Citations

Birt-Hogg-Dubé (BHD) syndrome is associated with an increased risk of multifocal renal tumors, including hybrid oncocytic tumor (HOT) and chromophobe renal cell carcinoma (chRCC). HOT exhibits heterogenous histologic features overlapping with chRCC and benign renal oncocytoma, posing challenges in diagnosis of HOT and renal tumor entities resembling HOT. In this study, we performed integrative analysis of bulk and single-cell RNA sequencing data from renal tumors and normal kidney tissues, and nominated candidate biomarkers of HOT, L1CAM, and LINC01187, which are also lineage-specific markers labeling the principal cell and intercalated cell lineages of the distal nephron, respectively. Our findings indicate the principal cell lineage marker L1CAM and intercalated cell lineage marker LINC01187 to be expressed mutually exclusively in a unique checkered pattern in BHD-associated HOTs, and these 2 lineage markers collectively capture the 2 distinct tumor epithelial populations seen to co-exist morphologically in HOTs. We further confirmed that the unique checkered expression pattern of L1CAM and LINC01187 distinguished HOT from chRCC, renal oncocytoma, and other major and rare renal cell carcinoma subtypes. We also characterized the histopathologic features and immunophenotypic features of oncocytosis in the background kidney of patients with BHD, as well as the intertumor and intratumor heterogeneity seen within HOT. We suggest that L1CAM and LINC01187 can serve as stand-alone diagnostic markers or as a panel for the diagnosis of HOT. These lineage markers will inform future studies on the evolution and interaction between the 2 transcriptionally distinct tumor epithelial populations in such tumors.

Abstract Disclosure: S. Tella: None. J.C. Morris: None. M. Rivera: None. S. Gupta: None. M.M. Ryder: None. Introduction: NUT carcinoma is a rare subtype of squamous cell carcinoma defined by NUTM1 rearrangements encoding NUT-fusion oncoproteins with a very dismal prognosis. Recent data showed that NUTM1 rearranged carcinoma of the thyroid can mimic other thyroid primaries but NSD3-NUTM1 fusions aid in the diagnosis. Other than systemic chemotherapy with ifosfamide-based therapies and bromodomain inhibitors, therapeutic options for metastatic NUTM1 rearranged carcinoma of thyroid are limited. Herein, we describe a case of NUTM1 carcinoma of thyroid that had an impressive response to lenvatinib. Clinical case: We describe a case of a 41-year-old Caucasian male who presented to urgent care with acute onset aphonia and was eventually diagnosed with poorly differentiated thyroid carcinoma of size 4 x 4 cms extending into the internal jugular vein. He was managed with partial resection of the mass, internal jugular vein ligation, and a right select neck dissection. He had R2 resection due to possible vascular or airway invasion. As such, he received external beam radiation 70 Gy with 35 fractions with concurrent cisplatin followed by 200 mci radio-iodine therapy. The radiation therapy was complicated by the formation of a tracheo-esophageal fistula. A follow-up FDG-avid PET scan demonstrated residual thyroid bed disease and oligometastatic disease in bones. He received a couple of Stereotactic Body Radiation Therapy twice for bone lesions. Unfortunately, he had rapid disease progression with large pleural-based nodules and histology showed poorly differentiated thyroid carcinoma. NUT1 IHC stain was negative. We simultaneously obtained next-generation sequencing (NGS) and started him on lenvatinib 20 mg. Interestingly, NGS showed NSD3-NUTM1 fusions favoring the diagnosis of NUTM1 rearranged carcinoma of thyroid. He had an impressive partial response to lenvatinib within 4 weeks of initiation of lenvatinib and the response is sustained on 8-month follow-up. He tolerated lenvatinib reasonably without any unexpected complications. In an event of progression, we intend to consider a clinical trial, chemotherapy in combination with bromodomain inhibitors, or another anti-angiogenic tyrosine kinase inhibitor therapy. Conclusions: We noticed an impressive and sustained partial response in NUTM1 rearranged carcinoma of thyroid with anti-angiogenic multi-kinase inhibitor, lenvatinib opening new avenues of targeted therapy in the therapeutic landscape of NUT1 carcinomas. Importantly, NUT1 stain could be negative and NGS is the gold standard for accurate diagnosis. Although our study is limited by one case report, we hope that by highlighting the spectrum of this disease that more cases will be recognized, facilitating risk stratification and improved treatment, and further enabling future studies to explore the clinical management and biology of NUTM1 rearranged carcinoma of thyroid. Presentation: Thursday, June 15, 2023

Abstract Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty‐seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non‐metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate‐sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS / RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non‐metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS / RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ ‐related germ cell tumors rather than non‐germ cell neoplasia in situ ‐derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Histopathology has remained a cornerstone for biomedical tissue assessment for over a century, with a resource-intensive workflow involving biopsy or excision, gross examination, sampling, tissue processing to snap frozen or formalin-fixed paraffin-embedded blocks, sectioning, staining, optical imaging, and microscopic assessment. Emerging chemical imaging approaches, including stimulated Raman scattering (SRS) microscopy, can directly measure inherent molecular composition in tissue (thereby dispensing with the need for tissue processing, sectioning, and using dyes) and can use artificial intelligence (AI) algorithms to provide high-quality images. Here we show the integration of SRS microscopy in a pathology workflow to rapidly record chemical information from minimally processed fresh-frozen prostate tissue. Instead of using thin sections, we record data from intact thick tissues and use optical sectioning to generate images from multiple planes. We use a deep learning–based processing pipeline to generate virtual hematoxylin and eosin images. Next, we extend the computational method to generate archival-quality images in minutes, which are equivalent to those obtained from hours/days-long formalin-fixed, paraffin-embedded processing. We assessed the quality of images from the perspective of enabling pathologists to make decisions, demonstrating that the virtual stained image quality was diagnostically useful and the interpathologist agreement on prostate cancer grade was not impacted. Finally, because this method does not wash away lipids and small molecules, we assessed the utility of lipid chemical composition in determining grade. Together, the combination of chemical imaging and AI provides novel capabilities for rapid assessments in pathology by reducing the complexity and burden of current workflows. Significance: Archival-quality (formalin-fixed paraffin-embedded), thin-section diagnostic images are obtained from thick-cut, fresh-frozen prostate tissues without dyes or stains to expedite cancer histopathology by combining SRS microscopy and machine learning.

Abstract Background Tumor cell phenotypes and anti-tumor immune responses are shaped by local metabolite availability, but intratumoral metabolite heterogeneity (IMH) and its phenotypic consequences remain poorly understood. In vitro mechanistic studies have demonstrated that the anti-tumor activity of lymphoid and myeloid cell populations is mediated by metabolite availability and signaling in the TME, raising the possibility that the immune response and metabolism of ccRCC tumors coevolve and jointly influence the likelihood that a patient responds to therapy.. However, both the broad patterns of coordination between metabolite abundance and TME cellular composition, as well as the precise cell populations producing metabolic phenotypes of interest, remain unknown. Methods To study IMH, we multiregionally profiled the metabolome, transcriptome, and genome of 187 tumor/normal regions from 31 clear cell renal cell carcinoma (ccRCC) patients. Using these measurements and additional multimodal metabolomic/transcriptomic profiling of ccRCC and other diseases, we developed computational models that can be used to understand RNA-metabolite covariation and ultimately impute metabolite levels from RNA sequencing data. Results Analysis of intratumoral metabolite-RNA covariation revealed that the immune composition of the microenvironment, and especially the abundance of myeloid cells, drove intratumoral metabolite variation. Motivated by the strength of RNA-metabolite covariation and the clinical significance of RNA biomarkers in ccRCC, we deployed and benchmarked a machine learning method (MIRTH) to impute metabolite levels directly from RNA sequencing data of primary and metastatic ccRCC tumors. We inferred metabolomic profiles from RNA sequencing data of ccRCC patients enrolled in 6 clinical trials, ultimately identifying specific metabolite biomarkers associated with response to anti-angiogenic agents. Conclusions Local metabolic phenotypes therefore emerge in tandem with the immune microenvironment and associate with therapeutic sensitivity. CDMRP DOD Funding: yes

Abstract Objectives There is a paucity of data on penile amyloidosis. We aimed to assess the frequency of different amyloid types in surgical specimens from the penis involved by amyloidosis and correlate relevant clinicopathologic parameters with proteomic findings. Methods Since 2008, our reference laboratory has performed liquid chromatography/tandem mass spectrometry (LC-MS/MS) for amyloid typing. The institutional pathology archive and reference laboratory database were queried to retrospectively identify all penile surgical pathology specimens with LC-MS/MS results between January 1, 2008, and November 23, 2022. Archived H&E-stained and Congo red–stained sections were re-reviewed. Results Twelve cases of penile amyloidosis were identified, which represented 0.35% (n = 3,456) of penile surgical specimens. AL-type amyloid was most frequent (n = 7), followed by keratin-type amyloid (n = 3) and ATTR (transthyretin)–type amyloid (n = 2). AL-type amyloid cases often showed diffuse dermal/lamina propria deposition, whereas all keratin-type amyloid cases were localized to the superficial dermis. Two cases with keratin-type amyloid had concomitant cutaneous findings (penile intraepithelial neoplasia and condyloma). Conclusions This series, the largest to date, demonstrates that penile amyloidosis has a heterogeneous proteomic landscape. To the best of our knowledge, this is the first study describing ATTR (transthyretin)–type penile amyloid.

548 Background: PD-L1 and B7-H3 have been found to be overexpressed in urothelial carcinoma (UC) of the urinary bladder. Recent studies have also demonstrated that B7-H3 and PD-L1 can promote resistance to platinum-based drugs but the predictive value of B7-H3 expression in patients treated with platinum-based chemotherapy is unknown. This study aims to investigate the association of PD-L1 and B7-H3 tumor expression with oncological outcomes in patients who underwent radical cystectomy (RC) and received subsequent adjuvant chemotherapy. Methods: Immunohistochemistry was performed on paraffin-embedded sections from bladder and lymph node specimens of 81 patients who had RC for bladder cancer. PD-L1 and B7-H3 expression on tumor cells was assessed by immunohistochemistry in both primary tumors and lymph node specimens. Association with clinicopathologic outcomes was determined using Fisher’s exact test and postoperative survival using Kaplan–Meier survival curves and Cox regression model. Results: B7-H3 expression in cystectomy specimens was more common than PD-L1 expression (72.8% vs. 35.8%). For both markers, no association was found with pathologic tumor stage, lymph node (LN) status, and histological subtype. Similar findings were observed for double-positive tumors (PD-L1+B7-H3+). Concordance between the primary tumor and patient-matched lymph nodes was found in 76.2% and 54.1% of patients for PD-L1 and B7-H3, respectively. PD-L1 tumor expression was not associated with oncologic outcomes. However, B7-H3 expression was associated with recurrence-free survival (HR: 2.38, 95% CI 1.06–5.31, p = 0.035) and cancer-specific survival (HR: 2.67, 95% CI 1.18–6.04, p = 0.019). Conclusions: In our single institutional study, B7-H3 is highly expressed in patients with UC treated with adjuvant chemotherapy and it was associated with decreased recurrence-free survival and cancer-specific survival. Pending further validation in larger cohorts, B7-H3 expression may function as a predictor of response to adjuvant chemotherapy and thus be useful in patient and regimen selection.

Context.— Epithelioid angiomyolipomas (eAMLs) are rare tumors of the kidney that occur in patients with tuberous sclerosis complex or in a sporadic setting; a subset of these tumors exhibit metastatic behavior. Objective.— To analyze molecular profiling data to identify pathogenic alterations in rare cases of metastatic eAML, and to identify immunohistochemistry (IHC)-based surrogate markers. Design.— Molecular profiling data from the American Association for Cancer Research GENIE registry was accessed for 23 patients with angiomyolipomas, and 9 of 16 patients with eAMLs in our institutional registry were evaluated with next-generation sequencing. IHC was performed to screen for alterations of P53, RB, and ATRX for all 16 institutional cases. Results.— Combined alterations of 5 tumor-suppressor genes (TP53, ATRX, RB1, APC, and NF1) were identified using next-generation sequencing in 7 of 8 (88%) patients with metastatic disease compared to a single patient with nonmetastatic disease (RB1 variant of uncertain significance; 1 of 24, 4%). No cases with abnormal IHC results were identified in 11 patients with nonmetastatic disease compared to 3 of 5 patients with metastatic disease. Conclusions.— Our results show that the majority of metastatic eAMLs have mutations of 5 tumor-suppressor genes (TP53, ATRX, RB1, APC, and NF1), while these are rare in patients with nonmetastatic disease. Furthermore, IHC for P53, RB, and ATRX may serve as a screen for a subset of these alterations in resource-limited settings. These findings, if validated in larger data sets, have the potential to predict metastatic behavior in eAMLs.