The expression of LINE1-MET chimeric transcript identifies a subgroup of aggressive breast cancers.

Demethylation of the long interspersed nuclear element (LINE-1; L1) antisense promoter can result in transcription of neighboring sequences as for the L1-MET transcript produced by the L1 placed in the second intron of MET. To define the role of L1-MET, we investigated the sequence and the transcription of L1-MET in vitro models and heterogeneous breast cancers, previously reported to show other L1-derived transcripts. L1-MET expressing cell lines were initially identified in silico and investigated for L1-MET promoter methylation, cDNA sequence and cell fraction mRNA. The transcriptional level of L1-MET and MET were then evaluated in breast specimens, including 9 cancer cell lines, 41 carcinomas of different subtypes, and 11 normal tissues. In addition to a L1-MET transcript ending at MET exon 21, six novel L1-MET splice variants were identified. Normal breast tissues were negative for the L1-MET expression, whereas the triple-negative breast cancer (TNBC) and the high-grade carcinomas were enriched with the L1-MET mRNA (p = 0.005 and p = 0.018, respectively). In cancer cells and tissues the L1-MET expression was associated with its promoter hypomethylation (ρ = -0.8 and -0.9, respectively). No correlation was found between L1-MET and MET mRNA although L1-MET expressing tumors with higher L1-MET/MET ratio were negative for the MET protein expression (p = 0.006). Besides providing the first identification and detailed description of L1-MET in breast cancer, we clearly demonstrate that higher levels of this transcript specifically recognize a subset of more aggressive carcinomas, mainly TNBC. We suggest the possible evaluation of L1-MET in the challenging diagnosis of early TNBCs.

Oxidative DNA damage induces hypomethylation in a compromised base excision repair colorectal tumourigenesis.

BACKGROUND: A compromised base excision repair (BER) promotes carcinogenesis by accumulating oxidative DNA-damaged products as observed in MUTYH-associated polyposis, a hereditary colorectal cancer syndrome marked by adenomas and cancers with an accumulation of 8-oxoguanine. Remarkably, DNA global demethylation has been shown to be mediated by BER, suggesting a relevant interplay with early colorectal tumourigenesis. To check this hypothesis, we investigated a cohort of 49 adenomas and 10 carcinomas, derived from 17 MUTYH-associated polyposis patients; as adenoma controls, we used a set of 36 familial adenomatous polyposis and 24 sporadic polyps. METHODS: Samples were analysed for their mutational and epigenetic status, measured as global LINE-1 (long interspersed nuclear element) and gene-specific LINE-1 MET methylation by mass spectrometry and pyrosequencing. RESULTS: MUTYH-associated polyposis adenomas were strikingly more hypomethylated than familial adenomatous and sporadic polyps for both DNA demethylation markers (P=0.032 and P=0.007 for LINE-1; P=0.004 and P<0.0001 for LINE-1 MET, respectively) with levels comparable to those of the carcinomas derived from the same patients. They also had mutations due mainly to KRAS/NRAS p.G12C, which was absent in the controls (P<0.0001 for both sets). CONCLUSIONS: Our results show that DNA demethylation, together with specific KRAS/NRAS mutations, drives the early steps of oxidative damage colorectal tumourigenesis.

Real-world histopathological approach to malignancy of undefined primary origin (MUO) to diagnose cancers of unknown primary (CUPs).

The aim of this study is to envisage a streamlined pathological workup to rule out CUPs in patients presenting with MUOs. Sixty-four MUOs were classified using standard histopathology. Clinical data, immunocytochemical markers, and results of molecular analysis were recorded. MUOs were histologically subdivided in clear-cut carcinomas (40 adenocarcinomas, 11 squamous, and 3 neuroendocrine carcinomas) and unclear-carcinoma features (5 undifferentiated and 5 sarcomatoid tumors). Cytohistology of 7/40 adenocarcinomas suggested an early metastatic cancer per se. In 33/40 adenocarcinomas, CK7/CK20 expression pattern, gender, and metastasis sites influenced tissue-specific marker selection. In 23/40 adenocarcinomas, a "putative-immunophenotype" of tissue of origin addressed clinical-diagnostic examinations, identifying 9 early metastatic cancers. Cell lineage markers were used to confirm squamous and neuroendocrine differentiation. Pan-cytokeratins were used to confirm the epithelial nature of poorly differentiated tumors, followed by tissue and cell lineage markers, which identified one melanoma. In total, 47/64 MUOs (73.4%) were confirmed CUP. Molecular analysis, feasible in 37/47 CUPs (78.7%), had no diagnostic impact. Twenty CUP patients, mainly with squamous carcinomas and adenocarcinomas with putative-gynecologic-immunophenotypes, presented with only lymph node metastases and had longer median time to progression and overall survival (< 0.001), compared with patients with other metastatic patterns. We propose a simplified histology-driven workup which could efficiently rule out CUPs and identify early metastatic cancer.

Coexisting cancer stem cells with heterogeneous gene amplifications, transcriptional profiles, and malignancy are isolated from single glioblastomas.

Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.

Integrative genomic and transcriptomic analyses illuminate the ontology of HER2-low breast carcinomas.

BACKGROUND: The "HER2-low" nomenclature identifies breast carcinomas (BCs) displaying a HER2 score of 1+/2+ in immunohistochemistry and lacking ERBB2 amplification. Whether HER2-low BCs (HLBCs) constitute a distinct entity is debated. METHODS: We performed DNA and RNA high-throughput analysis on 99 HLBC samples (n = 34 cases with HER2 score 1+/HLBC-1, n = 15 cases with HER2 score 2+ and ERBB2 not amplified/HLBC-2N, and n = 50 cases with score 2+ and ERBB2 copy number in the equivocal range/HLBC-2E). We compared the mutation rates with data from 1317 samples in the Memorial Sloan-Kettering Cancer Center (MSKCC) BC cohort and gene expression data with those from an internal cohort of HER2-negative and HER2-positive BCs. RESULTS: The most represented mutations affected PIK3CA (31/99, 31%), GATA3 (18/99, 18%), TP53 (17/99, 17%), and ERBB2 (8/99, 8%, private to HLBC-2E). Tumor mutational burden was significantly higher in HLBC-1 compared to HLBC-2E/N (P = 0.04). Comparison of mutation spectra revealed that HLBCs were different from both HER2-negative and HER2-positive BCs, with HLBC-1 resembling more HER2-negative tumors and HLBC-2 mutationally related to HER2-addicted tumors. Potentially actionable alterations (annotated by using OncoKB/ESCAT classes) affected 52 patients. Intra-group gene expression revealed overlapping features between HLBC-1 and control HER2-negative BCs, whereas the HLBC-2E tumors showed the highest diversity overall. The RNA-based class discovery analysis unveiled four subsets of tumors with (i) lymphocyte activation, (ii) unique enrichment in HER2-related features, (iii) stromal remodeling alterations, and (iv) actionability of PIK3CA mutations (LAURA classification). CONCLUSIONS: HLBCs harbor distinct genomic features when compared with HER2-positive and HER2-negative BCs; however, differences across IHC classes were also unveiled thus dissecting the full picture of heterogeneity across HER2-low disease. The HLBC-2E category harbors most distinctive features, whereas HLBC-1 seems superimposable to HER2-negative disease. Further studies are needed to ascertain whether the four genomic-driver classes of the LAURA classification hold prognostic and/or predictive implications.

A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient.

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α-fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

Cancer of unknown primary stem-like cells model multi-organ metastasis and unveil liability to MEK inhibition.

Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples.

Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.

Colorectal Adenomatous Polyposis: Heterogeneity of Susceptibility Gene Mutations and Phenotypes in a Cohort of Italian Patients.

AIMS: Colorectal adenomatous polyposis entailing cancer predisposition is caused by constitutional mutations in different genes. APC is associated with the familial adenomatous polyposis (FAP/AFAP) and MUTYH with the MUTYH-associated polyposis (MAP), while POLE and POLD1 mutations cause the polymerase proofreading-associated polyposis (PPAP). METHODS: We screened for mutations in patients with multiple adenomas/FAP: 121 patients were analyzed for APC and MUTYH mutations, and 36 patients were also evaluated for POLE and POLD1 gene mutations. RESULTS: We found 20 FAP/AFAP, 15 MAP, and no PPAP subjects: pathogenic mutations proved to be heterogeneous, and included 5 APC and 1 MUTYH novel mutations. The mutation detection rate was significantly different between patients with 5-100 polyps and those with >100 polyps (p = 8.154 × 10-7), with APC mutations being associated with an aggressive phenotype (p = 1.279 × 10-9). Mean age at diagnosis was lower in FAP/AFAP compared to MAP (p = 3.055 × 10-4). Mutation-negative probands showed a mean age at diagnosis that was significantly higher than FAP/AFAP (p = 3.46986 × 10-7) and included 45.3% of patients with <30 polyps and 70.9% of patients with no family history. CONCLUSIONS: This study enlarges the APC and MUTYH mutational spectra, and also evaluated variants of uncertain significance, including the MUTYH p.Gln338His mutation. Moreover this study underscores the phenotypic heterogeneity and genotype-phenotype correlations in a cohort of Italian patients.