Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6.

Malignant pleural mesothelioma (MPM) remains an incurable disease. This is partly due to the lack of experimental models that fully recapitulate the complexity and heterogeneity of MPM, a major challenge for therapeutic management of the disease. In addition, the contribution of the MPM microenvironment is relevant for the adaptive response to therapy. We established mesothelioma patient-derived organoid (mPDO) cultures from MPM pleural effusions and tested their response to pemetrexed and cisplatin. We aimed to evaluate the contribution of mesothelioma-associated fibroblasts (MAFs) to the response to pemetrexed and cisplatin (P+C). Organoid cultures were obtained from eight MPM patients using specific growth media and conditions to expand pleural effusion-derived cells. Flow cytometry was used to verify the similarity of the organoid cultures to the original samples. MAFs were isolated and co-cultured with mPDOs, and the addition of MAFs reduced the sensitivity of mPDOs to P+C. Organoid formation and expression of cancer stem cell markers such as ABCG2, NANOG, and CD44 were altered by conditioned media from treated MAFs. We identified IL-6 as the major contributor to the attenuated response to chemotherapy. IL-6 secretion by MAFs is correlated with increased resistance of mPDOs to pemetrexed and cisplatin.

Effects of KEAP1 Silencing on NRF2 and NOTCH Pathways in SCLC Cell Lines.

The KEAP1/NRF2 pathway is a master regulator of several redox-sensitive genes implicated in the resistance of tumor cells against therapeutic drugs. The dysfunction of the KEAP1/NRF2 system has been correlated with neoplastic patients' outcomes and responses to conventional therapies. In lung tumors, the growth and the progression of cancer cells may also involve the intersection between the molecular NRF2/KEAP1 axis and other pathways, including NOTCH, with implications for antioxidant protection, survival of cancer cells, and drug resistance to therapies. At present, the data concerning the mechanism of aberrant NRF2/NOTCH crosstalk as well as its genetic and epigenetic basis in SCLC are incomplete. To better clarify this point and elucidate the contribution of NRF2/NOTCH crosstalk deregulation in tumorigenesis of SCLC, we investigated genetic and epigenetic dysfunctions of the KEAP1 gene in a subset of SCLC cell lines. Moreover, we assessed its impact on SCLC cells' response to conventional chemotherapies (etoposide, cisplatin, and their combination) and NOTCH inhibitor treatments using DAPT, a γ-secretase inhibitor (GSI). We demonstrated that the KEAP1/NRF2 axis is epigenetically controlled in SCLC cell lines and that silencing of KEAP1 by siRNA induced the upregulation of NRF2 with a consequent increase in SCLC cells' chemoresistance under cisplatin and etoposide treatment. Moreover, KEAP1 modulation also interfered with NOTCH1, HES1, and DLL3 transcription. Our preliminary data provide new insights about the downstream effects of KEAP1 dysfunction on NRF2 and NOTCH deregulation in this type of tumor and corroborate the hypothesis of a cooperation of these two pathways in the tumorigenesis of SCLC.

Harnessing the value of TCTP in breast cancer treatment resistance: an opportunity for personalized therapy.

Early identification of breast cancer (BC) patients at a high risk of progression may aid in therapeutic and prognostic aims. This is especially true for metastatic disease, which is responsible for most cancer-related deaths. Growing evidence indicates that the translationally controlled tumor protein (TCTP) may be a clinically relevant marker for identifying poorly differentiated aggressive BC tumors. TCTP is an intriguing protein with pleiotropic functions, which is involved in multiple signaling pathways. TCTP may also be involved in stress response, cell growth and proliferation-related processes, underlying its potential role in the initiation of metastatic growth. Thus, TCTP marks specific cancer cell sub-populations with pronounced stress adaptation, stem-like and immune-evasive properties. Therefore, we have shown that in vivo phospho-TCTP levels correlate with the response of BC cells to anti-HER2 agents. In this review, we discuss the clinical relevance of TCTP for personalized therapy, specific TCTP-targeting strategies, and currently available therapeutic agents. We propose TCTP as an actionable clinically relevant target that could potentially improve patient outcomes.

Interrogating colorectal cancer metastasis to liver: a search for clinically viable compounds and mechanistic insights in colorectal cancer Patient Derived Organoids.

BACKGROUND: Approximately 20-50% of patients presenting with localized colorectal cancer progress to stage IV metastatic disease (mCRC) following initial treatment and this is a major prognostic determinant. Here, we have interrogated a heterogeneous set of primary colorectal cancer (CRC), liver CRC metastases and adjacent liver tissue to identify molecular determinants of the colon to liver spreading. Screening Food and Drug Administration (FDA) approved drugs for their ability to interfere with an identified colon to liver metastasis signature may help filling an unmet therapeutic need. METHODS: RNA sequencing of primary colorectal cancer specimens vs adjacent liver tissue vs synchronous and asynchronous liver metastases. Pathways enrichment analyses. The Library of Integrated Network-based Cellular Signatures (LINCS)-based and Connectivity Map (CMAP)-mediated identification of FDA-approved compounds capable to interfere with a 22 gene signature from primary CRC and liver metastases. Testing the identified compounds on CRC-Patient Derived Organoid (PDO) cultures. Microscopy and Fluorescence Activated Cell Sorting (FACS) based analysis of the treated PDOs. RESULTS: We have found that liver metastases acquire features of the adjacent liver tissue while partially losing those of the primary tumors they derived from. We have identified a 22-gene signature differentially expressed among primary tumors and metastases and validated in public databases. A pharmacogenomic screening for FDA-approved compounds capable of interfering with this signature has been performed. We have validated some of the identified representative compounds in CRC-Patient Derived Organoid cultures (PDOs) and found that pentoxyfilline and, to a minor extent, dexketoprofen and desloratadine, can variably interfere with number, size and viability of the CRC -PDOs in a patient-specific way. We explored the pentoxifylline mechanism of action and found that pentoxifylline treatment attenuated the 5-FU elicited increase of ALDHhigh cells by attenuating the IL-6 mediated STAT3 (tyr705) phosphorylation. CONCLUSIONS: Pentoxifylline synergizes with 5-Fluorouracil (5-FU) in attenuating organoid formation. It does so by interfering with an IL-6-STAT3 axis leading to the emergence of chemoresistant ALDHhigh cell subpopulations in 5-FU treated PDOs. A larger cohort of CRC-PDOs will be required to validate and expand on the findings of this proof-of-concept study.

Mapping Non-Coding RNAs in Space and Time: Another Weapon to Dissect Intra-Tumor Heterogeneity in Cancer Progression.

It is increasingly clear that Intratumor heterogeneity (ITH) fuels tumor evolution, matching the concept of cancer as a heterogeneous ecosystem of spatially and temporally modulated cell subpopulations, which exploits dynamic strategies to hijack local and systemic resources and tissue(s) space [...].

Butein-instigated miR-186-5p-dependent modulation of TWIST1 affects resistance to cisplatin and bioenergetics of Malignant Pleural Mesothelioma cells.

Aim: Malignant pleural mesothelioma is a chemoresistant tumor, and biphasic and sarcomatoid histologies portend the worst prognosis for malignant pleural mesothelioma (MPM) patients. We obtained the microRNA expression profile of three biphasic-sarcomatoid MPM cell lines to identify commonly expressed microRNAs and evaluate the effect of butein, a chemo-sensitizing compound, on this microRNA subset. Methods: Nanostring-based microRNA profiling and analysis through the ROSALIND platform were employed to identify the commonly modulated microRNAs and their targets. MicroRNA-mimic transfection, Luciferase assay, and Western blotting were employed to show specific perturbation of TWIST1 levels by miR-186-5p. Sphere-forming assays, invasion assay, and metabolic profiling were used to assess the biological consequences of the butein-instigated miR-186-5p-mediated perturbation of TWIST1 levels. TGCA analysis was used to search for the correlation between TWIST1 and miR-186-5p levels in biphasic and epithelioid MPM specimens. Results: We identified a set of perturbed microRNAs, common to three biphasic/sarcomatoid MPM cell lines, after butein treatment. When focusing on miR-186-5p, we unraveled a butein-ignited and miR-186-5p-mediated modulation of TWIST1 levels which affected the 3D anchorage-independent growth, cisplatin resistance, invasion, and bioenergetics of the MPM cell lines tested. We showed that miR-186-5p and TWIST1 levels are anti-correlated in biphasic MPM specimens from TCGA. Conclusion: We unraveled a novel mechanism of action of butein, which attenuated the pro-tumorigenic features of MPM at least through a miR-186-5p-TWIST1 axis. We suggest that those activities converge into the chemo-sensitizing effect of this compound and may be of translational relevance.

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing.

DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the "methylation_analysis" plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.

Breast cancer metastasis: Is it a matter of OMICS and proper ex-vivo models?

Genomics has greatly increased the understanding of the study of breast cancer (BC) and has shaped the concept of intra-tumor heterogeneity, currently recognized as a propelling force for cancer progression. In this context, knowledge and understanding of metastatic breast cancer (mBC) has somehow lagged behind that of primary breast cancer. This may be explained by the relative scarcity of matched mBC samples, however it is possible that the mutation spectrum obtained from primary BC does not capture the full complexity of the metastatic disease. Here, we provide a few examples supporting this possibility, from public databases. We evoke the need to perform an integrated multi-OMICS characterization of mBC, to obtain a broad understanding of this complex disease, whose evolution cannot be explained solely by genomics. Pertinent to this, we suggest that rather an infrequent use of Patient-Derived -Tumor-Organoids (PDTOs) may be influenced by assuming that the metastatic conditions of PDTOs growth (mPDTOs) should be similar to those of the tissue of origin. We challenge this view by suggesting that the use of "target-organ inspired" growth conditions for mPDTOs, may better fit the emerging knowledge of metastatic disease. Thus, the integrated use of multi-OMICS and of clinically relevant mPDTOs may allow a further understanding of such disease and foster therapeutically relevant advances. We believe that our points may be valid for other solid cancers.

Characterizing the tumor microenvironment in rare renal cancer histological types.

The tumor microenvironment (TME), including immune cells, cancer-associated fibroblasts, endothelial cells, adjacent normal cells, and others, plays a crucial role in influencing tumor behavior and progression. Here, we characterized the TME in 83 primary renal tumors and matched metastatic or recurrence tissue samples (n = 15) from papillary renal cell carcinoma (pRCC) types 1 (n = 20) and 2 (n = 49), collecting duct carcinomas (CDC; n = 14), and high-grade urothelial carcinomas (HGUC; n = 5). We investigated 10 different markers of immune infiltration, vasculature, cell proliferation, and epithelial-to-mesenchymal transition by using machine learning image analysis in conjunction with immunohistochemistry. Marker expression was compared by Mann-Whitney and Kruskal-Wallis tests and correlations across markers using Spearman's rank correlation coefficient. Multivariable Poisson regression analysis was used to compare marker expression between histological types, while accounting for variation in tissue size. Several immune markers showed different rates of expression across histological types of renal carcinoma. Using pRCC1 as reference, the incidence rate ratio (IRR) of CD3+ T cells (IRR [95% confidence interval, CI] = 2.48 [1.53-4.01]) and CD20+ B cells (IRR [95% CI] = 4.38 [1.22-5.58]) was statistically significantly higher in CDC. In contrast, CD68+ macrophages predominated in pRCC1 (IRR [95% CI] = 2.35 [1.42-3.9]). Spatial analysis revealed CD3+ T-cell and CD20+ B-cell expressions in CDC to be higher at the proximal (p < 0.0001) and distal (p < 0.0001) tumor periphery than within the central tumor core. In contrast, expression of CD68+ macrophages in pRCC2 was higher in the tumor center compared to the proximal (p = 0.0451) tumor periphery and pRCC1 showed a distance-dependent reduction, from the central tumor, in CD68+ macrophages with the lowest expression of CD68 marker at the distal tumor periphery (p = 0.004). This study provides novel insights into the TME of rare kidney cancer types, which are often understudied. Our findings of differences in marker expression and localization by histological subtype could have implications for tumor progression and response to immunotherapies or other targeted therapies.

Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma.

Background High resistance to therapy and poor prognosis characterizes malignant pleural mesothelioma (MPM). In fact, the current lines of treatment, based on platinum and pemetrexed, have limited impact on the survival of MPM patients. Adaptive response to therapy-induced stress involves complex rearrangements of the MPM secretome, mediated by the acquisition of a senescence-associated-secretory-phenotype (SASP). This fuels the emergence of chemoresistant cell subpopulations, with specific gene expression traits and protumorigenic features. The SASP-driven rearrangement of MPM secretome takes days to weeks to occur. Thus, we have searched for early mediators of such adaptive process and focused on metabolites differentially released in mesothelioma vs mesothelial cell culture media, after treatment with pemetrexed. METHODS: Mass spectrometry-based (LC/MS and GC/MS) identification of extracellular metabolites and unbiased statistical analysis were performed on the spent media of mesothelial and mesothelioma cell lines, at steady state and after a pulse with pharmacologically relevant doses of the drug. ELISA based evaluation of arachidonic acid (AA) levels and enzyme inhibition assays were used to explore the role of cPLA2 in AA release and that of LOX/COX-mediated processing of AA. QRT-PCR, flow cytometry analysis of ALDH expressing cells and 3D spheroid growth assays were employed to assess the role of AA at mediating chemoresistance features of MPM. ELISA based detection of p65 and IkBalpha were used to interrogate the NFkB pathway activation in AA-treated cells. RESULTS: We first validated what is known or expected from the mechanism of action of the antifolate. Further, we found increased levels of PUFAs and, more specifically, arachidonic acid (AA), in the transformed cell lines treated with pemetrexed. We showed that pharmacologically relevant doses of AA tightly recapitulated the rearrangement of cell subpopulations and the gene expression changes happening in pemetrexed -treated cultures and related to chemoresistance. Further, we showed that release of AA following pemetrexed treatment was due to cPLA2 and that AA signaling impinged on NFkB activation and largely affected anchorage-independent, 3D growth and the resistance of the MPM 3D cultures to the drug. CONCLUSIONS: AA is an early mediator of the adaptive response to pem in chemoresistant MPM and, possibly, other malignancies.

Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature.

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.

Impact of Histology and Tumor Grade on Clinical Outcomes Beyond 5 Years of Follow-Up in a Large Cohort of Renal Cell Carcinomas.

INTRODUCTION: The optimal length for clinical follow-up of renal cell carcinoma (RCC) patients is unclear. We evaluated the impact of ISUP/WHO tumor grade and histological subtype on short- and long-term survival and risk of recurrence/metastasis in a large cohort of RCC patients. PATIENTS AND METHODS: We studied 1679 RCC patients from a single referral center in Italy. Adjusted hazard ratios for overall survival were estimated using Cox regression models. Adjusted absolute risk of developing recurrence or metastasis was computed considering competing risks of mortality. RESULTS: During up to 13 years of follow-up, 175 (10.4%) RCC patients died, of whom 92% beyond 5 years. Hazard ratio of grade IV clear cell carcinomas (ccRCC) was 3.82 compared to grade II. Notably, 33% of recurrences and 56% of distant metastases occurred beyond 5 years of follow-up. The estimated probabilities of recurrence/metastasis were 15% and 45% within and beyond 5 years of follow-up, respectively. After 5 years, the absolute risk of recurrences increased also for papillary renal cell carcinoma type I (35.2%) and grade I ccRCC (17%). CONCLUSION: After 5 years of follow-up, both risk of mortality and recurrences or metastases were high and were modified by histological types and tumor grade. These data strongly support histology- and grade-tailored surveillance strategies and long-term follow-up for RCC patients.

Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development.

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.

EphA2 and EGFR: Friends in Life, Partners in Crime. Can EphA2 Be a Predictive Biomarker of Response to Anti-EGFR Agents?

The Eph receptors represent the largest group among Receptor Tyrosine kinase (RTK) families. The Eph/ephrin signaling axis plays center stage during development, and the deep perturbation of signaling consequent to its dysregulation in cancer reveals the multiplicity and complexity underlying its function. In the last decades, they have emerged as key players in solid tumors, including colorectal cancer (CRC); however, what causes EphA2 to switch between tumor-suppressive and tumor-promoting function is still an active theater of investigation. This review summarizes the recent advances in understanding EphA2 function in cancer, with detail on the molecular determinants of the oncogene-tumor suppressor switch function of EphA2. We describe tumor context-specific examples of EphA2 signaling and the emerging role EphA2 plays in supporting cancer-stem-cell-like populations and overcoming therapy-induced stress. In such a frame, we detail the interaction of the EphA2 and EGFR pathway in solid tumors, including colorectal cancer. We discuss the contribution of the EphA2 oncogenic signaling to the resistance to EGFR blocking agents, including cetuximab and TKIs.

Combined analysis of miR-200 family and its significance for breast cancer.

While the molecular functions of miR-200 family have been deeply investigated, a role for these miRNAs as breast cancer biomarkers remains largely unexplored. In the attempt to clarify this, we profiled the miR-200 family members expression in a large cohort of breast cancer cases with a long follow-up (H-CSS cohort) and in TCGA-BRCA cohort. Overall, miR-200 family was found upregulated in breast tumors with respect to normal breast tissues while downregulated in more aggressive breast cancer molecular subtypes (i.e. Luminal B, HER2 and triple negative), consistently with their function as repressors of the epithelial-to-mesenchymal transition (EMT). In particular miR-141-3p was found differentially expressed in breast cancer molecular subtypes in both H-CSS and TCGA-BRCA cohorts, and the combined analysis of all miR-200 family members demonstrated a slight predictive accuracy on H-CSS cancer specific survival at 12 years (survival c-statistic: 0.646; 95%CI 0.538-0.754).

Metformin: Metabolic Rewiring Faces Tumor Heterogeneity.

Tumor heterogeneity impinges on all the aspects of tumor history, from onset to metastasis and relapse. It is growingly recognized as a propelling force for tumor adaptation to environmental and micro-environmental cues. Metabolic heterogeneity perfectly falls into this process. It strongly contributes to the metabolic plasticity which characterizes cancer cell subpopulations-capable of adaptive switching under stress conditions, between aerobic glycolysis and oxidative phosphorylation-in both a convergent and divergent modality. The mitochondria appear at center-stage in this adaptive process and thus, targeting mitochondria in cancer may prove of therapeutic value. Metformin is the oldest and most used anti-diabetic medication and its relationship with cancer has witnessed rises and falls in the last 30 years. We believe it is useful to revisit the main mechanisms of action of metformin in light of the emerging views on tumor heterogeneity. We first analyze the most consolidated view of its mitochondrial mechanism of action and then we frame the latter in the context of tumor adaptive strategies, cancer stem cell selection, metabolic zonation of tumors and the tumor microenvironment. This may provide a more critical point of view and, to some extent, may help to shed light on some of the controversial evidence for metformin's anticancer action.

Assessment of HER2 Protein Overexpression and Gene Amplification in Renal Collecting Duct Carcinoma: Therapeutic Implication.

Collecting duct carcinoma (CDC) is rare and aggressive histology of kidney cancers. Although different therapeutic approaches have been tested, the 2-year survival remains very poor. Since CDC exhibits overlapping features with urothelial carcinoma, the analysis of shared molecular alterations could provide new insights into the understanding of this rare disease and also therapeutic options. We collected 26 CDC cases, and we assessed HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in-situ hybridization (FISH) according to 2018 ASCO/CAP HER2-testing recommendations. Six out of twenty-six (23%) tumors showed HER2 positive staining. In particular, 3+ score was present in 2/6 cases (33%), 2+ in 3/6 cases (50%) and 1+ in 1/6 cases (17%). The 6 HER2+ tumors were also analyzed by FISH to assess gene copy number. One out of six CDC with IHC 3+ was also HER2 amplified, showing an average HER2 copy number ≥4.0 (10.85) and a HER2/CEP17 ratio ≥ (5.63), while the 5/6 cases were HER2 negative. Based on the 2018 ASCO/CAP guidelines overall, 2/26 CDC cases (8%) were HER2+. The present study provides evidence for testing, in future studies, HER2 to assess its clinical value as a novel target for the treatment of this highly malignant cancer.

Hsa-miR-155-5p Up-Regulation in Breast Cancer and Its Relevance for Treatment With Poly[ADP-Ribose] Polymerase 1 (PARP-1) Inhibitors.

miR-155-5p is a well-known oncogenic microRNA, showing frequent overexpression in human malignancies, including breast cancer. Here, we show that high miR-155-5p levels are associated with unfavorable prognostic factors in two independent breast cancer cohorts (CSS cohort, n = 283; and TCGA-BRCA dataset, n = 1,095). Consistently, miR-155-5p results as differentially expressed in the breast cancer subgroups identified by the surrogate molecular classification in the CSS cohort and the PAM50 classifier in TCGA-BRCA dataset, with the TNBC and HER2-amplified tumors carrying the highest levels. Since the analysis of TCGA-BC dataset also demonstrated a significant association between miR-155-5p levels and the presence of mutations in homologous recombination (HR) genes, we hypothesized that miR-155-5p might affect cell response to the PARP-1 inhibitor Olaparib. As expected, miR-155-5p ectopic overexpression followed by Olaparib administration resulted in a greater reduction of cell viability as compared to Olaparib administration alone, suggesting that miR-155-5p might induce a synthetic lethal effect in cancer cells when coupled with PARP-1-inhibition. Overall, our data point to a role of miR-155-5p in homologous recombination deficiency and suggest miR-155-5p might be useful in predicting response to PARP1 inhibitors in the clinical setting.

The genomic and epigenomic evolutionary history of papillary renal cell carcinomas.

Intratumor heterogeneity (ITH) and tumor evolution have been well described for clear cell renal cell carcinomas (ccRCC), but they are less studied for other kidney cancer subtypes. Here we investigate ITH and clonal evolution of papillary renal cell carcinoma (pRCC) and rarer kidney cancer subtypes, integrating whole-genome sequencing and DNA methylation data. In 29 tumors, up to 10 samples from the center to the periphery of each tumor, and metastatic samples in 2 cases, enable phylogenetic analysis of spatial features of clonal expansion, which shows congruent patterns of genomic and epigenomic evolution. In contrast to previous studies of ccRCC, in pRCC, driver gene mutations and most arm-level somatic copy number alterations (SCNAs) are clonal. These findings suggest that a single biopsy would be sufficient to identify the important genetic drivers and that targeting large-scale SCNAs may improve pRCC treatment, which is currently poor. While type 1 pRCC displays near absence of structural variants (SVs), the more aggressive type 2 pRCC and the rarer subtypes have numerous SVs, which should be pursued for prognostic significance.