Extragonadal germ cell tumors: A clinicopathologic study with emphasis on molecular features, clinical outcomes and associated secondary malignancies.

Extragonadal germ cell tumors (EGCTs) are rare, representing <5% of all germ cell tumors (GCTs). Whilst EGCTs share morphological and immunohistochemical features with their gonadal counterparts, they tend to be more aggressive and are frequently associated with secondary somatic malignancies. The aim of our study was to evaluate the clinical, morphological and immunohistochemical features, and to analyze tumors for chromosomal abnormalities of 12p, in addition to any novel genetic alterations, in a series of EGCTs. Seventy-seven EGCTs were included. Anterior mediastinum was the most common anatomic site, followed by central nervous system, retroperitoneum, sacroccygeal area, and neck. Whole genome SNP array identified isochromosome 12p in 26% of tumors. Additional cytogenetic abnormalities included the presence of gain of chr 21 in 37% of tumors. Somatic-type malignancies were identified in 8% of patients. Disease progression (metastasis and/or recurrence) was documented in 8 patients, most of whom died from their relapse. Three patients who died of disease had somatic-type malignancies. Mediastinal seminomas had a significantly better overall survival when compared to mediastinal non-seminomatous GCTs. Our study demonstrates that EGCTs share similar histologic features, but diverse clinical outcomes compared to their gonadal counterparts. Outcomes vary according to anatomic location and histologic subtypes. Our data corroborate that somatic-type malignancies are frequently encountered in mediastinal EGCTs and that their presence portends a poorer prognosis.

Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay.

While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.

Performance of fluorescence in situ hybridization in biliary brushings with equivocal cytology: an institutional experience.

INTRODUCTION: Biliary brushing (BB) cytology has a sensitivity of 15%-65% and specificity approaching 100% for detecting malignancy. Fluorescence in-situ hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of malignancies with reported sensitivity of 43%-84%. We sought to evaluate the performance of FISH in BB with equivocal cytology at our institution. MATERIALS AND METHODS: Patients with atypical and suspicious BB with concurrent diagnostic FISH performed at our institution from 2014 to 2021 were identified through a query of our pathology database. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed for pathology results and outcomes. Patients were classified malignant if they had positive pathology or documented clinical impression of malignancy and benign if they had negative pathology and/or documented benign clinical course for at least 12 months. RESULTS: We identified 254 equivocal BB (238 atypical/16 suspicious) with concurrent FISH results from 191 patients (105 benign, 86 malignant). 12% (22/191) of patients were FISH positive. Twenty-four percent (21/86) of patients with malignancy had positive FISH but were nonspecific for pancreaticobiliary/ampullary adenocarcinomas. Almost all positive FISH were associated with malignancy (21/22; 95%). There was 1 positive FISH in a patient with primary sclerosing cholangitis who had a benign outcome. CONCLUSIONS: The small number of positive FISH results in BB with equivocal cytology raises the question of the optimal criteria for malignancy. Using only polysomy could result in lower sensitivity.

Continuous Wound Irrigation and Intraoperative Methadone Decreases Opioid Use and Shortens Length of Stay After CRS/HIPEC.

BACKGROUND: Epidural analgesia is resource and labor intense and may limit postoperative management options and delay discharge. This study compared postoperative outcomes after cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) with epidural analgesia versus continuous wound infusion system (CWIS) with/without intraoperative methadone. METHODS: A single-institution, retrospective chart review was performed including all patients undergoing open CRS/HIPEC from 2018 to 2021. Patient demographics, surgical characteristics, length of stay, and in-hospital analgesic use were reviewed. In-hospital opioid exposure in morphine milligram equivalents (MME) was calculated. Multivariate analysis (MVA) for mean total and daily opioid exposure was conducted. RESULTS: A total of 157 patients were included. Fifty-three (34%) had epidural analgesia, 96 (61%) had CWIS, and 79 (50%) received methadone. Length of stay was significantly shorter with CWIS + methadone versus epidural (7 vs. 8 days, p < 0.01). MVA showed significantly lower mean total and daily opioid exposure with CWIS+methadone versus epidural (total: 252.8 ± 17.7 MME vs. 486.8 ± 86.6 MME; odds ratio [OR] 0.72, 95% confidence interval [CI] 0.52-0.98, p = 0.04; Daily: 32.8 ± 2.0 MME vs. 51.9 ± 5.7 MME, OR 0.72, 95% CI 0.52-0.99, p ≤ 0.05). The CWIS-only group (n = 17) had a significantly lower median oral opioid exposure versus epidural (135 MME vs. 7.5 MME, p < 0.001) and longer length of stay versus CWIS + methadone (9 vs. 7 days, p = 0.04), There were no CWIS or methadone-associated complications and one epidural abscess. CONCLUSIONS: CWIS + methadone safely offers better pain control with less in-hospital opioid use, shorter length of stay, and decreased resource utilization compared with epidural analgesia in patients undergoing CRS-HIPEC.

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Myelodysplasia Cutis.

CONTEXT.­: Myelodysplasia cutis is an emerging concept in cutaneous neoplasia. Many of these cases were previously included under the umbrella of histiocytoid Sweet syndrome. However, with the advent of next-generation sequencing, cutaneous involvement by myelodysplastic syndrome is being increasingly recognized. OBJECTIVE.­: To review histiocytoid Sweet syndrome and myelodysplasia cutis and discuss our current understanding of these entities. Additionally, to discuss how next-generation sequencing can be applied in the evaluation of cutaneous infiltrates of immature histiocytoid cells. DATA SOURCES.­: The English-language literature from 2005 to 2023 on the topic of histiocytoid Sweet syndrome and myelodysplasia cutis was reviewed. CONCLUSIONS.­: Biopsy specimens showing infiltrates of histiocytoid, immature myeloid cells may represent cutaneous involvement by myelodysplastic syndrome. Close clinical correlation is recommended in these cases. Recent studies suggest that next-generation sequencing is useful in separating myelodysplasia cutis from true histiocytoid Sweet syndrome. This distinction has important implications for patients.

Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

Molecular Analysis of Liquid Vitreous Biopsy Reveals Occult Lymphoma Following Cytology-Negative Biopsies of the Brain and Vitreous.

PURPOSE: Primary central nervous system lymphoma (PCNSL) is a rare but deadly malignancy that principally affects adults in the fifth and sixth decades of life. Despite diagnostic advances in analyses of cerebral spinal fluid and neuroimaging, definitive diagnosis of PCNSL requires primary brain tissue biopsy. While small neurosurgical biopsy volumes are pursued to minimize removal of normal brain tissue, the spatial margins to precisely biopsy pathologic tissue are narrow and can result in missed diagnoses. Furthermore, prior steroid treatment can significantly reduce tumor burden increasing the likelihood of a non-diagnostic biopsy. METHODS: A retrospective case report from a tertiary referral center using a combination of neuroradiological studies, sterotactic tissue biopsy, and molecular testing for genome mutations. RESULTS: A 72-year-old woman with strong suspicion for PCNSL clinically and radiologically, but cerebral spinal fluid and primary brain tissue biopsy were negative for tumor. However, vitreous liquid biopsy molecular testing for a MYD88 mutation as well as B-cell clonality (IGH/IGK rearrangement) were positive, indicating the presence of secondary vitreoretinal lymphoma from PCNSL. Only after autopsy of her brain was histopathological and immunohistochemical evidence of PCNSL confirmed. CONCLUSION: This case illustrates the unique contribution of liquid biopsy neuropathology-oriented molecular testing in a challenging case with high clinical suspicion of PCNSL in which gold-standard diagnostic testing failed to yield a diagnosis.

Takotsubo Cardiomyopathy (TCM) After Uncomplicated Paraesophageal Hernia Repair: A Case Report and Review on Postoperative TCM.

Takotsubo cardiomyopathy (TCM) is a rare stress-induced condition that appears rarely in suspected acute myocardial infarction cases. It causes unexplained left ventricular failure, but most cases are reversible with supportive treatment. In this report, we present the case of a 70-year-old female who developed acute hypotension after a laparoscopic Toupet fundoplication on postoperative day one, requiring care in the surgical intensive care unit. Following consultation with the cardiology service and further imaging and tests, she was diagnosed with TCM. This report outlines the potential mechanisms and management of TCM in the intensive care unit, emphasizing the importance of prompt diagnosis and treatment.

Lessons from 801 clinical TFE3/TFEB fluorescence in situ hybridization assays performed on renal cell carcinoma suspicious for MiTF family aberrations.

OBJECTIVES: Fluorescence in situ hybridization (FISH) assays for the detection of chromosomal rearrangements involving TFE3 and TFEB are considered the gold standard for the diagnosis of MiTF family altered renal cell carcinoma (MiTF-RCC). We reviewed 801 clinical TFE3/TFEB FISH assays performed at our tertiary-level institution between 2014 and 2023 on kidney tumors suspicious at the morphologic or biomarker level for MiTF aberrations. METHODS: We summarized and analyzed clinical information, TFE3/TFEB FISH results, and available biomarker staining results in a cohort of 453 consecutive kidney tumor cases suspicious for MiTF-RCC. RESULTS: In total, 61 of 434 (14%) kidney tumors were confirmed for TFE3 translocation; 10 of 367 cases (2.7%) were confirmed for TFEB translocation. Since TFEB amplification interpretation was implemented in our service line, 20 of 306 cases (6.5%) were diagnosed with TFEB amplification. Importantly, TFE3 and TFEB rearrangements were never co-detected within the same kidney tumor. Patients with TFEB amplification were significantly older (P < .001) than patients with TFE3 or TFEB translocation. Kidney tumors with TFEB amplification were seen to be at least 3 times as common as those with TFEB translocation. CONCLUSIONS: Clinical TFE3/TFEB FISH assays successfully identified and confirmed rare MiTF-RCC with TFE3 and TFEB rearrangements. Although morphologic and biomarker features associated with a kidney tumor may be suggestive of MiTF-RCC, clinical TFE3/TFEB FISH assays are crucial for a confirmation and definitive subclassification of patients with MiTF-RCC.

Endometrial Carcinomas With Subclonal Loss of Mismatch Repair Proteins: A Clinicopathologic and Genomic Study.

Subclonal loss of mismatch repair (MMR) proteins has been described in a small subset of endometrial carcinomas (ECs), but the genomic basis for this phenomenon has received limited attention. Herein, we retrospectively evaluated all ECs with MMR immunohistochemistry (n=285) for subclonal loss, and in those (n=6), performed a detailed clinicopathologic and genomic comparison of the MMR-deficient and MMR-proficient components. Three tumors were FIGO stage IA, and one each stage IB, II, and IIIC2. Patterns of subclonal loss were as follows: (1) 3 FIGO grade 1 endometrioid carcinomas with subclonal MLH1/PMS2, MLH1 promoter hypermethylation, and no MMR gene mutations; (2) POLE -mutated FIGO grade 3 endometrioid carcinoma with subclonal PMS2, and PMS2 and MSH6 mutations limited to the MMR-deficient component; (3) dedifferentiated carcinoma with subclonal MSH2/MSH6, as well as complete loss of MLH1/PMS2, MLH1 promoter hypermethylation, and PMS2 and MSH6 mutations in both components; (4) dedifferentiated carcinoma with subclonal MSH6, and somatic and germline MSH6 mutations in both components, but with a higher allele frequency in MMR-deficient foci. Recurrences occurred in 2 patients, one consisted of the MMR-proficient component from a FIGO 1 endometrioid carcinoma, while the other was from the MSH6 -mutated dedifferentiated endometrioid carcinoma. At the last follow-up (median: 44 mo), 4 patients were alive and disease-free and 2 were alive with disease. In summary, subclonal MMR loss reflects subclonal and often complex genomic and epigenetic alterations, which may have therapeutic implications and therefore must be reported when present. In addition, subclonal loss can occur in both POLE -mutated and Lynch syndrome-associated ECs.

ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia.

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6-RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6-RUNX1+/- like B-ALL "signature" genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6-RUNX1+/- like B-ALL. SIGNIFICANCE: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1.

Aqueous Humor-Derived MYD88 L265P Mutation Analysis in Vitreoretinal Lymphoma: A Potential Less Invasive Method for Diagnosis and Treatment Response Assessment.

PURPOSE: To investigate whether MYD88 L265P mutation, which is frequently present in vitreoretinal lymphoma, can be detected in aqueous humor, a specimen that can be obtained in a clinic setting, potentially mitigating the need for more invasive vitrectomy procedures, and whether this approach can be used to monitor treatment response. DESIGN: Observational case series. SUBJECTS: Patients who were diagnosed with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma or biopsy-confirmed vitritis. METHODS: We evaluated aqueous humor-derived (AHD) MYD88 L265P mutation during vitreous biopsy or at the initial presentation in the clinic if vitreous biopsy was not feasible. Demographic or clinical features of patients were retrospectively reviewed. Aqueous humor-derived MYD88 L265P mutation was re-evaluated after patients completed a course of intravitreal methotrexate and rituximab injection therapy. The NM_002468.4: c.794T>C (p.L265P) mutation in the MYD88 gene was evaluated in AHD cellular and cell-free DNA using allele-specific polymerase chain reaction. MAIN OUTCOME MEASURES: Detection of AHD MYD88 L265P mutation at the initial diagnosis and to monitor the treatment response. RESULTS: Aqueous humor from 18 eyes of 14 patients with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma and 3 eyes of 3 patients with biopsy-confirmed vitritis were evaluated. Aqueous humor-derived MYD88 L265P mutation was detected in cell-based and cell-free DNA from 15 (83%) of 18 eyes with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma but not identified in any of the 3 eyes with vitritis. The mutation was less readily detectable in cellular DNA (10 of 18) compared with cell-free DNA (15 of 18). Furthermore, aqueous sampling after intravitreal methotrexate and rituximab injection therapy revealed absence of this mutation after complete response in 7 eyes. The mutation was detected in 1 eye that developed recurrence in a posttreatment window of 6 months. After a mean of follow-up of 9 months, there was no clinical evidence of vitreoretinal lymphoma recurrence in the 7 eyes with no detectable AHD MYD88 L265P mutation. CONCLUSIONS: This investigational study suggests that AHD MYD88 L265P can be detected in eyes with lymphoma and may thus serve as a surrogate, less invasive biopsy in the diagnosis and follow-up of vitreoretinal lymphoma, particularly when cell-free DNA is evaluated.

GATA-3 is a proto-oncogene in T-cell lymphoproliferative neoplasms.

Neoplasms originating from thymic T-cell progenitors and post-thymic mature T-cell subsets account for a minority of lymphoproliferative neoplasms. These T-cell derived neoplasms, while molecularly and genetically heterogeneous, exploit transcription factors and signaling pathways that are critically important in normal T-cell biology, including those implicated in antigen-, costimulatory-, and cytokine-receptor signaling. The transcription factor GATA-3 regulates the growth and proliferation of both immature and mature T cells and has recently been implicated in T-cell neoplasms, including the most common mature T-cell lymphoma observed in much of the Western world. Here we show that GATA-3 is a proto-oncogene across the spectrum of T-cell neoplasms, including those derived from T-cell progenitors and their mature progeny, and further define the transcriptional programs that are GATA-3 dependent, which include therapeutically targetable gene products. The discovery that p300-dependent acetylation regulates GATA-3 mediated transcription by attenuating DNA binding has novel therapeutic implications. As most patients afflicted with GATA-3 driven T-cell neoplasms will succumb to their disease within a few years of diagnosis, these findings suggest opportunities to improve outcomes for these patients.

Notch Signaling Promotes Mature T-Cell Lymphomagenesis.

Peripheral T-cell lymphomas (PTCL) are agressive lymphomas that develop from mature T cells. The most common PTCLs are genetically, molecularly, and clinically diverse and are generally associated with dismal outcomes. While Notch signaling plays a critically important role in both the development of immature T cells and their malignant transformation, its role in PTCL is poorly understood, despite the increasingly appreciated function of Notch in regulating the proliferation and differentiation of mature T cells. Here, we demonstrate that Notch receptors and their Delta-like family ligands (DLL1/DLL4) play a pathogenic role in PTCL. Notch1 activation was observed in common PTCL subtypes, including PTCL-not otherwise specified (NOS). In a large cohort of PTCL-NOS biopsies, Notch1 activation was significantly associated with surrogate markers of proliferation. Complementary genetically engineered mouse models and spontaneous PTCL models were used to functionally examine the role of Notch signaling, and Notch1/Notch2 blockade and pan-Notch blockade using dominant-negative MAML significantly impaired the proliferation of malignant T cells and PTCL progression in these models. Treatment with DLL1/DLL4 blocking antibodies established that Notch signaling is ligand-dependent. Together, these findings reveal a role for ligand-dependent Notch signaling in driving peripheral T-cell lymphomagenesis. SIGNIFICANCE: This work demonstrates that ligand-dependent Notch activation promotes the growth and proliferation of mature T-cell lymphomas, providing new therapeutic strategies for this group of aggressive lymphomas.

SOCS1 Haploinsufficiency Presenting as Severe Enthesitis, Bone Marrow Hypocellularity, and Refractory Thrombocytopenia in a Pediatric Patient with Subsequent Response to JAK Inhibition.

Haploinsufficiency of suppressor of cytokine signaling 1 (SOCS1) is a recently discovered autoinflammatory disorder with significant rheumatologic, immunologic, and hematologic manifestations. Here we report a case of SOCS1 haploinsufficiency in a 5-year-old child with profound arthralgias and immune-mediated thrombocytopenia unmasked by SARS-CoV-2 infection. Her clinical manifestations were accompanied by excessive B cell activity, eosinophilia, and elevated IgE levels. Uniquely, this is the first report of SOCS1 haploinsufficiency in the setting of a chromosomal deletion resulting in complete loss of a single SOCS1 gene with additional clinical findings of bone marrow hypocellularity and radiologic evidence of severe enthesitis. Immunologic profiling showed a prominent interferon signature in the patient's peripheral blood mononuclear cells, which were also hypersensitive to stimulation by type I and type II interferons. The patient showed excellent clinical and functional laboratory response to tofacitinib, a Janus kinase inhibitor that disrupts interferon signaling. Our case highlights the need to utilize a multidisciplinary diagnostic approach and consider a comprehensive genetic evaluation for inborn errors of immunity in patients with an atypical immune-mediated thrombocytopenia phenotype.

Anthropogenic Zinc Exposure Increases Mortality and Antioxidant Gene Expression in Monarch Butterflies with Low Access to Dietary Macronutrients.

Biologists seek to understand why organisms vary in their abilities to tolerate anthropogenic contaminants, such as heavy metals. However, few studies have considered how tolerance may be affected by condition-moderating factors such as dietary resource availability. For instance, the availability of crucial limiting macronutrients, such as nitrogen and phosphorous, can vary across space and time either naturally or due to anthropogenic nutrient inputs (e.g., agricultural fertilizers or vehicle emissions). Organisms developing in more macronutrient-rich environments should be of higher overall condition, displaying a greater ability to tolerate metal contaminants. In monarch butterflies (Danaus plexippus), we factorially manipulated dietary macronutrient availability and exposure to zinc, a common metal contaminant in urban habitats that can be toxic but also has nutritional properties. We tested whether (1) the ability to survive zinc exposure depends on dietary macronutrient availability and (2) whether individuals exposed to elevated zinc levels display higher expression of antioxidant genes, given the roles of antioxidants in combatting metal-induced oxidative stress. Exposure to elevated zinc reduced survival only for monarchs developing on a low-macronutrient diet. However, for monarchs developing on a high-macronutrient diet, elevated zinc exposure tended to increase survival. In addition, monarchs exposed to elevated zinc displayed higher expression of antioxidant genes when developing on the low-macronutrient diet but lower expression when developing on the high-macronutrient diet. Altogether, our study shows that organismal survival and oxidative stress responses to anthropogenic zinc contamination depend on the availability of macronutrient resources in the developmental environment. In addition, our results suggest the hypothesis that whether zinc acts as a toxicant or a nutrient may depend on macronutrient supply. Environ Toxicol Chem 2022;41:1286-1296. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

TERT Promoter Mutations in Keratinizing and Nonkeratinizing Squamous Metaplasia of the Urinary Tract.

We identified urothelial tract biopsy and resection specimens with keratinizing squamous metaplasia (KSM), nonkeratinizing squamous metaplasia (NKSM), and urothelial and squamous carcinomas over a 20-yr period, focusing on cases with neurogenic lower urinary tract dysfunction (NLUTD) and/or those with spatial or temporal variation in sampling. TERT promoter mutations as assessed via allele-specific polymerase chain reaction were surprisingly common in our testing cohort, identified not only in 15 (94%) invasive cancer foci but also in 13 (68%) examples of KSM and seven (70%) examples of NKSM. TERT promoter mutations were present in 23 foci from NLUTD specimens and 11 foci from bladder diverticula, including in foci of KSM, NKSM, and unremarkable urothelium from cases with no clinical association with previous, concurrent, or subsequent cancer. Our demonstration of temporally and spatially persistent TERT promoter mutation in examples of KSM and NKSM in cases of bladder cancer and in morphologically benign cases with neurogenic dysfunction suggests a molecular mechanism by which such pre-neoplastic lesions can potentially progress and develop into overt carcinoma. Given the interest in TERT promoter mutations as a potential biomarker for the development of bladder cancer, these findings possibly explain the association between conditions with chronic urinary bladder injury (such as the natural history of NLUTD) and higher risk of bladder cancer. TERT promoter mutations may represent an early event in bladder cancer tumorogenesis, and our findings expand on the clinical ramifications and predictive value of TERT promoter mutations in this context. PATIENT SUMMARY: Mutations in the TERT gene are the most common genetic changes in bladder cancer. We found that these mutations are also sometimes present in patients with chronic bladder irritation such as neurogenic bladder dysfunction and changes to the lining of the bladder that pathologists would consider "benign." This finding might explain why such conditions are associated with the development of bladder cancer.

Targeting Lymphoma-associated Macrophage Expansion via CSF1R/JAK Inhibition is a Therapeutic Vulnerability in Peripheral T-cell Lymphomas.

The reciprocal relationship between malignant T cells and lymphoma-associated macrophages (LAM) within the tumor microenvironment (TME) is unique, as LAMs are well poised to provide ligands for antigen, costimulatory, and cytokine receptors that promote T-cell lymphoma growth. Conversely, malignant T cells promote the functional polarization and homeostatic survival of LAM. Therefore, we sought to determine the extent to which LAMs are a therapeutic vulnerability in these lymphomas, and to identify effective therapeutic strategies for their depletion. We utilized complementary genetically engineered mouse models and primary peripheral T-cell lymphoma (PTCL) specimens to quantify LAM expansion and proliferation. A high-throughput screen was performed to identify targeted agents that effectively deplete LAM within the context of PTCL. We observed that LAMs are dominant constituents of the TME in PTCL. Furthermore, their dominance was explained, at least in part, by their proliferation and expansion in response to PTCL-derived cytokines. Importantly, LAMs are a true dependency in these lymphomas, as their depletion significantly impaired PTCL progression. These findings were extrapolated to a large cohort of human PTCL specimens where LAM proliferation was observed. A high-throughput screen demonstrated that PTCL-derived cytokines led to relative resistance to CSF1R selective inhibitors, and culminated in the identification of dual CSF1R/JAK inhibition as a novel therapeutic strategy to deplete LAM in these aggressive lymphomas. Malignant T cells promote the expansion and proliferation of LAM, which are a bone fide dependency in these lymphomas, and are effectively depleted with a dual CSF1R/JAK inhibitor. Significance: LAMs are a therapeutic vulnerability, as their depletion impairs T-cell lymphoma disease progression. Pacritinib, a dual CSF1R/JAK inhibitor, effectively impaired LAM viability and expansion, prolonged survival in preclinical T-cell lymphoma models, and is currently being investigated as a novel therapeutic approach in these lymphomas.