treatment should be of oncologic concern. These findings can affect the design of new clinical trials and the risk-stratified application of conventional or novel treatments.

First-in-Human Intrathoracic Implantation of Multidrug-Eluting Microdevices for In Situ Chemotherapeutic Sensitivity Testing as Proof of Concept in Nonsmall Cell Lung Cancer.

OBJECTIVE: To evaluate the safety and feasibility of implantation and retrieval of a novel implantable microdevice (IMD) in NSCLC patients undergoing operative resection. BACKGROUND: Adjuvant therapy has limited impact on postsurgical outcomes in NSCLC due to the inability to predict optimal treatment regimens. METHODS: An IMD measuring 6.5 mm by 0.7 mm, containing micro-reservoirs allowing for high-throughput localized drug delivery, was developed and loaded with 12 chemotherapeutic agents. Five patients with peripheral lung lesions larger than 1.0 cm were enrolled in this phase 1 clinical study. IMDs were inserted into tumors intraoperatively under direct vision, removed with the resected specimen, and retrieved in pathology. Surrounding tissues were sectioned, stained, and analyzed for tissue drug response to the IMD-delivered microdoses of these agents by a variety of pharmacodynamic markers. RESULTS: A total of 14 IMDs were implanted intraoperatively with 13 (93%) successfully retrieved. After technique refinement, IMDs were reliably inserted and retrieved in open, Video-Assisted Thoracoscopic Surgery, and robotic cases. No severe adverse reactions were observed. The one retained IMD has remained in place without movement or any adverse effects. Analysis of patient blood revealed no detection of chemotherapeutic agents. We observed differential sensitivities of patient tumors to the drugs on the IMD. CONCLUSIONS: A multi-drug IMD can be safely inserted and retrieved into lung tumors during a variety of surgical approaches. Future studies will encompass preoperative placement to better examine specific tumor responsiveness to therapeutic agents, allowing clinicians to tailor treatment regimens to the microenvironment of each patient.

Prognostic Significance of Percentage and Size of Dedifferentiation in Dedifferentiated Chondrosarcoma.

Dedifferentiated chondrosarcoma is rare, aggressive, and microscopically bimorphic. How pathologic features such as the amounts of dedifferentiation affect prognosis remains unclear. We evaluated the percentages and sizes of dedifferentiation in a consecutive institutional series of dedifferentiated chondrosarcomas from 1999 to 2021. The statistical analysis included cox proportional hazard models and log-rank tests. Of the 67 patients (26 women, 41 men; age, 39 to >89 [median 61] years; 2 with Ollier disease), 58 presented de novo; 9 were identified with conventional chondrosarcomas 0.6-13.2 years (median, 5.5 years) prior. Pathologic fracture and distant metastases were noted in 27 and 7 patients at presentation. The tumors involved the femur (n = 27), pelvis (n = 22), humerus (n = 7), tibia (n = 4), scapula/ribs (n = 4), spine (n = 2), and clivus (n = 1). In the 56 resections, the tumors ranged in size from 3.5 to 46.0 cm (median, 11.5 cm) and contained 1%-99.5% (median, 70%) dedifferentiated components that ranged in size from 0.6 to 24.0 cm (median, 7.3 cm). No correlation was noted between total size and percentage of dedifferentiation. The dedifferentiated components were typically fibrosarcomatous or osteosarcomatous, whereas the associated cartilaginous components were predominantly grade 1-2, rarely enchondromas or grade 3. The entire cohort's median overall survival and progression-free survival were 11.8 and 5.4 months, respectively. In the resected cohort, although the total size was not prognostic, the percentage of dedifferentiation ≥20% and size of dedifferentiation >3.0 cm each predicted worse overall survival (9.9 vs 72.5 months; HR, 3.76; 95% CI, 1.27-11.14; P = .02; 8.7 vs 58.9 months; HR, 3.03; 95% CI, 1.21-7.57; P = .02, respectively) and progression-free survival (5.3 vs 62.1 months; HR, 3.05; 95% CI, 1.13-8.28; P = .03; 5.3 vs 56.6 months; HR, 2.50; 95% CI, 1.06-5.88; P = .04, respectively). In conclusion, both the percentages and sizes of dedifferentiation were better prognostic predictors than total tumor sizes in dedifferentiated chondrosarcomas, highlighting the utility of their pathologic evaluations.

Molecular and immunohistochemical testing of bone tumours: review and update.

Primary bone tumours can pose diagnostic problems due to their overlapping radiologic and histologic features. Given the recent advancement in our understanding of the biology of bone tumours, multiple immunohistochemical and molecular markers have been devised to aid in their diagnosis. This review provides brief updates on select bone tumours, including chondrosarcomas, benign chondrogenic tumours, osteosarcomas, benign osteogenic tumours, fibroosseous lesions, vascular tumours, osteoclastic giant cell-rich or cystic tumours, chordoma, adamantinoma, small round blue cell sarcomas, and others. We discuss their salient molecular features and novel immunohistochemical correlates, along with some tips to avoid common diagnostic pitfalls.

Concordance of ASCL1, NEUROD1 and POU2F3 transcription factor-based subtype assignment in paired tumour samples from small cell lung carcinoma.

AIMS: Small cell lung carcinoma (SCLC) can be classified into transcription factor-based subtypes (ASCL1, NeuroD1, POU2F3). While in-vitro studies suggest intratumoral heterogeneity in the expression of these markers, how SCLC subtypes vary over time and among locations in patients remains unclear. METHODS AND RESULTS: We searched a consecutive series of patients at our institution in 2006-22 for those with greater than one available formalin-fixed paraffin-embedded SCLC sample in multiple sites and/or time-points. Immunohistochemistry for ASCL1, NeuroD1 and POU2F3 was performed and evaluated using H-scores, with subtype assigned based on the positive marker (H-score threshold >10) with the highest H-score. The 179 samples (75, lung; 51, lymph nodes; 53, non-nodal metastases) from 84 patients (74 with two, 10 with more than two samples) included 98 (54.7%) ASCL1-dominant, 47 (26.3%) NeuroD1-dominant, 15 (8.4%) POU2F3-dominant, 17 (9.5%) triple-negative and two (1.1%) ASCL1/NeuroD1 co-dominant samples. NeuroD1-dominant subtype was enriched in non-lung locations. Subtype concordance from pairwise comparison was 71.4% overall and 89.7% after accounting for ASCL1/NeuroD1-dual expressors and technical factors including <500 cells/slide, H-score thresholds and sample decalcification. No significant difference in subtype concordance was noted with a longer time lapse or with extrathoracic versus intrathoracic samples in this cohort. CONCLUSIONS: After accounting for technical factors, transcription factor-based subtyping was discordant among multiple SCLC samples in ~10% of patients, regardless of sample locations and time lapse. Our findings highlighted the spatiotemporal heterogeneity of SCLC in clinical samples and potential challenges, including technical and biological factors, that might limit concordance in SCLC transcription factor-based subtyping.

Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

Implementation and Clinical Adoption of Precision Oncology Workflows Across a Healthcare Network.

BACKGROUND: Precision oncology relies on molecular diagnostics, and the value-proposition of modern healthcare networks promises a higher standard of care across partner sites. We present the results of a clinical pilot to standardize precision oncology workflows. METHODS: Workflows are defined as the development, roll-out, and updating of disease-specific molecular order sets. We tracked the timeline, composition, and effort of consensus meetings to define the combination of molecular tests. To assess clinical impact, we examined order set adoption over a two-year period (before and after roll-out) across all gastrointestinal and hepatopancreatobiliary (GI) malignancies, and by provider location within the network. RESULTS: Development of 12 disease center-specific order sets took ~9 months, and the average number of tests per indication changed from 2.9 to 2.8 (P = .74). After roll-out, we identified significant increases in requests for GI patients (17%; P < .001), compliance with testing recommendations (9%; P < .001), and the fraction of "abnormal" results (6%; P < .001). Of 1088 GI patients, only 3 received targeted agents based on findings derived from non-recommended orders (1 before and 2 after roll-out); indicating that our practice did not negatively affect patient treatments. Preliminary analysis showed 99% compliance by providers in network sites, confirming the adoption of the order sets across the network. CONCLUSION: Our study details the effort of establishing precision oncology workflows, the adoption pattern, and the absence of harm from the reduction of non-recommended orders. Establishing a modifiable communication tool for molecular testing is an essential component to optimize patient care via precision oncology.

Lymphocyte-activation gene 3 in non-small-cell lung carcinomas: correlations with clinicopathologic features and prognostic significance.

Lymphocyte-activation gene 3 (LAG-3) modulates the tumor microenvironment through immunosuppressive effects. Its associations with clinicopathologic parameters and prognostic significance in non-small-cell lung carcinomas remain unclear. We examined LAG-3 expression in 368 resected non-small-cell lung carcinomas (including 218 adenocarcinomas and 150 squamous-cell carcinomas) using tissue microarrays, with normalization to CD8+ T-cell count (LAG-3/CD8 index), and correlated LAG-3, CD8, and LAG-3/CD8 index with clinicopathologic features, molecular status, and survival. LAG-3 expression in the immune cells (ranged 0.35-540.1 cells/mm²) was identified in 92% of non-small-cell lung carcinomas. In adenocarcinomas and squamous-cell carcinomas, LAG-3 expression correlated with CD8+ T-cell count and PD-L1 expression. In adenocarcinomas, high LAG-3 expression (defined as >median) was additionally associated with smoking history, high T stage, aggressive pathologic features (solid-predominant histologic pattern, lymphovascular invasion, and nodal metastasis), and lack of EGFR mutation. In the entire resected tumor cohort and in adenocarcinomas, high LAG-3 and LAG-3/CD8 index were each associated with worse overall survival. In squamous-cell carcinomas, high CD8 was associated with better overall survival. In an exploratory analysis of pretreatment samples from advanced non-small-cell lung carcinoma patients treated with pembrolizumab, high CD8 was predictive of improved overall and progression-free survival, while high LAG-3, but not high LAG-3/CD8 index, was associated with improved progression-free survival. In conclusion, the clinicopathologic correlations and prognostic impact of LAG-3 in non-small-cell lung carcinoma are histotype-dependent, highlighting differences in the immune microenvironment between adenocarcinomas and squamous-cell carcinomas. The predictive impact of LAG-3 warrants further investigation.

Assessing the Safety and Utility of Wound VAC Temporization of the Sarcoma or Benign Aggressive Tumor Bed Until Final Margins Are Achieved.

BACKGROUND: Local recurrence of microinvasive sarcoma or benign aggressive pathologies can be limb- and life-threatening. Although frozen pathology is reliable, tumor microinvasion can be subtle or missed, having an impact on surgical margins and postoperative radiation planning. The authors' service has begun to temporize the tumor bed after primary tumor excision with a wound vacuum-assisted closure (VAC) pending formal margin analysis, with coverage performed in the setting of final negative margins. METHODS: This retrospective analysis included all patients managed at a tertiary referral cancer center with VAC temporization after soft tissue sarcoma or benign aggressive tumor excision from 1 January 2000 to 1 January 2019 and at least 2 years of oncologic follow-up evaluation. The primary outcome was local recurrence. The secondary outcomes were distant recurrence, unplanned return to the operating room for wound/infectious indications, thromboembolic events, and tumor-related deaths. RESULTS: For 62 patients, VAC temporization was performed. The mean age of the patients was 62.2 ± 22.3 years (median 66.5 years; 95% confidence interval [CI] 61.7-72.5 years), and the mean age-adjusted Charlson Comorbidity Index was 5.3 ± 1.9. The most common tumor histology was myxofibrosarcoma (51.6%, 32/62). The mean volume was 124.8 ± 324.1 cm3, and 35.5% (22/62) of the cases were subfascial. Local recurrences occurred for 8.1% (5/62) of the patients. Three of these five patients had planned positive margins, and 17.7% (11/62) of the patients had an unplanned return to the operating room. No demographic or tumor factors were associated with unplanned surgery. CONCLUSIONS: The findings showed that VAC-temporized management of microinvasive sarcoma and benign aggressive pathologies yields favorable local recurrence and unplanned operating room rates suggestive of oncologic and technical safety. These findings will need validation in a future randomized controlled trial.

Vulvar angiomyofibroblastoma is molecularly defined by recurrent MTG1-CYP2E1 fusions.

Angiomyofibroblastoma (AMF), a rare benign vulvovaginal mesenchymal tumour, poses a diagnostic challenge due to histologic and immunohistochemical overlap with other vulvar mesenchymal tumours. Recently, MTG1-CYP2E1 fusion transcripts were reported in 5/5 AMFs; no other genetic alterations have been described to date. Herein, we sought to investigate the frequency of the MTG1-CYP2E1 fusion and the presence of other potential genetic alterations in a cohort of AMFs (n = 7, patient age range: 28-49 years). Tumours demonstrated classic morphologic features including alternating hypo/hypercellular areas, capillary channels surrounded by epithelioid/spindled tumour cells, and variable amounts of mature adipose tissue. reverse transcription-polymerase chain reaction (RT-PCR) for MTG1-CYP2E1 fusion, performed in all seven cases, showed the fusion transcript in five of six cases (one case with technical failure). Two tumours, including the one lacking the fusion, were subjected to targeted next-generation sequencing (104 genes) and a sarcoma fusion assay (28 genes); the fusion negative AMF also underwent RNA sequencing. No additional mutations, copy number alterations, or fusion genes were identified with the assays employed. We conclude that the majority of AMFs harbour recurrent MTG1-CYP2E1 fusion transcripts and identification of this fusion may aid in the diagnosis.

Large-scale analysis of BAP1 expression reveals novel associations with clinical and molecular features of malignant pleural mesothelioma.

BRCA1-associated protein-1 (BAP1) expression is commonly lost in several tumors including malignant pleural mesothelioma (MPM). Presence or absence of immunohistochemical BAP1 nuclear staining in tumor cells is currently used for differential diagnosis of MPM. In this study, a large cohort of 596 MPM tumors with available clinical data was analyzed to examine associations of BAP1 staining pattern with clinical and molecular features that may reflect the impact of BAP1 mutation on MPM biology. Cases were classified according to the BAP1 staining pattern of tumor cells. Exome and RNA-sequencing data were available for subsets of cases. Levels of mRNA encoding claudin 15 (CLDN15) and vimentin (VIM) were determined using RT-qPCR on 483 cases to estimate the relative proportions of epithelial-like and mesenchymal-like components in each tumor. Four BAP1 staining patterns were observed: single-pattern nuclear staining (36%), single-pattern cytoplasmic staining (25%), single-pattern absent staining (12%), and combinations of these staining patterns (27%). This study confirmed prior reports that nuclear BAP1 is more frequently associated with wild-type BAP1 and sarcomatoid histology. However, no associations between BAP1 staining pattern(s) and mutations in specific protein domains and/or mutation type were observed. BAP1 staining patterns were significantly associated (p < 0.001) with BAP1 gene expression, MPM histologic subtypes, molecular clusters, and markers of epithelial-to-mesenchymal transition. Frequent observation of combinations of BAP1 staining patterns in MPM tumors indicated intra-tumoral heterogeneity of BAP1 status. Cytoplasmic BAP1 staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM. In conclusion, novel significant associations among different BAP1 staining patterns and subgroups of MPM tumors were observed, suggesting that the role of BAP1 in tumor progression may be more complex than its presumed tumor suppressor function. Cytoplasmic staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM, potentially addressing a critical need in clinical decision-making in this disease. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.