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Pathological estimation of tumor necrosis after chemotherapy is essential for patients with osteosarcoma. This study reports the first fully automated tool to assess viable and necrotic tumor in osteosarcoma, employing advances in histopathology digitization and automated learning. We selected 40 digitized whole slide images representing the heterogeneity of osteosarcoma and chemotherapy response. With the goal of labeling the diverse regions of the digitized tissue into viable tumor, necrotic tumor, and non-tumor, we trained 13 machine-learning models and selected the top performing one (a Support Vector Machine) based on reported accuracy. We also developed a deep-learning architecture and trained it on the same data set. We computed the receiver-operator characteristic for discrimination of non-tumor from tumor followed by conditional discrimination of necrotic from viable tumor and found our models performing exceptionally well. We then used the trained models to identify regions of interest on image-tiles generated from test whole slide images. The classification output is visualized as a tumor-prediction map, displaying the extent of viable and necrotic tumor in the slide image. Thus, we lay the foundation for a complete tumor assessment pipeline from original histology images to tumor-prediction map generation. The proposed pipeline can also be adopted for other types of tumor.

Low-grade oncocytic tumor (LOT) of the kidney is a recently described entity with poorly understood pathogenesis. Using next-generation sequencing (NGS) and complementary approaches, we provide insight into its biology. We describe 22 LOT corresponding to 7 patients presenting with a median age of 75 years (range 63–86 years) and male to female ratio 2:5. All 22 tumors demonstrated prototypical microscopic features. Tumors were well-circumscribed and solid. They were composed of sheets of tumor cells in compact nests. Tumor cells had eosinophilic cytoplasm, round to oval nuclei (without nuclear membrane irregularities), focal subtle perinuclear halos, and occasional binucleation. Sharply delineated edematous stromal islands were often observed. Tumor cells were positive for PAX8, negative for CD117, and exhibited diffuse and strong cytokeratin-7 expression. Six patients presented with pT1 tumors. At a median follow-up of 29 months, four patients were alive without recurrence (three patients had died from unrelated causes). All tumors were originally classified as chromophobe renal cell carcinoma, eosinophilic variant (chRCC-eo). While none of the patients presented with known syndromic features, one patient with multiple bilateral LOTs was subsequently found to have a likely pathogenic germline TSC1 mutation. Somatic, likely activating, mutations in MTOR and RHEB were identified in all other evaluable LOTs. As assessed by phospho-S6 and phospho-4E-BP1, mTOR complex 1 (mTORC1) was activated across all cases but to different extent. MTOR mutant LOT exhibited lower levels of mTORC1 activation, possibly related to mTORC1 dimerization and the preservation of a wild-type MTOR copy (retained chromosome 1). Supporting its distinction from related entities, gene expression analyses showed that LOT clustered separately from classic chRCC, chRCC-eo, and RO. In summary, converging mTORC1 pathway mutations, mTORC1 complex activation, and a distinctive gene expression signature along with characteristic phenotypic features support LOT designation as a distinct entity with both syndromic and non-syndromic cases associated with an indolent course.

Background COVID-19 is caused by SARS-CoV2, which primarily produces respiratory disease. Although liver enzyme abnormalities are common in patients with COVID-19, little is known about histological changes in the liver. Most of the available information is based on autopsy studies, with limited liver biopsy studies in living patients. Methods Liver biopsies performed between March 2021 and September 2021 in patients with a history of COVID-19 were retrieved, and 17 biopsies were reviewed. Additionally, immunohistochemical staining for the SARS-CoV2 nucleocapsid protein was performed in all cases. The clinical follow-up period was 36 months. Results After reviewing the clinical data, the biopsies were grouped into three cohorts: (1) those with no underlying liver disease, (2) those with preexisting liver disease, and (3) those with liver post-transplant allografts. In patients without underlying liver disease, there were small clusters of debris-laden macrophages in the portal areas and lobules, as highlighted by periodic acid-Schiff and diastase staining. Increased mitosis and binucleated hepatocytes were common, and rare micro-abscesses were also observed. These findings indicated mild non-specific hepatitis, and no severe inflammatory infiltrates were observed in portal areas or lobules. These features are highly suggestive of hepatitis resolution. Patients with previous liver disease showed microscopic features compatible with underlying liver disease, without any additional features that could be attributed to COVID-19. Posttransplant allograft biopsies showed no special features that could be described as occurring solely due to COVID-19. All patients tested negative on SARS-CoV2 nucleoprotein immunostains. Conclusions Liver biopsies performed after COVID-19 demonstrated a mildly resolved hepatitis-like pattern. 36 months after biopsy, all patients in Group 1 and all surviving patients in Group 2 and 3 presented normal liver enzyme levels. It is therefore, from this small case series, that the liver may not be the target of significant inflammatory damage in COVID-19 and that liver injuries are resolved over time. Key messages • Liver biopsies of patients with no prior liver disease following COVID-19 infection showed mild non-specific hepatitis, indicating recent liver injury. • Liver biopsy with underlying liver disease presents a different type of challenge due to its overlapping histological features with COVID-related hepatitis, and no specific features could be described as occurring solely due to COVID-19. • All patients were negative for SARS-CoV2 nucleoprotein, which raises the question of whether liver damage was not the direct result of viral infection but a reactive response to cytokines produced during the infection.

Pediatric clinical laboratories commonly measure tricarboxylic acid cycle intermediates for screening, diagnosis, and monitoring of specific inborn errors of metabolism, such as organic acidurias. In the past decade, the same tricarboxylic acid cycle metabolites have been implicated and studied in cancer. The accumulation of these metabolites in certain cancers not only serves as a biomarker but also directly contributes to cellular transformation, therefore earning them the designation of oncometabolites. -2-hydroxyglutarate, -2-hydroxyglutarate, succinate, and fumarate are the currently recognized oncometabolites. They are structurally similar and share metabolic proximity in the tricarboxylic acid cycle. As a result, they promote tumorigenesis in cancer cells through similar mechanisms. This review summarizes the currently understood common and distinct biological features of these compounds. In addition, we will review the current laboratory methodologies that can be used to quantify these metabolites and their downstream targets. Oncometabolites play an important role in cancer biology. The metabolic pathways that lead to the production of oncometabolites and the downstream signaling pathways that are activated by oncometabolites represent potential therapeutic targets. Clinical laboratories have a critical role to play in the management of oncometabolite-associated cancers through development and validation of sensitive and specific assays that measure oncometabolites and their downstream effectors. These assays can be used as screening tools and for follow-up to measure response to treatment, as well as to detect minimal residual disease and recurrence.

Anti-angiogenic (AA) therapy is a cornerstone of metastatic clear cell renal cell carcinoma (ccRCC) treatment, but not everyone responds, and predictive biomarkers are lacking. CD31, a marker of vasculature, is insufficient, and the Angioscore, an RNA-based angiogenesis quantification method, is costly, associated with delays, difficult to standardize, and does not account for tumor heterogeneity. Here, we developed an interpretable deep learning (DL) model that predicts the Angioscore directly from ubiquitous histopathology slides yielding a visual vascular network (H&E DL Angio). H&E DL Angio achieves a strong correlation with the Angioscore across multiple cohorts (spearman correlations of 0.77 and 0.73). Using this approach, we found that angiogenesis inversely correlates with grade and stage and is associated with driver mutation status. Importantly, DL Angio expediently predicts AA response in both a real-world and IMmotion150 trial cohorts, out-performing CD31, and closely approximating the Angioscore (c-index 0.66 vs 0.67) at a fraction of the cost. Angioscore is a transcriptome based predicter of treatment response in clear cell renal cell carcinoma. Here, the authors use deep learning to infer the Angioscore from histopathology slides using clinical trial data.

The spectrum of neoplasms associated with DICER1 variants continues to expand, with the recent addition of primary “ DICER1 -associated central nervous system sarcoma” (DCS). DCS is a high-grade malignancy predominantly affecting pediatric patients. Six pediatric DCS were identified through a combination of clinical diagnostic studies, archival inquiry, and interinstitutional collaboration. Clinical, histologic, immunohistologic, and molecular features were examined. Genomic findings in the 6 DCS were compared with those in 14 additional DICER1 -associated tumors sequenced with the same assay. The six patients presented at ages 3–15 years with CNS tumors located in the temporal ( n  = 2), parietal ( n  = 1), fronto-parietal ( n  = 1), and frontal ( n  = 2) lobes. All underwent surgical resection. Histologic examination demonstrated high-grade malignant spindle cell tumors with pleuropulmonary blastoma-like embryonic “organoid” features and focal rhabdomyoblastic differentiation; immature cartilage was seen in one case. Immunohistochemically, there was patchy desmin and myogenin staining, and patchy loss of H3K27me3, and within eosinophilic cytoplasmic globules, alfa-fetoprotein staining. Biallelic DICER1 variants were identified in all cases, with germline variants in two of five patients tested. DCS demonstrated genomic alterations enriched for Ras pathway activation and TP53 inactivation. Tumor mutational burden was significantly higher in the 6 DCS tumors than in 14 other DICER1 -associated tumors examined (mean 12.9 vs. 6.8 mutations/Mb, p  = 0.035). Postoperative care included radiation ( n  = 5) and chemotherapy ( n  = 3); at the last follow-up, three patients were alive without DCS, and three had died of disease. Our analysis expands the clinical, histologic, immunohistological, and molecular spectrum of DCS, identifying distinctive features that can aid in the diagnosis, multidisciplinary evaluation, and treatment of DCS.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6–19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3, inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3/HES3, that contributes to impaired myogenic differentiation and has prognostic significance in human disease. One of the most common cancers in children and adolescents is rhabdomyosarcoma, a cancer of soft tissue such as muscle, tendon or cartilage. The fusion of DNA on two different chromosomes causes the most aggressive form of rhabdomyosarcoma. The fused DNA produces an abnormal protein called PAX3-FOXO1. During normal muscle development, a subset of rapidly growing cells eventually slow down and form mature, working muscle cells. It is still unclear how exactly rhabdomyosarcoma develops, but it is thought that PAX3-FOXO1 stops muscle cells from maturing and the cells that grow out of control result in a tumor. Learning how PAX3-FOXO1 hijacks normal muscle development could lead to new treatments for rhabdomyosarcoma. One treatment approach is to slow the growth of a tumor and force the cells to mature. Then, young patients might avoid chemotherapy or radiation treatments and their side effects. Efforts to improve treatment for this type of cancer face several obstacles. Currently, only one vertebrate animal model of the disease is available to test drugs, and it is still not known how PAX3-FOXO1 causes healthy cells to become cancerous. It is also hard to turn off PAX3-FOXO1 itself, so scientists must find additional proteins that collaborate with it to target with drugs. Now, Kendall et al. show that genetically engineered zebrafish with human PAX3-FOXO1 develop rhabdomyosarcoma-like tumors. Experiments on these zebrafish showed that the protein turns on a gene called her3. Humans have a similar gene called HES3. In zebrafish or mouse cells, human HES3 interferes with muscle-cell maturation and allows cells that acquire PAX3-FOXO1 to persist during development instead of dying. It also increases the cell growth and cancerous behavior in human tumor cells. Kendall et al. further looked at HES3 levels in tumors collected from patients with rhabdomyosarcoma and found that having higher levels of HES3 increased the risk of death from the cancer. Human rhabdomyosarcoma tumors with high HES3 levels were also more likely to have certain cell-growth and cell-differentiation related proteins. Drugs that turn off or modify the activity of these proteins already exist. Testing these drugs that target processes such as cell growth in the zebrafish with rhabdomyosarcoma-like tumors may help scientists determine if they reduce tumor growth. If they do, additional trials could determine if they would help patients with rhabdomyosarcoma.

Mitochondria can function as signaling organelles, and part of this output leads to epigenetic remodeling. The full extent of this far-reaching interplay remains undefined. Here, we show that MYC transcriptionally activates IDH2 and increases alpha-ketoglutarate (αKG) levels. This regulatory step induces the activity of αKG-dependent DNA hydroxylases and RNA demethylases, thus reducing global DNA and RNA methylation. MYC, in a IDH2-dependent manner, also promotes the nuclear accumulation of TET1-TET2-TET3, FTO and ALKBH5. Notably, this subcellular movement correlated with the ability of MYC, in an IDH2-dependent manner, and, unexpectedly, of αKG to directly induce O-GlcNAcylation. Concordantly, modulation of the activity of OGT and OGA, enzymes that control the cycling of this non-canonical mono-glycosylation, largely recapitulated the effects of the MYC-IDH2-αKG axis on the subcellular movement of DNA and RNA demethylases. Together, we uncovered a hitherto unsuspected crosstalk between MYC, αKG and O-GlcNAcylation which could influence the epigenome and epitranscriptome homeostasis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models ( ASPSCR1-TFE3 , PRCC-TFE3 , SFPQ-TFE3 , RBM10-TFE3 , and MALAT1-TFEB ) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.