Upper tract urothelial carcinoma (UTUC) is an aggressive malignancy with limited molecular characterization in advanced disease. FGFR3 alterations are well established in low-grade urothelial carcinoma, but their prevalence, stability, and biological significance in locally advanced and metastatic UTUC remain only partially defined. We performed an integrated morphologic, immunohistochemical, and molecular analysis of 24 locally advanced and/or metastatic UTUC from 20 patients. FGFR3 status was assessed by RT-PCR across multiple tumor compartments, including biopsies, primary tumors, lymph-node metastases, and distant metastatic sites. Immunohistochemistry included CK20, CK5, GATA3, p53, and mismatch repair proteins. Targeted next-generation sequencing (NGS) was used to characterize co-occurring genomic alterations and to assess concordance with p53 immunophenotype. FGFR3 alterations were identified in 50% of patients and in 54.2% of analyzed tumors. FGFR3 status showed high intra-patient stability, with concordance between primary tumors and distant metastases in 90% of cases, whereas concordance with lymph node metastases was lower (50%), suggesting site-specific clonal divergence. Despite advanced stage, 92.3% of FGFR3-altered tumors displayed papillary urothelial carcinoma morphology, and most showed a luminal immunophenotype (61.5% by CK20/CK5 and 69.2% by GATA3/CK5). Targeted NGS revealed additional pathogenic alterations in 75% of patients, most frequently involving RTK/RAS/MAPK signaling (70%), cell-cycle regulation (25%), and PI3K/AKT pathway components (10%). TP53 mutations co-occurred with FGFR3 alterations in 60% of FGFR3-mutated patients and showed 90.4% concordance with p53 immunohistochemistry. Finally, a few cases exhibited complex, multi-site FGFR3 mutational patterns, consistent with intratumoral clonal evolutions. In conclusion, FGFR3 alterations are frequent and remarkably stable in advanced UTUC, even in high-grade and metastatic disease. These findings support the reliability of FGFR3 testing on limited diagnostic material and reinforce its relevance for therapeutic stratification. UTUC emerges as a molecularly dynamic disease in which early oncogenic drivers such as FGFR3 continue to shape tumor biology and therapeutic vulnerability at advanced stages.

Papillary renal neoplasm with reverse polarity (PRNRP) is a recently recognized renal tumor characterized by papillary architecture lined by a single layer of low-grade eosinophilic cells with apically located nuclei. In the latest WHO classification, they are not recognized as a distinct entity but rather as a morphological pattern of papillary renal cell carcinoma. To date, limited studies have compared PRNRP with eosinophilic/oncocytic papillary renal cell carcinoma (E/OPRCC), which they are not infrequently confused with, although a few previous comparisons with classic papillary renal cell carcinoma have already identified several distinguishing features. A comparative analysis of 15 of PRNRPs and 16 of E/OPRCC cases was conducted, evaluating their histopathological, immunophenotypic, interphase cytogenetic, and molecular profiles. PRNRPs demonstrated distinctive morphological features, including consistent apical nuclear positioning, absence of foamy macrophages in the papillary cores (p = 0.0001), and a lower nucleolar grade compared to E/OPRCCs (p = < 0.0001). Immunohistochemically, PRNRPs exhibited strong and uniform GATA3 expression and were negative for vimentin, CD10, and CD13, in contrast to E/OPRCCs. Cytogenetically, PRNRPs lacked trisomies of chromosomes 7 and 17, which were present in 40

Suppl. Figure 6. A. Left. Protein expression levels of phospho-ERK and ERK in HPAFII and PATU8902 cells treated with MEKi (50 nM) for 24 h. Right. Protein expression levels of phospho-AXL and AXL in HPAFII and PATU8902 cells treated with AXLi (0.2 μM) for 24 h. Quantification: pAXL levels normalized to total AXL levels and housekeeping protein levels. B. Heatmap of the Fa fraction of CAPAN2 PDAC human cell line treated with single MEKi, AXLi, or both at indicated concentrations for 72 h. C. 2D surface plot of Bliss synergy scores from B. Red regions indicate synergistic interactions.

Suppl. Figure 7. A. KRAS, TP53, SMAD4 genetic alteration in the 2 PDO characterized by DNA sequencing.

Suppl. Figure 1. A. Association analysis of LAMC2 expression with clinical variables using quartile 1 of H score (23.2) as cut-off. B. Correlation of LAMC2 expression and tumor volume in a tissue array of 119 PDAC samples. C and D. Survival analysis stratified by LAMC2 expression from ICGC and TCGA datasets.

Suppl. Figure 4. A. Correlation plot of mRNA expression levels of LAMC2 and AXL in the PDAC human data set from ICGC. B. Correlation plot of mRNA expression levels of LAMC2 and AXL in the PDAC human data set from TCGA.

Suppl. Figure 5. A. RNA expression of KRAS, AREG, DUSP6 and FOSL1 in PATU8902 and HPAFII cells expressing a TET-inducible KRAS shRNA or a control shRNA. B. Box and whiskers plot showing Mean Fluorescence Intensity (MFI) of Areg, Dusp6, Fosl1 upon KRas inactivation in iKRAS GEM. Error bars correspond to s.d. C. Heat map of downregulated genes in HPAFII cells expressing a tet-inducible LAMC2 shRNA compared to control group (shRNA GFP). D. LAMC2 and FOSL1 mRNA expression in PATU8902 and HPAFII cells expressing a tet-inducible LAMC2 shRNA or a control shRNA. Error bars correspond to s.d. E. mRNA expression of overlapping LAMC2 and FOSL1 downregulated genes (LAMC2 -ctrl-, FGFBP1, HMGA1, AURKA, HMMR, PLK1, SNAI2 and PITPNC1) in PATU8902 and HPAFII cells expressing a TET-inducible LAMC2 shRNA or a control shRNA. F. Protein expression of phospho-ERK, ERK. Phospho-EGFR, EGFR in PATU8902 and HPAFII cells expressing a TETinducible LAMC2 shRNAs or a control shRNA. Quantification: pEGFR levels normalized to total EGFR levels and housekeeping protein levels. G. Fosl1 and AXL protein expression in PATU8902 and HPAFII cells treated with AKTi (10μM) and vehicle control. Quantification: AXL and FOSL1 levels normalized to housekeeping protein levels.

Suppl. Figure 3. A. Immunoblot of LAMC2 upon Lamc2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines. B. Quantification of organoid number upon LAMC2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines.C. Quantification of organoid area (AU) in same cell lines as in B. Error bars correspond to s.d. D. Immunoblot of LAMC2 upon LAMC2 knockdown in HPAFII cell line. E. Quantification of organoid number and organoid area (AU) in HPAFII cell line expressing a TET-inducible shRNA against LAMC2 (bottom) or a control shRNA (top) cultured in 3D. F. Representative images of the subcutaneous tumors from PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. G. Representative images of the subcutaneous tumors from HPAFII cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. H and I. Representative IHC of LAMC2 expression in subcutaneous tumors from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice J. Representative images of the tumors derived from intrapancreatic injection of PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. K. Representative images of the subcutaneous tumors from PM12167 mouse cells expressing a shRNA against Lamc2 or a control shRNA at sacrifice. L and M. Representative images of the subcutaneous (sc) tumors from PATU8902 and HPAFII expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. ShRNA expression induced after tumor establishment at Day 8. N and O. Representative IHC of LAMC2 expression in subcutaneous tumors (Tx) from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA, shRNA expression was induced at day 8 of tumor establishment. P. Quantification of Ki67 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx. Q. Quantification of cleaved caspase3 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx.

This file includes the Suppl. Methods, Table (1) and References

Suppl. Figure 2. A. LAMC2 immunoblot in HPAFII after LAMC2 knockdown using 2 different sgRNAs. B. Cell proliferation assessed by MTS after LAMC2 knockdown using 2 sgRNAs. Error bars correspond to s.d. C. LAMC2 immunoblot in PM12167 after shRNA mediated Lamc2 knockdown. D. Cell proliferation assessed by MTS after shRNA mediated Lamc2 knockdown. Error bars correspond to s.d. E. Colony forming ability after shRNA mediated Lamc2 knockdown. Error bars correspond to s.d. F. Cell proliferation assessed by MTS, after shRNA-mediated LAMC2 knockdown in PATU8902 and HPAFII PDAC cell lines using a tet-inducible system. Error bars correspond to s.d.

ABSTRACT Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping out fibroblasts’ heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in the definition of PDAC CAF phenotypes. We identify the myCAF transcriptional phenotype as uniquely dependent on proficient MAPK signalling. In addition, CAFs displaying elevated MAPK activity are specifically anchored to basal-like/squamous PDAC cells and define tumour subdomains with reduced frequency of CD8+ T cells. We characterize the single-cell transcriptome of mouse PDAC tumours in response to MAPK inhibition and identify gene expression signatures of MAPK high CAFs, which suggest immunoregulatory functions. Accordingly, a gene expression signature of MAPK high CAFs correlates with poor prognosis in several human cancer conditions, including PDAC, and with reduced response to immune checkpoint inhibition in immune-reactive solid tumours. Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a new strategy for targeting of myofibroblastic CAFs in vivo .

Fumarate hydratase (FH)-deficient renal cell carcinomas are rare neoplasms characterized by wide morphologic heterogeneity and pathogenetic mutations in the FH gene. They often show aggressive behavior with rapid diffusion to distant organs, so novel therapeutic scenarios have been explored, including EGFR inhibitors and PD-L1 expression for targeted immunotherapy. Herein, we investigated a series of 11 primary FH-deficient renal cell carcinomas and 7 distant metastases to evaluate tumor heterogeneity even in metastatic sites and estimate the specific spread rates to various organs. Furthermore, the tumors were tested for immunohistochemical PD-L1 expression and EGFR mutations. Most metastatic cases involved the abdominal lymph nodes (4/7; 57%), followed by the peritoneum (3/7; 42%), the liver (2/7; 29%), and the lungs (1/7; 14%). Six metastatic localizations were histologically documented, revealing a morphologic heterogeneous architecture often differing from that of the corresponding primary renal tumor. Peritoneal involvement morphologically resembled a benign reactive mesothelial process or primary peritoneal mesothelioma, thus advocating to perform an accurate immunohistochemical panel, including PAX8 and FH, to reach a proper diagnosis. A pure low-grade succinate dehydrogenase-looking primary FH-deficient renal cell carcinoma was also recorded. As for therapy, significant PD-L1 labeling was found in 60% of primary renal tumors, whereas none of them carried pathogenetic EGFR mutations. Our data show that FH-deficient renal cell carcinoma may be morphologically heterogeneous in metastases as well, which involve the lymph nodes, the liver, and the peritoneum more frequently than other renal tumors. Due to the high frequency of this latter (42%), pathologists should always be concerned about ruling out mesothelial-derived mimickers, and the occurrence of rarer, primary, low-grade-looking types. Finally, contrary to EGFR mutations, PD-L1 expression could be a possible predictive biomarker for the therapy of these tumors.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells’ proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor.

AbstractPurpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Objective Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. Design Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. Results PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. Conclusion Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss.

Isocitrate dehydrogenase 1 (IDH1) is an evolutionarily conserved enzyme that catalyzes the interconversion of isocitrate to α-ketoglutarate with the concomitant reduction of NADP+ to NADPH. IDH1 has previously been shown to participate in lipid biosynthesis in various tissues such as the liver and adipose tissue. We examined the potential role of IDH1 in phospholipid metabolism in the brain. Here we show that IDH1 is highly expressed in the brain and astrocytes during embryonic development and the postnatal period and subsequently declines in adulthood. Silencing of IDH1 expression using siRNA in astrocytes isolated from E18.5 mouse cortices led to increased incorporation of [3H]-palmitate into the phosphatidylcholines (PCs) and decreased the incorporation of [3H]-palmitate into sphingomyelin and the phosphatidylethanolamines (PEs). In pulse-chase experiments, knock-down of IDH1 expression impaired the turnover of PCs and decreased the synthesis of PEs. The decrease in [3H]-palmitate incorporation into PEs when IDH1 was knocked-down in astrocytes was not due to impairments within the CDP-ethanolamine pathway or in the rate of decarboxylation of phosphatidylserine (PS). In conclusion, our results reveal a role for IDH1 in the synthesis/turnover of phospholipids in developing astrocytes and highlight the lipid alterations resulting from the loss of wild-type IDH1 activity.

Hypothesis: Poorly cohesive (PC) gastric cancer (GC) exhibits variable clinical behavior, being extremely aggressive in most cases but more indolent at times. We hypothesized that the integrative genomic and gene expression characterization of a PC GC series could help identifying molecular subtypes with potential clinical implications. Materials and Methods: 64 PC GCs were assessed for alterations in 409 genes and 30 cases were subjected to transcriptomic profiling of 20,815 genes. Results: A median of 8.2 mutations per Mb (interquartile range 6.9–10.4) was found and a tumor mutational load >10 muts/Mb was significantly associated with patients’ worse survival ( P =0.0024). The most frequent mutated genes were CDH1 and TP53 (each 32.8%) followed by PIK3CA (10.9%). In 15 samples (23.4%), at least 1 chromatin remodeling gene was mutated: KMT2D (5 cases); ARID1A and BAP1 (4 cases each); EZH2 , KMT2A , PBRM1 (1 case each). Eight samples (12.5%) had fusion genes involving CLDN18 gene. Gene expression profiling identified 4 different clusters: cluster A associated with epithelial to mesenchymal transition (EMT) signature; cluster B associated to proliferative signature and EMT; cluster C correlated to hedgehog signaling; cluster D showing no enrichment for any of the previous signatures. Notably, cluster A and B showed a worse prognosis compared with clusters C and D ( P =0.0095). Conclusion: integrated genomic and transcriptomic analysis suggest the existence of 4 molecular subtypes of PC GC with prognostic significance where EMT features are associated with a worse outcome.

Pancreatoblastoma (PB) is a rare tumor of the pancreas. In case of metastases, the treatment options are sparse and targeted approaches are not developed. We here evaluate MCL1 amplification as a putative target in PB. Thirteen samples from adult (10/13) and pediatric patients (3/13) were collected. Three of these samples had been previously subjected to whole-exome sequencing (2 cases) or whole-genome sequencing (1 case) within a precision oncology program (NCT/DKTK MASTER), and this analysis had shown copy number gains of MCL1 gene. We established a fluorescence in situ hybridization (FISH) test to assess the copy number alterations of MCL1 gene in 13 formalin-fixed paraffin-embedded PBs, including the 3 cases assessed by genome sequencing. FISH analysis showed the amplification of MCL1 in 2 cases (both were adult PB), one of which was a case with the highest copy number gain at genomic analysis. In both cases, the average gene copy number per cell was ≥ 5.7 and the MCL1/1p12 ratio was ≥ 2.4. Our data support MCL1 as a putative target in PB. Patients with MCL1-amplified PB might benefit from MCL1 inhibition. Sequencing data is useful to screen for amplification; however, the established FISH for MCL1 can help to determine the level and cellular heterogeneity of MCL1 amplification more accurately.

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.

Purpose of Review Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism used by some types of malignancies, including pancreatic neuroendocrine tumors, to overcome the issue of telomere shortening, thus supporting tumor growth and cell proliferation. This review is focused on the most important achievements and opportunities deriving from ALT assessment in PanNET onco-pathology, highlighting the most promising fields in which such biomarker could be implemented in clinical practice. Recent Findings In pancreatic neuroendocrine tumors (PanNET), ALT is strongly correlated with the mutational status of two chromatin remodeling genes, DAXX and ATRX . Recent advances in tumor biology permitted to uncover important roles of ALT in the landscape of PanNET, potentially relevant for introducing this biomarker into clinical practice. Indeed, ALT emerged as a reliable indicator of worse prognosis for PanNET, helping in clinical stratification and identification of “high-risk” patients. Furthermore, it is a very specific marker supporting the pancreatic origin of neuroendocrine neoplasms and can be used for improving the diagnostic workflow of patients presenting with neuroendocrine metastasis from unknown primary. The activation of this process can be determined by specific FISH analysis. Summary ALT should be introduced in clinical practice for identifying “high-risk” PanNET patients and improving their clinical management, and as a marker of pancreatic origin among neuroendocrine tumors.