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Figures S1-S3

Supplemental Tables

Table S1 shows Clinical features. Table S2 shows Patient/sample information and NGS statistics. Table S3 shows Methylation status of CpG sites of WNT targets with CTNNB1 mutation. Table S4 shows Chromosomal aberrations.

High level amplicons in ASCP genomes. The CNV profiles of A) A90 and B) PENN 040 included focal high level (log2 ratio >2.0) amplicons that targeted the MPL and the FRS2 loci respectively. The X and Y axes in the CGH plots represent chromosome and log2 ratios for each genome. Red shaded areas denote homozygous deletions.

Supplementary Figure legends

Figure S6 shows phylogenies based on point mutations with UV signature mutations removed.

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion–positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer.Significance:These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.

Supplementary Tables S1-S8 show the samples used for study (Table S1), the somatic single nucleotide variants, small insertions and small deletions (Table S2), the somatic copy number alterations anallyzed (Table S3), the gene list used to analyze somatic mutations (Table S4), putative driver genes, immunotherapy related genes, and mutation-targeted therapy genes affected by mutations in each case (Table S5), mutation signatures detected with a cosine score {greater than or equal to}0.80 (Table S6), Jaccard similarity coefficients (Table S7), and putative neoantigens in each case (Table S8).

Purpose:Melanoma is a biologically heterogeneous disease composed of distinct clinicopathologic subtypes that frequently resist treatment. To explore the evolution of treatment resistance and metastasis, we used a combination of temporal and multilesional tumor sampling in conjunction with whole-exome sequencing of 110 tumors collected from 7 patients with cutaneous (n = 3), uveal (n = 2), and acral (n = 2) melanoma subtypes.Experimental Design:Primary tumors, metastases collected longitudinally, and autopsy tissues were interrogated. All but 1 patient died because of melanoma progression.Results:For each patient, we generated phylogenies and quantified the extent of genetic diversity among tumors, specifically among putative somatic alterations affecting therapeutic resistance.Conclusions:In 4 patients who received immunotherapy, we found 1–3 putative acquired and intrinsic resistance mechanisms coexisting in the same patient, including mechanisms that were shared by all tumors within each patient, suggesting that future therapies directed at overcoming intrinsic resistance mechanisms may be broadly effective.

Figure S1 shows Histological findings of stepwise hepatocarcinogenesis and somatic substitution patterns. Figure S2 shows Activation of WNT targets. Figure S3 shows Homozygous deletion in 4q and 16q. Figure S4 shows Circos plot and transcription of the 5' portion of fusion genes that included TERT sequences. Figure S5 shows Upregulation mechanisms of TERT gene. Figure S6 shows Classification of HCC samples based on the methylation data.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, TERT was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including CTNNB1 and TP53, early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of CTNNB1 mutation status because β-catenin did not translocate into the nucleus due to the E-cadherin/β-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with CTNNB1 mutation associated with downregulation of CDH1 and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with TP53 or RB1 gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status.Significance:In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.

Locally injected lipid nanoparticle (LNP)-based mRNA vaccines migrate systemically, which could raise safety concerns. From a mechanistic viewpoint, whether local or systemic antigen expression contributes to the vaccine effects remains unclear. Herein, we localized the antigen protein expression using naked mRNA and drastically improved the delivery efficiency in the skin by jet injection. Consequently, jet-injected naked mRNA outperformed a widely-used LNP in humoral immunity induction at the highest tolerable mRNA doses of each formulation in mice. A mechanistic investigation suggests that antigenpresenting cells taking up antigens at the jet-injection site of naked mRNA migrate to draining lymph nodes, enabling robust immunization without systemic mRNA distribution. Ultimately, jet injection of SARS-CoV-2 spike mRNA provided efficient antibody responses, neutralizing potential and cellular immunity in rodents and non-human primates with no reactogenicity. Conclusively, naked mRNA jet injection is a robust, tolerable, and simple vaccine candidate.

Ten‐eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1‐upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast‐like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5‐hmC) profiling and found that 5‐hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4–monomethylated, where the H3K27‐acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast‐like HCC., Ten‐eleven translocation 1 (TET1), a methylcytosine dioxygenase of the DNA demethylation pathway, is overexpressed in the subgroup of hepatocellular carcinoma with the hepatoblast‐like expression pattern. Comprehensive epigenome profilings revealed the ectopic enhancer activation of HMGA2 through cytosine hydroxymethylation, H3K27 acetylation, H3K4 monomethylation, and promoter‐enhancer interaction in a TET1‐dependent manner.

Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve the admixture of normal and tumor cells, and/or of distinct tumor subclones; high throughput single-cell DNA sequencing circumvents these and brings cancer genomic studies to higher resolution. However, its application has been limited to liquid tumors or a small batch of solid tumors, mainly because of the lack of a scalable workflow to process solid tumor samples. Here we optimized a highly automated nuclei extraction workflow that achieved fast and reliable targeted single-nucleus DNA library preparation of 38 samples from 16 pancreatic adenocarcinoma (PDAC) patients, with an average library yield per sample of 2867 single nuclei. We demonstrate that this workflow not only performs well using low cellularity or low tumor purity samples but reveals novel genomic evolution patterns of PDAC as well.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, TERT was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including CTNNB1 and TP53, early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of CTNNB1 mutation status because β-catenin did not translocate into the nucleus due to the E-cadherin/β-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with CTNNB1 mutation associated with downregulation of CDH1 and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with TP53 or RB1 gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status. Significance: In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.

Entosis is a type of regulated cell death that promotes cancer cell competition. Though several studies have revealed the molecular mechanisms that govern entosis, the clinical and genetic correlates of entosis in human tumors is less well understood. Here we reviewed entotic cell-in-cell (CIC) patterns in a large single institution sequencing cohort (MSK IMPACT clinical sequencing cohort) of more than 1600 human pancreatic ductal adenocarcinoma (PDAC) samples to identify the genetic and clinical correlates of this cellular feature. After case selection, 516 conventional PDACs and 21 ASCs entered this study and ~45,000 HPFs (median 80 HPFs per sample) were reviewed; 549 entotic-CICs were detected through our cohort. We observed that entotic-CIC occurred more frequently in liver metastasis compared with primary in PDAC. Moreover, poorly differentiated adenocarcinoma or adenosquamous carcinoma had more entotic-CIC than well or moderately differentiated adenocarcinoma. With respect to genetic features TP53 mutations, KRAS amplification, and MYC amplification were significantly associated with entosis in PDAC tissues. From a clinical standpoint entotic CICs were independently associated with a poor prognosis by multivariate Cox regression analysis when considering all cases or primary PDACs specifically. These results provide a contextual basis for understanding entosis in PDAC, a highly aggressive cancer for which molecular insights are needed to improve survival.

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes., Distant metastases from pancreatic cancer patients were previously reported by the authors to be dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Here the authors report a novel metabolic adaptation that that stably activates PGD to reprogram metastatic chromatin.

Surgery is the only curative option for stage I/II pancreatic cancer; nonetheless, most patients will experience a recurrence after surgery and die of their disease. To identify novel opportunities for management of recurrent pancreatic cancer, we performed whole-exome or targeted sequencing of 10 resected primary cancers and matched intrapancreatic recurrences or distant metastases. We identified that recurrent disease after adjuvant or first-line platinum therapy corresponds to an increased mutational burden. Recurrent disease is enriched for genetic alterations predicted to activate MAPK/ERK and PI3K–AKT signaling and develops from a monophyletic or polyphyletic origin. Treatment-induced genetic bottlenecks lead to a modified genetic landscape and subclonal heterogeneity for driver gene alterations in part due to intermetastatic seeding. In 1 patient what was believed to be recurrent disease was an independent (second) primary tumor. These findings suggest routine post-treatment sampling may have value in the management of recurrent pancreatic cancer. Significance: The biological features or clinical vulnerabilities of recurrent pancreatic cancer after pancreaticoduodenectomy are unknown. Using whole-exome sequencing we find that recurrent disease has a distinct genomic landscape, intermetastatic genetic heterogeneity, diverse clonal origins, and higher mutational burden than found for treatment-naïve disease. See related commentary by Bednar and Pasca di Magliano, p. 762. This article is highlighted in the In This Issue feature, p. 747

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Background:The clinical course of hepatocellular carcinoma (HCC) is complicated, because it often recurs and shows multiple lesions, some of which progress to a more malignant form, shortening the life of the patient. The hepatocyte growth factor receptor c-Met has been shown to play an important role in the pathogenesis of HCC, but the influence of c-Met expression on the clinical course of HCC remains to be fully elucidated. Methods:We randomly selected and included 600 tumor specimens obtained from the primary and recurrent lesions of 319 HCC cases between 1995 and 2007. The expression of c-Met was determined by immunohistochemistry using archived formalin-fixed paraffin-embedded samples. We analyzed the correlation between c-Met expression and clinical parameters, including survival. In addition, we examined c-Met expression in the malignant transition of HCC in all cases including recurrent lesions. Results:Survival analysis using the multivariate Cox proportional-regression model revealed that the prognosis was significantly better in the primary cases with high c-Met expression than in those with low c-Met expression (hazard ratio 0.159, 95% confidence interval 0.065–0.391; p < 0.001). During the course of recurrence, some cases with high c-Met expression returned to low c-Met expression. Among 40 cases with high c-Met expression, 29 survived more than 2 years after detecting the high c-Met expression. Conclusion:High expression of c-Met may be a prognostic factor for a good, rather than a poor, HCC prognosis. The involvement of c-Met expression in the malignant transition of recurrent HCC is obscure.

Metabolism is a critical regulator of cell fate determination. Recently, the significance of metabolic reprogramming in environmental adaptation during tumorigenesis has attracted much attention in cancer research. Recurrent mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes have been identified in several cancers, including intrahepatic cholangiocarcinoma (ICC). Mutant IDHs convert α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which affects the activity of multiple α-KG-dependent dioxygenases including histone lysine demethylases. Although mutant IDH can be detected even in the early stages of neoplasia, how IDH mutations function as oncogenic drivers remains unclear. In this study, we aimed to address the biological effects of IDH1 mutation using intrahepatic biliary organoids (IBOs). We demonstrated that mutant IDH1 increased the formation of IBOs as well as accelerated glucose metabolism. Gene expression analysis and ChIP results revealed the upregulation of platelet isoform of phosphofructokinase-1 (PFKP), which is a rate-limiting glycolytic enzyme, through the alteration of histone modification. Knockdown of the Pfkp gene alleviated the mutant IDH1-induced increase in IBO formation. Notably, the high expression of PFKP was observed more frequently in patients with IDH-mutant ICC compared to in those with wild-type IDH (p

Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their “cell of origin” derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification., While hepatoblastoma is the most common pediatric liver cancer, its molecular background has not been fully characterised. Here, the authors perform genomic and epigenomic profiling of 163 untreated pediatric liver tumours and suggest the upregulation of ASCL2 and methylation patterns of IGF2 promoters in driving hepatoblast carcinogenesis.

Background Adenosquamous carcinoma of the prostate (ASCP) is an extremely rare and aggressive prostate cancer variant, whose genomic characteristics have not been elucidated. Although liquid biopsy of circulating tumor cells (CTCs) is an emerging topic in oncology, no study has assessed CTCs in patients with ASCP. Case presentation. A 76-year-old man presented with discomfort in his urethra. His prostate-specific antigen (PSA) level was 13.37 ng/mL. A computed tomography (CT) scan indicated a prostate mass with multiple lymph node and lung metastases. The patient underwent transurethral resection of the prostate and prostatic needle biopsy; both specimens demonstrated Gleason grade group 5 acinar adenocarcinoma of the prostate. Bone scintigraphy indicated bone metastasis in the ischium. Combined androgen blockade was implemented, and his serum PSA level rapidly decreased to 0.01 ng/mL. However, a CT scan 6 months after the initial diagnosis revealed worsening of the disease. The patient therefore underwent repeated prostatic needle biopsy; its specimen demonstrated prostatic adenocarcinoma together with squamous carcinoma components. As immunohistochemical analyses showed the tumor cells to be negative for CD56, chromogranin A, synaptophysin, and PSA, the definitive diagnosis was ASCP. Although the patient underwent chemotherapy (docetaxel and cabazitaxel), he died of the disease 3 months after the diagnosis of ASCP, or 13 months after the initial diagnosis of prostatic adenocarcinoma. His PSA values remained ≤ 0.2 ng/mL. CTCs from the patient’s blood (collected before starting docetaxel) were analyzed and genomically assessed. It showed 5 cytokeratin (CK)+ CTCs, 14 CK− CTCs, and 8 CTC clusters, per 10 mL. Next-generation sequencing identified a total of 14 mutations in 8 oncogenes or tumor suppressor genes: PIK3CB, APC, CDKN2A, PTEN, BRCA2, RB1, TP53, and CDK12. Of 14 mutations, 9 (64%) were detected on CK− CTCs and 5 (36%) were detected on CK+ CTCs. Conclusions This is the first report of CTC analysis and genomic assessment in ASCP. Although the prognosis of ASCP is dismal due to lack of effective treatment, genomic analysis of CTCs might lead to effective treatment options and improved survival.

Introduction: Graft-versus-host-disease (GVHD) arises from the inflammatory cascade triggered by alloreactive T cell following allogeneic hematopoietic cell transplantation (allo-HCT). The tissues most frequently involved by GVHD include the small and large intestines, skin, and liver. Prior studies of T cell receptor (TCR) sequencing in diagnostic biopsies and blood have identified dominant T cell clones in GVHD-affected tissues that were not abundant in circulation or shared across multiple patients, but this has not been studied in sites poorly accessible to biopsy nor in lymphoid tissues in humans. We hypothesized that the GVHD-affected tissues have distinct TCR repertoires reflecting the differential expression of allo-antigens at different anatomic sites. Methods: We performed rapid autopsies on patients whose post allo-HCT course was complicated by GVHD to profile the TCR repertoire in tissues inaccessible to biopsy. Tissues were obtained from seven patients (HLA-identical allografts with a variety of graft sources and GVHD-prophylaxis regimens), all with active GVHD and/or on immunosuppression for GVHD control. Spleen, liver, skin, and multiple sites along the gastrointestinal (GI) tract were sampled, when possible, from the esophagus to the rectum. When available, blood and bone marrow mononuclear cells were also viably preserved. T cell receptors were sequenced from 38 different snap-frozen tissues from five patients via genomic DNA based next-generation sequencing of the TCR-beta CDR3 (ImmunoSeq, Adaptive Biotechnologies). Four out of five subjects sequenced had skin and GI GVHD, one only GI. In parallel, TCRs were sequenced from GVHD-affected tissues from a major and minor mouse model of GVHD: BALB/c 7-14 days after HCT with C57BL/6 T cells and C57BL/6 mice > 30 days after HCT with LP/J T cells. Results: Sequences representing 264,678 productive TCRs were recovered from the human autopsy samples with over 100 unique clones for nearly all tissues (mean 1539; standard deviation 1626). We found virtually no TCR clones defined by nucleotide sequence shared across patients, even in the context of shared HLA haplotypes (4/5 patients shared HLA-A*02-01). This is consistent with prior studies of diagnostic biopsies describing a paucity of clones shared across patients. We observed greater repertoire overlap between sites within the GI tract compared to other tissues in a given patient, as measured by the Jensen Shannon Diversity (JSD) index, especially compared to the skin, another GVHD-affected tissue. Despite differences in global repertoires across tissues, some clones were shared across all samples for a given patient, with at least one clone present among the top twenty clones by frequency for each tissue with sufficient sampling. A strikingly similar pattern of repertoire sharing across tissues was observed in the TCR repertoires of a major and minor mouse model of GVHD. While tissues within the GI tract and mesenteric lymph nodes shared the most clones in the mice, there were also dominant clones shared across tissues outside the GI tract. Individual mice with GVHD had highly divergent repertoires from each other in spite of the markedly controlled setting including inbred mice with the same donor T cell pool for each transplant, consistent with the notion that T cells that mediate GVHD may be present at very low frequency in the pool of naïve T cells in the allograft. Conclusion: This work characterizes T cell repertoire in GVHD in human tissues that have not been previously available, including lymphoid tissues and small intestine samples. Combined with the mouse data, this study reveals the relationships between TCR repertoires across different tissues, highlighting the striking diversity of clones driving the GVHD process across tissues and individuals. Although some even dominant clones are shared across all tissues within an individual, each sampled site had a tissue-specific clonal composition. While GVHD-directed therapy focuses primarily on the inhibition of the T cells themselves, additional attention must be devoted to understanding the pattern of target tissue-specific antigen expression in order to identify the key drivers of GVHD. Disclosures Giardina: Seres Therapeutics: Other: salary support. Slingerland:Seres Therapeutics: Other: salary support. Jenq:Kaleido Biosciences: Membership on an entity's Board of Directors or advisory committees; Karius Dx: Speakers Bureau; Merck: Consultancy; MicrobiomeDx: Consultancy; Prolacta: Consultancy; Seres Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: January 1, 2040. Giralt:Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; PFIZER: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinuum: Other: Advisory board, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Other: Advisory Board, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees; SPECTRUM Pharma: Membership on an entity's Board of Directors or advisory committees; Jensenn: Membership on an entity's Board of Directors or advisory committees, Research Funding; JAZZ Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; MILTENYI: Research Funding; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; OMEROS: Research Funding. Perales:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Medigene: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding; Kite/Gilead: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Cidara Therapeutics: Other; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees, Other; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Iacobuzio-Donahue:BMS: Research Funding. van den Brink:Seres Therapeutics: Consultancy, Patents & Royalties, Research Funding; DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Forty-Seven, Inc: Consultancy; Juno Therapeutics: Patents & Royalties; WindMIL Therapeutics: Honoraria; Rheos: Honoraria; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Magenta: Honoraria; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Peled:Seres Therapeutics: Patents & Royalties, Research Funding; Davolterra: Consultancy.

We present two cases of diffusely decreased liver attenuation on post-mortem computed tomography (CT) which is lower than that on ante-mortem CT. Cases include a 37-year-old female whose cause of death was related to myelodysplastic syndrome (Case #1) and an 88-year-old female whose cause of death was related to malignant lymphoma (Case #2). We compared post-mortem CT (PMCT) liver attenuations with those on ante-mortem CT (AMCT). For Case #1, average AMCT values of the right/left liver were 46.1/50.0 Hounsfield Units (HU) 2 days ante-mortem and PMCT values of the right/left liver were 33.9/38.4 HU 3 h post-mortem. For Case #2, average AMCT values were 61.4/54.9 HU 5 days ante-mortem and PMCT values of the right/left liver were 38.2/40.6 HU 2 h post-mortem. In both Case # 1 and #2, decreased liver attenuations were found on PMCT. Further, autopsy revealed diffuse hepatocellular hemorrhage, deficits, and necrosis in Case #1, and liver infiltration of lymphoma in Case #2. These pathological findings were considered to be related to diffusely decreased liver attenuation on PMCT in conjunction with remarkable deterioration of hepatobiliary function before death. The observed decreases in liver attenuation on PMCT may be attributed mainly to hepatic pathological changes just prior to death. PMCT liver attenuation does not necessarily reflect the ante-mortem state some time before death, and it is therefore necessary to be careful when presuming the ante-mortem hepatic pathological conditions from only PMCT. This attenuation change should be taken into account during PMCT interpretation.

Metastatic pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths with very few treatment options and low-success rates. Generally speaking metastatic PDAC remains incurable. One reason behind PDAC morbidity and mortality is intratumor heterogeneity which allows metastatic dispersion and treatment resistance. We hypothesize that given multiple selective bottlenecks during metastatic progression, only a set of non-random gene programs are required for tumor cells to metastasize to different organs. To identify gene programs that become selected during PDAC metastatic progression we collected multiple (>20) metastatic tissue samples from different local and distant organs, from two clinically non-redundant human rapid-autopsies as part of the Human Tumor Atlas Network. We generated single-nuclei RNA-seq and Multiplexed Ion Beam Imaging data from these samples. Using trajectory analysis we inferred gene program dynamics between primary and metastatic samples and found an epithelial-to-mesenchymal-to-epithelial axis general to the metastases, suggesting that epithelial-mesenchymal plasticity is needed for cells to colonize other tissues. However, different organs have vastly different cell type compositions and may represent significantly different evolutionary bottlenecks. We employed archetype analysis as a tool to distinguish optimized phenotypes that may be shared or unique across colonized tissues. Archetype analysis revealed multiple gene programs, some of which are specific to a particular tissue and others ubiquitous. An epithelial-to-mesenchymal gene program was found to be shared across all samples at varying proportions, together with an extracellular matrix deposition/interaction program. Other gene programs identified include angiogenesis, hypoxia, cell cycle, immune interaction, lipid metabolism, autophagy/stress response, and cell migration. Some of these programs are present in various different metastases while others are unique to a specific site (e.g. Lipid metabolism in peritoneal metastasis). Together these programs shed light into organ tropism, metastatic modes of spread, adaptation to local tumor microenvironments, and cell-cell interactions with stromal cells. Further validation of these optimized phenotypes and integration of their spatial context will provide a deeper molecular understanding of metastatic PDAC and provide a source for data-driven therapeutic targets. [A. J-S. and Y.X contributed equally to this work.] Citation Format: Alejandro Jiménez-Sánchez, Yubin Xie, Roshan Sharma, Tin Yi Chu, Vincent Liu, Wungki Park, Akimasa Hayashi, Shigeaki Umeda, Linas Mazutis, Tal Nawy, Christine Iacobuzio-Donahue, Dana Pe’er. Optimal tumor metastatic gene programs in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 279.

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion–positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. Significance: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.

Trimethylation of histone H3 at lysine 27 (H3K27me3) acts as a transcriptional repressor of target genes. Recent immunohistochemical studies have reported a loss of H3K27me3 modification in diffuse (especially 1p/19q-codeleted) gliomas. However, we did not observe H3K27me3 loss in diffuse gliomas using routine immunostaining conditions for the detection of H3K27me3 loss in malignant peripheral nerve sheath tumors (MPNSTs). Therefore, we conducted immunohistochemical analysis of surgically resected specimens to understand the differences in the H3K27me3 status in MPNSTs and diffuse gliomas and evaluate the diagnostic utility of H3K27me3 immunohistochemistry. Staining with a standard 1:200 dilution of the C36B11 antibody showed a complete loss of H3K27me3 in 5 out of 11 MPNSTs, whereas most diffuse gliomas (149/151, 98.7%) showed diffuse immunoreactivity. At a 1:2000 antibody dilution, 12.6% (19/151) of the diffuse gliomas showed H3K27me3 loss, which was significantly associated with 1p/19q codeletion (P

Purpose: Melanoma is a biologically heterogeneous disease composed of distinct clinicopathologic subtypes that frequently resist treatment. To explore the evolution of treatment resistance and metastasis, we used a combination of temporal and multilesional tumor sampling in conjunction with whole-exome sequencing of 110 tumors collected from 7 patients with cutaneous (n = 3), uveal (n = 2), and acral (n = 2) melanoma subtypes. Experimental Design: Primary tumors, metastases collected longitudinally, and autopsy tissues were interrogated. All but 1 patient died because of melanoma progression. Results: For each patient, we generated phylogenies and quantified the extent of genetic diversity among tumors, specifically among putative somatic alterations affecting therapeutic resistance. Conclusions: In 4 patients who received immunotherapy, we found 1–3 putative acquired and intrinsic resistance mechanisms coexisting in the same patient, including mechanisms that were shared by all tumors within each patient, suggesting that future therapies directed at overcoming intrinsic resistance mechanisms may be broadly effective.

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer. Iacobuzio-Donahue and colleagues use integrated transcriptomic, histologic and mutational data to analyze squamous features of pancreatic ductal adenocarcinoma (PDAC), further refining the understanding of heterogeneity and evolution in PDAC.

Summary In the female genital tract, extrauterine leiomyomas such as those that arise in the ovary and paraovarian/paratubal regions are rare. Currently, little is known about the background genetic changes in such adnexal leiomyomas. Recent studies have found that the MED12 mutation is common in uterine leiomyomas, which suggests that such mutations may play an oncogenic role in smooth muscle neoplasms in females. Herein, we examined a series of ovarian and other adnexal leiomyomas in terms of MED12 mutational status to investigate possible MED12 involvement in the pathogenesis of extrauterine smooth muscle tumors. We evaluated 10 cases of adnexal leiomyomas (5 ovarian, 3 paraovarian, and 2 paratubal) and 49 cases of ovarian sex cord–stromal tumors as controls. We performed polymerase chain reaction followed by direct sequencing of exon 2 of MED12, and immunohistochemical staining for smooth muscle actin and desmin. We identified somatic MED12 mutations in 90% (9/10) of the adnexal leiomyomas. None of the sex cord–stromal tumors in the control group harbored MED12 mutations. Diffuse immunoreactivity for both smooth muscle actin and desmin was characteristic of adnexal leiomyomas only. Thus, we conclude that ovarian leiomyomas are distinct from sex cord–stromal tumors. MED12 mutations are key molecular features of ovarian and other adnexal leiomyomas. We speculate that the pathogenesis of adnexal leiomyoma is similar to that of its uterine counterpart.

CLDN18-ARHGAP26/6 fusions have been identified in gastric cancers, with a predominance in diffuse-type gastric cancers (DGCs). Although in vitro experiments have suggested an oncogenic role for CLDN18-ARHGAP26/6 fusions, the exact frequencies and clinicopathological characteristics of the fusion-positive cases are poorly understood. We analyzed 254 cases of gastric cancer (172 diffuse-type and 82 intestinal-type) using RT-PCR and FISH, and also analyzed TCGA transcriptome datasets to identify genes that are related to the aggressive behaviors of fusion-positive cancers. Our assays identified 26 fusion-positive cases, 22 of which were DGCs (22/172, 12.8%). Unlike fusion-negative DGCs, almost all fusion-positive DGCs retained E-cadherin expression (P = 0.036). Fusion-positive DGCs also showed a higher prevalence of lymphatic and distant organ metastases, and these trends were only significant in the younger age group (< 60 years). In this group, the majority of cases with distant organ metastases (4 of 6 cases) were fusion-positive, and the multivariate regression analysis revealed that fusion status was an independent predictive marker for distant organ metastases (P = 0.002). In the TCGA dataset analysis, carbonic anhydrase 9 was postulated to be a potential modulator of the age-specific effects of the fusion protein, compatible with the immunohistochemical analysis of our cohort. Therefore, CLDN18-ARHGAP26/6 fusion-positive DGCs are considered biologically distinct entities that will require more advanced therapeutic options.

A 68-year-old male who had undergone low anterior resection for primary rectal cancer 19 months ago presented with multiple CLM at Couinaud's segments IV, V, and VIII. There was no apparent macroscopic intrabiliary growth on preoperative computed tomography and gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging (EOB-MRI). However, the hepatobiliary phase of EOB-MRI revealed peritumoral low signal intensity in lesions in segments V and VIII, which indicates vascular invasion around hepatocellular carcinoma. Contrast-enhanced intraoperative ultrasound (CE-IOUS) clearly determined the extent of macroscopic glissonean growth from lesions in segments V and VIII, and more extensive resection was performed than was planned. Analysis of the resected specimens from segments V and VIII confirmed the presence of macroscopic intrabiliary growth with microscopic portal vein invasion. All three CLM were histopathologically diagnosed as well-to-moderately differentiated adenocarcinoma, and R0 resection was verified. Postoperative recovery was uneventful, and the patient was alive without evidence of recurrence 12 months after hepatic resection. CE-IOUS should be considered at the time of CLM resection, as it might enable more accurate detection of macroscopic intrabiliary growth of CLM, and enable resection with safer margins.

Background Only a few studies have reported the resection and reconstruction of major hepatic veins during hepatectomy. Here we present our strategy and techniques for venous reconstruction with cryopreserved homologous veins, and describe the surgical outcome. Methods Among 2387 hepatectomy patients, 39 patients who required hepatic venous reconstruction were reviewed retrospectively. Venous reconstruction was performed to secure a non-congested liver remnant volume of at least 40% of the total liver volume. Results There was no operative mortality, and the severe morbidity rate was 5% in this series. A total of 41 veins were reconstructed; 30 with homologous veins (73.2%) and 11 with autologous veins (26.8%), with the middle hepatic vein being the most frequent (n=23, 56%). Interposition grafting was performed more often (P=0.003), the length of the venous resection was longer (P=0.007), and pathologic wall infiltration of the vein was revealed more often (P=0.002) in the homologous graft group than in the autologous graft group. The 1-, 2-, and 3-year overall patency of the reconstructed veins was 55.4%, 46.3%, and 46.3%, respectively. Conclusions Aggressive venous reconstruction during hepatectomy using cryopreserved homologous veins is a feasible option with satisfactory short-term outcomes, and may be warranted to improve operative safety. This article is protected by copyright. All rights reserved.

Temsirolimus is an inhibitor of mammalian target of rapamycin and interstitial lung disease (ILD) is known to be one of the adverse events associated with temsirolimus, which usually improves rapidly after discontinuation of the drug and rarely worsens thereafter. Herein, we report a case of delayed exacerbation of ILD after discontinuation of temsirolimus for metastatic renal cell carcinoma in an 86-year-old male with chronic ILD. The patient developed gradually worsening dyspnea five weeks after an initiation of temsirolimus and was admitted to our facility. On his admission, although a pulmonary function test revealed a decreased diffusion capacity, there was no obvious progression of ILD on HRCT scan. His dyspnea once improved after discontinuation of temsirolimus, but it recurred and acute exacerbation of ILD was diagnosed 40 days after his last administration of temsirolimus. He received high-dose steroid therapy, however, he deteriorated and died. Histopathological examination of the lungs at autopsy revealed overlapping diffuse alveolar damage with chronic interstitial changes. In the present case, since there were no specific factors that could have caused acute exacerbation of ILD except for temsirolimus, it was considered to contribute to the exacerbation of underlying ILD. In conclusion, physicians should be aware of the possibility of temsirolimus-induced ILD not only while the medication is administered, but also even after it is discontinued. It is important to carefully interview the patient and to recognize the value of physiological tests, such as respiratory function tests and blood gas analysis, as well as imaging findings on HRCT.