Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.

Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers.

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

HER2 overexpression and anti-HER2 agents represent probably the best story of success of individualized therapy in breast cancer. Due to the important therapeutic implications, the issue under the spotlight has been, since ever, the correct identification of true HER2 positivity on tissue specimens. Eligibility to anti-HER2 agents is strictly dependent on the demonstration of HER2 overexpression (by immunohistochemistry) or of HER2 gene amplification by in situ techniques (fluorescence in situ hybridization, FISH), however there are controversial issues involving cases with "equivocal" HER2 status based on conventional techniques (about 20% of specimens). In terms of HER2 expression a major debate is the presence of full-length and truncated forms of the protein and controversial clinical data have been reported on the therapeutic implications of these HER2 fragments. In terms of HER2 gene assessment, the occurrence of amplification of the chromosome 17 centromeric region (CEP17) has been proven responsible for misleading HER2 FISH results, precluding anti-HER2 based therapy to some patients. Finally HER2 activating mutations have been recently described as a biological mechanisms alternative to HER2 gene amplification. In this review we will focus on the controversies that pathologists and oncologists routinely face in the attempt to design the most tailored treatment for breast cancer patients. We will focus on the HER2 gene and on the protein, both at technical and interpretational levels.

We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

We propose a new multi-network-based strategy to integrate different layers of genomic information and use them in a coordinate way to identify driving cancer genes. The multi-networks that we consider combine transcription factor co-targeting, microRNA co-targeting, protein-protein interaction and gene co-expression networks. The rationale behind this choice is that gene co-expression and protein-protein interactions require a tight coregulation of the partners and that such a fine tuned regulation can be obtained only combining both the transcriptional and post-transcriptional layers of regulation. To extract the relevant biological information from the multi-network we studied its partition into communities. To this end we applied a consensus clustering algorithm based on state of art community detection methods. Even if our procedure is valid in principle for any pathology in this work we concentrate on gastric, lung, pancreas and colorectal cancer and identified from the enrichment analysis of the multi-network communities a set of candidate driver cancer genes. Some of them were already known oncogenes while a few are new. The combination of the different layers of information allowed us to extract from the multi-network indications on the regulatory pattern and functional role of both the already known and the new candidate driver genes.

Colorectal cancer (CRC) transcriptional subtypes have been recently identified by gene expression profiling. Here we describe an analytical pipeline, microRNA master regulator analysis (MMRA), developed to search for microRNAs potentially driving CRC subtypes. Starting from a microRNA-mRNA tumour expression data set, MMRA identifies candidate regulator microRNAs by assessing their subtype-specific expression, target enrichment in subtype mRNA signatures and network analysis-based contribution to subtype gene expression. When applied to a CRC data set of 450 samples, assigned to subtypes by 3 different transcriptional classifiers, MMRA identifies 24 candidate microRNAs, in most cases downregulated in the stem/serrated/mesenchymal (SSM) poor prognosis subtype. Functional validation in CRC cell lines confirms downregulation of the SSM subtype by miR-194, miR-200b, miR-203 and miR-429, which share target genes and pathways mediating this effect. These results show that, by combining statistical tests, target prediction and network analysis, MMRA effectively identifies microRNAs functionally associated to cancer subtypes.

Many microarray experiments search for genes with differential expression between a common "reference" group and multiple "test" groups. In such cases currently employed statistical approaches based on t-tests or close derivatives have limited efficacy, mainly because estimation of the standard error is done on only two groups at a time. Alternative approaches based on ANOVA correctly capture within-group variance from all the groups, but then do not confront single test groups with the reference. Ideally, a t-test better suited for this type of data would compare each test group with the reference, but use within-group variance calculated from all the groups.

In routine practice, nuclear pleomorphism of tumours is assessed by haematoxylin staining of the membrane-bound heterochromatin. However, decoration of the nuclear envelope (NE) through the immunofluorescence staining of NE proteins such as lamin B and emerin can provide a more objective appreciation of the nuclear shape. In breast cancer, nuclear pleomorphism is one of the least reproducible parameters to score histological grade, thus we sought to use NE proteins to improve the reproducibility of nuclear grading. First, immuno-fluorescence staining of NE as well as confocal microscopy and three-dimensional reconstruction of nuclei in cultured cells showed a smooth and uniform NE of normal breast epithelium in contrast to an irregular foldings of the membrane and the presence of deep invaginations leading to the formation of an intranuclear scaffold of NE-bound tubules in breast cancer cells. Following the above methods and criteria, we recorded the degree of NE pleomorphism (NEP) in a series of 273 invasive breast cancers tested by immunofluorescence. A uniform nuclear shape with few irregularities (low NEP) was observed in 135 cases or, alternatively, marked folds of the NE and an intranuclear tubular scaffold (high NEP cases) were observed in 138 cases. The latter features were significantly correlated (P-value <0.002) with lymph node metastases in 54 histological grade 1 and in 173 cancers with low mitotic count. Decoration of the NE might thus be regarded as a novel diagnostic parameter to define the grade of malignancy, which parallels and enhances that provided by routine histological procedures.

Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 × ) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of ~90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues.

In the absence of identified therapeutic targets, chemotherapy is the main systemic treatment option for triple-negative breast cancer (TNBC). The achievement of a pathological complete response (pCR) after neoadjuvant chemotherapy leads to good outcome, whereas patients not achieving a pCR are at high risk of relapse. Various trials have evaluated the inclusion of platinum in neoadjuvant chemotherapy regimens for TNBC, leading to non-univocal results. The panel of the Italian Association of Medical Oncology (AIOM) Guidelines on Breast Cancer developed a clinical recommendation on the addition of platinum to anthracycline/taxane-based neoadjuvant chemotherapy for TNBC by using the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) methodology and the Evidence to Decision framework (EtD). Five studies were eligible. The panel identified the following outcomes of benefit: pCR (critical), disease/event-free survival (DFS/EFS, critical), and overall survival (OS, critical). The panel identified febrile neutropenia (critical), serious adverse events (critical), anemia grade 3-4 (important), thrombocytopenia grade 3-4 (important) as outcomes of harms. The probability of pCR was higher in the platinum-based chemotherapy group versus control group (RR = 1.45, 95%CI 1.28-1.64); however, no impact on long-term outcome was observed. Neoadjuvant treatment regimens containing platinum resulted in a non-significant increase in the risk of febrile neutropenia and in a significant increase in the risk serious adverse events, G3-G4 anemia and G3-G4 thrombocytopenia: 11.3% versus 0.8%, RR = 15.66 (95%CI 6.38-38.44). The panel judged uncertain/favorable the benefit/harms balance. The panel's final recommendation was conditional in favor of the inclusion of platinum in anthracycline/taxane-based neoadjuvant regimens for TNBC.

The accuracy of histopathological diagnosis is strictly reliant on adequate tissue preservation, which is completely dependent on pre-analytical variables. Among these variables, the time interval between the end of surgical excision to the onset of fixation (the cold ischemia time) may adversely affect preservation of tissue morphology, influencing the interpretation and reproducibility of diagnosis. During this time interval, the activation of enzymes may produce autolysis and degradation of antigens and nucleic acids, thus potentially affecting immunocytochemical and molecular results. Several studies have described under-vacuum at 4 °C storage of fresh surgical specimens as a safe and reliable method to control cold ischemia and preserve fresh tissues, as well as to standardize fixation times and implement tissue-banking. This review article gives a systematic overview of the advantages and drawbacks of the use of under-vacuum tissue preservation and cooling in surgical pathology, highlighting the impact this procedure may have on diagnostic and experimental pathology. It also documents our experience acquired within daily practice and national and international projects.

The role of nipple discharge cytology (NDc) in the surgical management of breast cancer patients is unclear. We aimed: (i) to evaluate the effect of malignant NDc on the surgical approach to the nipple-areola complex, and (ii) to verify the association between malignant NDc and nipple malignancy.

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Overexpression of miR-100 in stem cells derived from basal-like breast cancers causes loss of stemness, induction of luminal breast cancer markers and response to endocrine therapy. We, therefore, explored miR-100 as a novel biomarker in patients with luminal breast cancer.

The MET oncogene encodes a tyrosine kinase receptor involved in the control of a complex network of biological responses that include protection from apoptosis and stimulation of cell growth during embryogenesis, tissue regeneration, and cancer progression. We previously developed an antagonist antibody (DN30) inducing the physical removal of the receptor from the cell surface and resulting in suppression of the biological responses to MET. In its bivalent form, the antibody displayed a residual agonist activity, due to dimerization of the lingering receptors, and partial activation of the downstream signaling cascade. The balance between the two opposing activities is variable in different biological systems and is hardly predictable. In this study, we generated and characterized two single-chain antibody fragments derived from DN30, sharing the same variable regions but including linkers different in length and composition. The two engineered molecules bind MET with high affinity but induce different biological responses. One behaves as a MET-antagonist, promoting programmed cell death in MET "addicted" cancer cells. The other acts as a hepatocyte growth factor (HGF)-mimetic, protecting normal cells from doxorubicin-induced apoptosis. Thus, by engineering the same receptor antibody, it is possible to generate molecules enhancing or inhibiting apoptosis either to kill cancer cells or to protect healthy tissues from the injuries of chemotherapy.

Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature (stdFFPE) and at 4°C (coldFFPE), respectively. Two independent cohorts were prospectively collected, cohort A (collected 6 years prior to the study, n = 21), cohort B (collected at time of the study, n = 17). DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in coldFFPE compared to stdFFPE samples (mean values: 0.69 vs. 0.36, p < 0.0001) and stdFFPE breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between coldFFPE tissues of both cohorts; conversely, DNA integrity of stdFFPE was significantly lower in cohort A compared to cohort B (p < 0.0001). Of note, QC scores of stdFFPE (but not of coldFFPE) samples were significantly reduced following 6 months of storage (p = 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.

Metaplastic breast carcinoma (MBC) and uterine carcinosarcoma (UCS) are rare aggressive cancers, characterized by an admixture of adenocarcinoma and areas displaying mesenchymal/sarcomatoid differentiation. We sought to define whether MBCs and UCSs harbor similar patterns of genetic alterations, and whether the different histologic components of MBCs and UCSs are clonally related. Whole-exome sequencing (WES) data from MBCs (n = 35) and UCSs (n = 57, The Cancer Genome Atlas) were reanalyzed to define somatic genetic alterations, altered signaling pathways, mutational signatures, and genomic features of homologous recombination DNA repair deficiency (HRD). In addition, the carcinomatous and sarcomatous components of an additional cohort of MBCs (n = 11) and UCSs (n = 6) were microdissected separately and subjected to WES, and their clonal relatedness was assessed. MBCs and UCSs harbored recurrent genetic alterations affecting TP53, PIK3CA, and PTEN, similar patterns of gene copy number alterations, and an enrichment in alterations affecting the epithelial-to-mesenchymal transition (EMT)-related Wnt and Notch signaling pathways. Differences were observed, however, including a significantly higher prevalence of FAT3 and FAT1 somatic mutations in MBCs compared to UCSs, and conversely, UCSs significantly more frequently harbored somatic mutations affecting FBXW7 and PPP2R1A as well as HER2 amplification than MBCs. Genomic features of HRD and biallelic alterations affecting bona fide HRD-related genes were found to be more prevalent in MBCs than in UCSs. The distinct histologic components of MBCs and UCSs were clonally related in all cases, with the sarcoma component likely stemming from a minor subclone of the carcinoma component in the samples with interpretable chronology of clonal evolution. Despite the similar histologic features and pathways affected by genetic alterations, UCSs differ from MBCs on the basis of FBXW7 and PPP2R1A mutations, HER2 amplification, and lack of HRD, supporting the notion that these entities are more than mere phenocopies of the same tumor type in different anatomical sites.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is caused by somatic mosaic NRAS variants and characterized by melanocytic/sebaceous naevi, eye, and brain malformations, and FGF23-mediated hypophosphatemic rickets. The MEK inhibitor Trametinib, acting on the RAS/MAPK pathway, is a candidate for CSHS therapy. A 4-year-old boy with seborrheic nevus, eye choristoma, multiple hamartomas, brain malformation, pleural lymphangioma and chylothorax developed severe hypophosphatemic rickets unresponsive to phosphate supplementation. The c.182A > G;p.(Gln61Arg) somatic NRAS variant found in DNA from nevus biopsy allowed diagnosing CSHS. We administered Trametinib for 15 months investigating the transcriptional effects at different time points by whole blood RNA-seq. Treatment resulted in prompt normalization of phosphatemia and phosphaturia, catch-up growth, chylothorax regression, improvement of bone mineral density, reduction of epidermal nevus and hamartomas. Global RNA sequencing on peripheral blood mononucleate cells showed transcriptional changes under MEK inhibition consisting in a strong sustained downregulation of signatures related to RAS/MAPK, PI3 kinase, WNT and YAP/TAZ pathways, reverting previously defined transcriptomic signatures. CSHS was effectively treated with a MEK inhibitor with almost complete recovery of rickets and partial regression of the phenotype. We identified "core" genes modulated by MEK inhibition potentially serving as surrogate markers of Trametinib action.

Approximately 20% of patients with RAS wild-type metastatic colorectal cancer (mCRC) experience objective responses to the anti-EGFR antibody cetuximab, but disease eradication is seldom achieved. The extent of tumor shrinkage correlates with long-term outcome. We aimed to find rational combinations that potentiate cetuximab efficacy by disrupting adaptive dependencies on antiapoptotic molecules (BCL2, BCL-XL, MCL1).