Molecular Characterization of Juxtaglomerular Cell Tumors: Evidence of Alterations in MAPK-RAS Pathway.

Juxtaglomerular cell tumor (JGCT) is a rare neoplasm, part of the family of mesenchymal tumors of the kidney. Although the pathophysiological and clinical correlates of JGCT are well known, as these tumors are an important cause of early-onset arterial hypertension refractory to medical treatment, their molecular background is unknown, with only few small studies investigating their karyotype. Herein we describe a multi-institutional cohort of JGCTs diagnosed by experienced genitourinary pathologists, evaluating clinical presentation and outcome, morphologic diversity, and, importantly, the molecular features. Ten JGCTs were collected from 9 institutions, studied by immunohistochemistry, and submitted to whole exome sequencing. Our findings highlight the morphologic heterogeneity of JGCT, which can mimic several kidney tumor entities. Three cases showed concerning histologic features, but the patient course was unremarkable, which suggests that morphologic evaluation alone cannot reliably predict the clinical behavior. Gain-of-function variants in RAS GTPases were detected in JGCTs, with no evidence of additional recurrent genomic alterations. In conclusion, we present the largest series of JGCT characterized by whole exome sequencing, highlighting the putative role of the MAPK-RAS pathway.

Inhibition of MALT1 and BCL2 Induces Synergistic Antitumor Activity in Models of B-Cell Lymphoma.

The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling.

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.

REST-repressed lncRNA LINC01801 induces neuroendocrine differentiation in prostate cancer via transcriptional activation of autophagy.

The association between REST reduction and the development of neuroendocrine prostate cancer (NEPC), a novel drug-resistant and lethal variant of castration-resistant prostate cancer (CRPC), is well established. To better understand the mechanisms underlying this process, we aimed to identify REST-repressed long noncoding RNAs (lncRNAs) that promote neuroendocrine differentiation (NED), thus facilitating targeted therapy-induced resistance. In this study, we used data from REST knockdown RNA sequencing combined with siRNA screening to determine that LINC01801 was upregulated and played a crucial role in NED in prostate cancer (PCa). Using The Cancer Genome Atlas (TCGA) prostate adenocarcinoma database and CRPC samples collected in our laboratory, we demonstrated that LINC01801 expression is upregulated in NEPC. Functional experiments revealed that overexpression of LINC01801 had a slight stimulatory effect on the NED of LNCaP cells, while downregulation of LINC01801 significantly inhibited the induction of NED. Mechanistically, LINC01801 is transcriptionally repressed by REST, and transcriptomic analysis revealed that LINC01801 preferentially affects the autophagy pathway. LINC01801 was found to function as a competing endogenous RNA (ceRNA) to regulate the expression of autophagy-related genes by sponging hsa-miR-6889-3p in prostate cancer cells. In conclusion, our data expand the current knowledge of REST-induced NED and highlight the contribution of the REST-LINC01801-hsa-miR-6889-3p axis to autophagic induction, which may provide promising avenues for therapeutic opportunities.

Klotho Overexpression Is Frequently Associated With Upstream Rearrangements in Fusion-Negative Phosphaturic Mesenchymal Tumors of Bone and Sinonasal Tract.

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.

Touch print smear of testicular tissue with thionine stain for intraoperative diagnosis in azoospermic men.

PURPOSE: To compare the diagnostic value of testicular tissue touch print smear (TPS) conducted on azoospermic patients with results obtained from histopathology and in vitro fertility (IVF) lab findings. METHODS: Microdissection testicular sperm extraction was performed on a group of 148 azoospermic patients and testicular samples obtained intraoperatively. Using TPS, the samples were smeared onto a sterile slide, followed with staining using thionine. The testis tissue bulk samples were also transferred to the IVF lab, and determinations of sperm presence or absence obtained from IVF lab tests were compared with the TPS sample results. Needle testis biopsy was separately performed on a group of 360 azoospermic patients, and results of pathohistology review on the biopsies were further compared with determinations of spermatogenesis stage obtained from TPS for those patients. RESULTS: When compared with IVF lab results, TPS was found to have 100% (126/126) positive predictive value and 95.5% (25/26) negative predictive value for predicting sperm presence or absence, respectively. Furthermore, TPS was further found to have a 93.6% correlation (337 of 360 biopsies) with results of histological diagnoses performed by needle biopsy. Results from histology and TPS for the detection of sperm presence were concordant in 96.1% (346/360) of biopsies. Diagnosis of SCO by TPS shows the highest correlation with histopathology (98.6%), followed by complete spermatogenesis (97.5%), early maturation arrest (78.9%), and late maturation arrest (27.3%). CONCLUSIONS: The results support the continued use of TPS in testicular tissue analysis for more rapid assessment of spermatogenesis and for detection of spermatozoa in azoospermic subjects.

Constant p.L424H Mutation in GTF2I in Micronodular Thymomas With Lymphoid Stroma: Evidence Supporting Close Relationship With Type A and AB Thymomas.

Micronodular thymoma with lymphoid stroma is a rare thymic neoplasm characterized by discrete nodules of epithelial tumor cells separated by abundant lymphoid stroma. The genetic features of micronodular thymoma with lymphoid stroma remain largely unexplored. Owing to the interference of abundant intratumoral, nonneoplastic lymphoid cells, a highly sensitive approach is necessary to study genetic changes in these tumors. In this study, we used a highly sensitive next-generation sequencing assay using the molecular barcoding Ion AmpliSeq HD technology to study the most commonly mutated genes in thymomas, including GTF2I, HRAS, NRAS, KRAS, and TP53. A total of 12 cases of micronodular thymomas with lymphoid stroma were tested, and 2 cases also had areas of type A thymoma in their tumor bed. Two micronodular thymic carcinomas with lymphoid stroma, a histological mimic of micronodular thymoma, were also included for comparison. Recurrent p.L424H mutations in GTF2I were found in all the cases of micronodular thymoma with lymphoid stroma but not in the cases of micronodular thymic carcinomas. In addition, 3 cases of micronodular thymoma with lymphoid stroma also had concomitant HRAS and/or KRAS mutations. Our study showed that p.L424H mutations in GTF2I is a constant genetic feature of micronodular thymoma with lymphoid stroma. This finding strongly suggests that micronodular thymoma with lymphoid stroma is closely related to type A and AB thymomas because they all share p.L424H mutations in GTF2I.

Interobserver reproducibility of cribriform cancer in prostate needle biopsies and validation of International Society of Urological Pathology criteria.

AIMS: There is strong evidence that cribriform morphology indicates a worse prognosis of prostatic adenocarcinoma. Our aim was to investigate its interobserver reproducibility in prostate needle biopsies. METHODS AND RESULTS: A panel of nine prostate pathology experts from five continents independently reviewed 304 digitised biopsies for cribriform cancer according to recent International Society of Urological Pathology criteria. The biopsies were collected from a series of 702 biopsies that were reviewed by one of the panellists for enrichment of high-grade cancer and potentially cribriform structures. A 2/3 consensus diagnosis of cribriform and noncribriform cancer was reached in 90% (272/304) of the biopsies with a mean kappa value of 0.56 (95% confidence interval 0.52-0.61). The prevalence of consensus cribriform cancers was estimated to 4%, 12%, 21%, and 20% of Gleason scores 7 (3 + 4), 7 (4 + 3), 8, and 9-10, respectively. More than two cribriform structures per level or a largest cribriform mass with ≥9 lumina or a diameter of ≥0.5 mm predicted a consensus diagnosis of cribriform cancer in 88% (70/80), 84% (87/103), and 90% (56/62), respectively, and noncribriform cancer in 3% (2/80), 5% (5/103), and 2% (1/62), respectively (all P < 0.01). CONCLUSION: Cribriform prostate cancer was seen in a minority of needle biopsies with high-grade cancer. Stringent diagnostic criteria enabled the identification of cribriform patterns and the generation of a large set of consensus cases for standardisation.

LOT and HOT or not. The proliferation of clinically insignificant and poorly characterised types of renal neoplasia.

The classification of malignant tumours is influenced by both immunohistochemical and molecular genetic findings. This is highlighted in the latest World Health Organization classification of renal neoplasia, which has a tumour category of 'tumours that are molecularly defined'. This implies that the defining molecular features are integral to tumourigenesis, which may not necessarily be the case. Renal oncocytoma is recognised as a benign tumour with variable morphology and immunoexpression. A variant of these tumours is hybrid oncocytic chromophobe tumour, which has features of both oncocytoma and chromophobe renal cell carcinoma and may, on rare occasions, show malignant behaviour. Recent reports have proposed two further entities with eosinophilic cytoplasm and varying nuclear pleomorphism, designated low grade oncocytic tumour (LOT) and eosinophilic vacuolated tumour (EVT), formally known as high grade oncocytic tumour (HOT). The diagnosis of these apparently benign tumours was made on the basis of morphological and immunohistochemical features. More recently it has been claimed that the mutations in the mTOR pathway are also a diagnostic feature and it is further suggested that these mutations are key to the pathogenesis of these tumours. As is seen in oncocytoma, immunohistochemical expression of tumours included in series of LOT and EVT is variable. The mutations in the mTOR pathway, where detected, were not constant, with any combination of mTOR, TSC1 and/or TSC2 being involved. A major issue is that in many of the studies full comparative genomic hybridisation results are not presented. In addition it is well recognised that mTOR mutations are seen in a variety of renal tumours. In view of these conflicting results, the rarity of these tumours and their apparent benign nature, raises questions as to why these tumours should be considered specific entities.

Molecular Characterization of Metanephric Adenoma, Epithelial Wilms Tumor, and Overlap Lesions: An Integrated Whole-exome and Transcriptome Sequencing Analysis.

Metanephric adenoma (MA) and Wilms tumor (WT) represent 2 prototypes of primary renal neoplasms closely resembling embryonal renal tubules. Tumors with overlapping features may occur, requiring differential diagnoses between the 2. Evidence of divergent oncogenic pathways has been reported, suggesting that MA is driven by BRAF mutation while most WT is of the BRAF wild-type. We collected 4 MA cases, 3 cases of monophasic epithelial WT, and 1 overlap metanephric tumor that contains both conventional MA and high-grade components similar to epithelial WT. Whole-exome sequencing and whole transcriptome sequencing were performed to discover mutations, somatic copy number variation, and differential expression. The findings were compared with those of WT of the TARGET database (WT-TARGET). BRAF V600E mutation was detected in all MAs as well as the overlap tumor but was undetectable in all epithelial WTs and WT-TARGET. The overlap tumor showed an additional pathogenic mutation of SETD2. Three frequent gene mutations observed in WT-TARGET were not common in epithelial WT, in which the mutations appeared sporadic. The profiles of recurrent copy number variations were all different among MA, epithelial WT, and WT-TARGET. Differential expression and unsupervised hierarchical cluster analyses revealed distinct clusters of the 3 categories. Remarkably, the overlap tumor coclustered with MA, separated from epithelial WT and WT-TARGET. The distinctiveness of MA and WT were demonstrated corresponding to BRAF-mutated and non-BRAF-mutated pathways from the molecular perspective. BRAF assay has diagnostic implication for overlap tumors.

Discovery of Non-Nucleotide Small-Molecule STING Agonists via Chemotype Hybridization.

The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.

Changes in Tongue Area, Pharyngeal Area, and Pharyngeal Airway Velocity after Correction of Mandibular Prognathism.

This study aimed to investigate the correlation between the amount of mandibular setback, and the related changes of the tongue area, pharyngeal area, and pharyngeal airflow velocity. Twenty-five patients treated for mandibular prognathism, and serial cephalograms were obtained (T1: preoperation, T2: more than one year postoperation). The postoperative area of the tongue, pharyngeal airway space, and pharyngeal airflow velocity were investigated. Statistical analysis was performed with the Student t-test and Pearson correlation. The amount of mandible setback was significant after surgery (12.8 mm; p < 0.001). The pharyngeal area was significantly reduced 115.5 mm2 (p = 0.046). There was a slight reduction of the tongue area (43.2 mm2; p = 0.305) and an increase of pharyngeal airflow velocity (0.3 m/s; p = 0.133). The Pearson correlation coefficient test showed no statistical significance among the amount of horizontal setback and vertical movement of the mandible, such as the reductions in the tongue area, the pharyngeal airway space, and the increase in pharyngeal airflow velocity. Larger amounts of mandibular setback caused a significant reduction of pharyngeal airway area, but without significant changes of the tongue area and pharyngeal airflow velocity.

Glutamate-cysteine ligase catalytic subunit as a therapeutic target in acute myeloid leukemia and solid tumors.

Acute myeloid leukemia (AML) is a highly heterogenous and aggressive disease with a poor prognosis, necessitating further improvements in treatment therapies. Recently, several targeted therapies have become available for specific AML populations. To identify potential new therapeutic targets for AML, we analyzed published genome wide CRISPR-based screens to generate a gene essentiality dataset across a panel of 14 human AML cell lines while eliminating common essential genes through integration analysis with core fitness genes among 324 human cancer cell lines and DepMap databases. The key glutathione metabolic enzyme, glutamate-cysteine ligase catalytic subunit (GCLC), met the selection threshold. Using CRISPR knockout, GCLC was confirmed to be essential for the cell growth, survival, clonogenicity, and leukemogenesis in AML cells but was comparatively dispensable for normal hematopoietic stem and progenitor cells (HSPCs), indicating that GCLC is a potential therapeutic target for AML. In addition, we evaluated the essentiality of GCLC in solid tumors and demonstrated that GCLC represents a synthetic lethal target for ARID1A-deficient ovarian and gastric cancers.

The Genitourinary Pathology Society Update on Classification and Grading of Flat and Papillary Urothelial Neoplasia With New Reporting Recommendations and Approach to Lesions With Mixed and Early Patterns of Neoplasia.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in bladder neoplasia with a focus on issues relevant to the practicing surgical pathologist for the understanding and effective reporting of bladder cancer, emphasizing particularly on the newly accumulated evidence post-2016 World Health Organization (WHO) classification. The work is presented in 2 manuscripts. Here, in the first, we revisit the nomenclature and classification system used for grading flat and papillary urothelial lesions centering on clinical relevance, and on dilemmas related to application in routine reporting. As patients of noninvasive bladder cancer frequently undergo cystoscopy and biopsy in their typically prolonged clinical course and for surveillance of disease, we discuss morphologies presented in these scenarios which may not have readily applicable diagnostic terms in the WHO classification. The topic of inverted patterns in urothelial neoplasia, particularly when prominent or exclusive, and beyond inverted papilloma has not been addressed formally in the WHO classification. Herein we provide a through review and suggest guidelines for when and how to report such lesions. In promulgating these GUPS recommendations, we aim to provide clarity on the clinical application of these not so uncommon diagnostically challenging situations encountered in routine practice, while also importantly advocating consistent terminology which would inform future work.

The Genitourinary Pathology Society Update on Classification of Variant Histologies, T1 Substaging, Molecular Taxonomy, and Immunotherapy and PD-L1 Testing Implications of Urothelial Cancers.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in bladder cancer focusing on important topics of high interest for the practicing surgical pathologist and urologist. This review represents the second of 2 manuscripts ensuing from this effort. Herein, we address the effective reporting of bladder cancer, focusing particularly on newly published data since the last 2016 World Health Organization (WHO) classification. In addition, this review focuses on the importance of reporting bladder cancer with divergent differentiation and variant (subtypes of urothelial carcinoma) histologies and the potential impact on patient care. We provide new recommendations for reporting pT1 staging in diagnostic pathology. Furthermore, we explore molecular evolution and classification, emphasizing aspects that impact the understanding of important concepts relevant to reporting and management of patients.

Ossifying low grade endometrial stromal sarcoma with PHF1-BRD8 fusion.

Low-grade endometrial stromal sarcoma and ossifying fibromyxoid tumors are two types of mesenchymal tumors that share no similarities in terms of site, sex, and morphological characteristics. They are rare, low grade tumors of uncertain lineage, with no definite immunological markers. Interestingly, a common PHF1 gene- related rearrangement was observed in these two tumors. Here, we report a case of endometrial stromal sarcoma with distinct ossification. Microscopically, the tumor is composed of bland-looking ovoid cells with low cellularity in the fibromyxoid stroma. Foci of metaplastic bone formation were noted. Using a combination of FISH, transcriptome sequencing, and molecular techniques, we identified a new PHF1-BRD8 fusion transcript, which was previously described, but in its reciprocal fusion form. This case expands the current understanding of low-grade endometrial stromal sarcoma and emphasizes the importance of molecular characterization of unique fusion, which may be related to its distinct morphological features and the possibly chemosensitive target.

DNA-Model-Based Design and Execution of Some Fused Benzodiazepine Hybrid Payloads for Antibody-Drug Conjugate Modality.

A new series with the tetrahydroisoquinoline-fused benzodiazepine (TBD) ring system combined with the surrogates of (1-methyl-1H-pyrrol-3-yl)benzene ("MPB") payloads were designed and executed for conjugation with a monoclonal antibody for anticancer therapeutics. DNA models helped in rationally identifying modifications of the "MPB" binding component and guided structure-activity relationship generation. This hybrid series of payloads exhibited excellent in vitro activity when tested against a panel of various cancer cell lines. One of the payloads was appended with a lysosome-cleavable peptide linker and conjugated with an anti-mesothelin antibody via a site-specific conjugation method mediated by the enzyme bacterial transglutaminase (BTGase). Antibody-drug conjugate (ADC) 50 demonstrated good plasma stability and lysosomal cleavage. A single intravenous dose of ADC 50 (5 or 10 nmol/kg) showed robust efficacy in an N87 gastric cancer xenograft model.

Clinicopathological and molecular characterisation of papillary renal neoplasm with reverse polarity and its renal papillary adenoma analogue.

AIMS: Papillary renal neoplasm with reverse polarity (PRNRP) is a newly defined entity with distinct histomorphology and recurrent KRAS mutation. It has been estimated to constitute 4% of previously diagnosed papillary renal cell carcinoma (PRCC). Renal papillary adenoma (PA) is suggested to be the precursor of PRCC. This study aimed to investigate the association between PRNRP and PA, particularly the morphologically similar type D PA. METHODS AND RESULTS: Nephrectomy specimens of PRCC and PA from our 10-year pathology archives were retrieved and reviewed. GATA3 immunohistochemistry and RAS/BRAF testing were performed in all cases reclassified as PRNRP and all PAs with sufficient materials. Overall, PRNRP accounted for 9.1% (10 of 110) of PRCC and there was no recurrence/metastasis with a mean follow-up period of 39 months. Three novel morphological features were described, including clear cell change, mast cell infiltration and metaplastic ossification. Nine of the 10 PRNRPs showed diffuse and strong GATA3 expression and KRAS missense mutations at codon 12. One case exhibited moderate GATA3 staining on 80% of the tumour cells and RAS/BRAF wild-type. In a total of 73 PAs, 11 were classified as type D. GATA3 expression was significantly more frequent in type D versus non-type D PAs (100 versus 35%, P < 0.01). KRAS missense mutations were identified in six of eight (75%) of the type D PAs but none of the 18 non-type D PAs. CONCLUSIONS: Type D PA was different from other types of PA and represented an analogue or a small-sized PRNRP for their identical morphology, immunophenotype and molecular signature.