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Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic and lethal disease. Gasdermins are primarily associated with necrosis via membrane permeabilization and pyroptosis, a lytic pro‐inflammatory type of cell death. In this study, GSDMC upregulation during PDAC progression is reported. GSDMC directly induces genes related to stemness, EMT, and immune evasion. Targeting Gsdmc in murine PDAC models reprograms the immunosuppressive tumor microenvironment, rescuing the recruitment of anti‐tumor immune cells through CXCL9. This not only results in diminished tumor initiation, growth and metastasis, but also enhances the response to KRASG12D inhibition and PD‐1 checkpoint blockade, respectively. Mechanistically, it is discovered that ADAM17 cleaves GSDMC, releasing nuclear fragments binding to promoter regions of stemness, metastasis, and immune evasion‐related genes. Pharmacological inhibition of GSDMC cleavage or prevention of its nuclear translocation is equally effective in suppressing GSDMC's downstream targets and inhibiting PDAC progression. The findings establish GSDMC as a potential therapeutic target for enhancing treatment response in this deadly disease.

Summary: Background: We studied the poorly-known dynamics of circulating DNA (cir-nDNA), as monitored prospectively over an extended post-surgery period, in patients with cancer. Methods: On patients with stage III colon cancer (N = 120), using personalised molecular tags we carried out the prospective, multicenter, blinded cohort study of the post-surgery serial analysis of cir-nDNA concentration. 74 patients were included and 357 plasma samples tested. Findings: During post-operative follow-up, the patients’ median cir-nDNA concentration was greater (P 18 months post-surgery, the data suggest that the persistence of NETs formation is not due to the adjuvant CT. Interpretation: (1), Given the inter-patient heterogeneity, the post-surgery cir-nDNA level cannot be considered a reliable value, and caution must be exercised when determining mutation allele frequency or the mutation status; and (2), specific studies must be undertaken to investigate the possible clinical impact of the persistent, low-grade inflammation resulting from elevated NETs levels, such as observed in these post-surgery patients, given that such levels are known to potentially induce adverse cardiovascular or thrombotic events. Funding: This work was supported by the H2020 European ERA-NET grant on Translational Cancer Research (TRANSCAN-2).

Abstract Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: ‘pseudo-diploid’ cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

Summary: Pancreatic ductal adenocarcinoma (PDAC) poses significant clinical challenges, often presenting as unresectable with limited biopsy options. Here, we show that circulating tumor cells (CTCs) offer a promising alternative, serving as a “liquid biopsy” that enables the generation of in vitro 3D models and highly aggressive in vivo models for functional and molecular studies in advanced PDAC. Within the retrieved CTC pool (median 65 CTCs/5 mL), we identify a subset (median content 8.9%) of CXCR4+ CTCs displaying heightened stemness and metabolic traits, reminiscent of circulating cancer stem cells. Through comprehensive analysis, we elucidate the importance of CTC-derived models for identifying potential targets and guiding treatment strategies. Screening of stemness-targeting compounds identified stearoyl-coenzyme A desaturase (SCD1) as a promising target for advanced PDAC. These results underscore the pivotal role of CTC-derived models in uncovering therapeutic avenues and ultimately advancing personalized care in PDAC.

Abstract Background the problem in early diagnosis of sporadic cancer is understanding the individual’s risk to develop disease. In response to this need, global scientific research is focusing on developing predictive models based on non-invasive screening tests. A tentative solution to the problem may be a cancer screening blood-based test able to discover those cell requirements triggering subclinical and clinical onset latency, at the stage when the cell disorder, i.e. atypical epithelial hyperplasia, is still in a subclinical stage of proliferative dysregulation. Methods a well-established procedure to identify proliferating circulating tumor cells was deployed to measure the cell proliferation of circulating non-haematological cells which may suggest tumor pathology. Moreover, the data collected were processed by a supervised machine learning model to make the prediction. Results the developed test combining circulating non-haematological cell proliferation data and artificial intelligence shows 98.8% of accuracy, 100% sensitivity, and 95% specificity. Conclusion this proof of concept study demonstrates that integration of innovative non invasive methods and predictive-models can be decisive in assessing the health status of an individual, and achieve cutting-edge results in cancer prevention and management.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin’s solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin’s fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

Multigenic tests represent an essential tool for the selection of adjuvant therapy in estrogen-positive/HER2-negative (ER + /HER2-) early breast cancer (BC). The workflow of these tests, either if they are externalized or carried out in-house, generates a workload for the pathology laboratories, that is often underestimated and may affect timely therapy initiation. Here, we describe the evolving role of pathology laboratories in using multigenic tests and, more in general, in providing adequate tissue for molecular analyses. Moreover, we propose a “reflex testing” model, in which pathologists, based on pre-specified and shared criteria, are expected to action multigene testing independently of multidisciplinary team discussion in ER + /HER2- BC patients, in order to optimize turnaround time and proper therapy intervention.

Abstract 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.

Abstract The onset of multiple and metachronous tumors in young patients induces to suspect the presence of genetic variants in genes associated with tumorigenesis. We describe here the unusual case of a 16-year-old patient who developed a synchronous bifocal colorectal adenocarcinoma with distant metastases. We provide high throughput molecular characterization with whole-exome sequencing (WES) and DNA targeted sequencing of different tumoral lesions and normal tissue samples that led to unveil a germline POLE mutation (p.Ser297Cys) coexisting with the PMS2 c.2174 + 1 G > A splicing mutation. This clinical scenario defines a “POLE-LYNCH” collision syndrome, which explains the ultra-mutator phenotype observed in the tumor lesions, and the presence of MMR deficiency-associated unusual signatures. The patient was successfully treated with immune checkpoint inhibitors but subsequently developed a high-grade urothelial carcinoma cured by surgery. We complement this analysis with a transcriptomic characterization of tumoral lesions with a panel targeting 770 genes related to the tumor microenvironment and immune evasion thus getting insight on cancer progression and response to immunotherapy.

Metabolic reprogramming upon SIRT6 loss induces tumour formation in the intestine but the mechanism is unclear. Here, the authors show that loss of SIRT6 leads to the expansion of epithelial cells with high pyruvate dehydrogenase kinase activity resulting in enhanced stem cell activity and tumour-initiating potential

Background: We report here for the first time, a comprehensive characterization of biological and clinical features of early-stage triple negative Invasive Lobular Carcinomas(TN-ILCs) Methods: We analyzed all consecutive patients with early-stage TN-ILC operated at two reference cancer-centers between 1994 and 2012.Primary objective was to assess the invasive disease-free survival(iDFS).Co-primary objective was to assess biological features of TN-ILCs, including molecular intrinsic subtypes based on PAM-50 assay, expression of androgen receptor (AR) and mutational status of ERBB2-gene.Additionally, DNA mutational status of an independent cohort of 45 TN-ILCs from three databases were analyzed, to confirm mutations in ERBB2-gene and to identify other recurrently mutated genes. Results: Among 4152 ILCs, 74(1.8%) were TN and were analyzed.The iDFS at 5 and 10 years of FUP were 50.4%(95%CI,38.0–61.6) and 37.2%(95%CI,25.5–48.8), respectively.The molecular subtype was defined through PAM50-classifier for 31 out of 74 TN-ILCs: 48% were Luminal-A(15/31), 3% luminal-B(1/31), 32% HER2-enriched (10/31), and only 16% basal-like(5/31).Luminal tumors expressed AR more frequently than non-luminal tumors (AR≥1% in 94% of luminal tumors versus 53% in non-luminal tumors; p-value = 0.001).20% of TN-ILCs analyzed(7/35), harbored a pathogenetic and actionable mutation in the ERBB2-gene. Analysis of the independent cohort of 45 TN-ILCs from three different databases, confirmed similar percentage of pathogenetic and actionable mutations in ERBB2-gene(20%; 9/45).Among the top 10 molecular pathways significantly enriched for recurrently mutated genes in TN-ILCs(FDR

Genomic surveillance of SARS-CoV-2 is crucial to monitor the spread of variants of concern. A new sequencing method enables cost-effective SARS-CoV-2 genomic surveillance at scale and is easily adaptable to other viruses.

Cancer of unknown primary (CUP) is a mysterious malignancy featuring metastatic dissemination in the absence of a recognizable primary tumor. By characterizing CUP cancer stem cells we show that self-sustained long-term propagation and sensitivity to MEK inhibition are CUP common features.

Abstract Background Many oncologists debate if lobular neoplasia (LN) is a risk factor or an obligatory precursor of more aggressive disease. This study has three aims: (i) describe the different treatment options (surgical resection vs observation), (ii) investigate the upgrade rate in surgically treated patients, and (iii) evaluate the long-term occurrences of aggressive disease in both operated and unoperated patients. Methods A series of 122 patients with LN bioptic diagnosis and follow-up information were selected. Clinical, radiological, and pathological data were collected from medical charts. At definitive histology, either invasive or ductal carcinoma in situ was considered upgraded lesions. Results Atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), and high-grade LN (HG-LN) were diagnosed in 44, 63, and 15 patients, respectively. The median follow-up was 9.5 years. Ninety-nine patients were surgically treated, while 23 underwent clinical-radiological follow-up. An upgrade was observed in 28/99 (28.3%). Age ≥ 54 years (OR 4.01, CI 1.42–11.29, p = 0.009), Breast Imaging-Reporting and Data System (BI-RADS) categories 4–5 (OR 3.76, CI 1.37–10.1, p = 0.010), and preoperatory HG-LN diagnosis (OR 8.76, 1.82–42.27, p = 0.007) were related to upgraded/aggressive disease. During follow-up, 8 patients developed an ipsilateral malignant lesion, four of whom were not initially operated (4/23, 17%). Conclusions BI-RADS categories 4–5, HG-LN diagnosis, and age ≥ 54 years were features associated with an upgrade at definitive surgery. Moreover, 17% of unoperated cases developed an aggressive disease, emphasizing that LN patients need close surveillance due to the long-term risk of breast cancer.

Abstract Single-cell RNA sequencing (scRNAseq) is an essential tool to investigate cellular heterogeneity. Thus, it would be of great interest being able to disclose biological information belonging to cell subpopulations, which can be defined by clustering analysis of scRNAseq data. In this manuscript, we report a tool that we developed for the functional mining of single cell clusters based on Sparsely-Connected Autoencoder (SCA). This tool allows uncovering hidden features associated with scRNAseq data. We implemented two new metrics, QCC (Quality Control of Cluster) and QCM (Quality Control of Model), which allow quantifying the ability of SCA to reconstruct valuable cell clusters and to evaluate the quality of the neural network achievements, respectively. Our data indicate that SCA encoded space, derived by different experimentally validated data (TF targets, miRNA targets, Kinase targets, and cancer-related immune signatures), can be used to grasp single cell cluster-specific functional features. In our implementation, SCA efficacy comes from its ability to reconstruct only specific clusters, thus indicating only those clusters where the SCA encoding space is a key element for cells aggregation. SCA analysis is implemented as module in rCASC framework and it is supported by a GUI to simplify it usage for biologists and medical personnel.

Abstract Background HER2-targeting agents have dramatically changed the therapeutic landscape of HER2+ advanced breast cancer (ABC). Within a short time frame, the rapid introduction of new therapeutics has led to the approval of pertuzumab combined with trastuzumab and a taxane in first-line, and trastuzumab emtansine (T-DM1) in second-line. Thereby, evidence of T-DM1 efficacy following trastuzumab/pertuzumab combination is limited, with data from some retrospective reports suggesting lower activity. The purpose of the present study is to investigate T-DM1 efficacy in pertuzumab-pretreated and pertuzumab naïve HER2 positive ABC patients. We also aimed to provide evidence on the exposure to different drugs sequences including pertuzumab and T-DM1 in HER2 positive cell lines. Methods The biology of HER2 was investigated in vitro through sequential exposure of resistant HER2 + breast cancer cell lines to trastuzumab, pertuzumab, and their combination. In vitro experiments were paralleled by the analysis of data from 555 HER2 + ABC patients treated with T-DM1 and evaluation of T-DM1 efficacy in the 371 patients who received it in second line. Survival estimates were graphically displayed in Kaplan Meier curves, compared by log rank test and, when possibile, confirmed in multivariate models. Results We herein show evidence of lower activity of T-DM1 in two HER2+ breast cancer cell lines resistant to trastuzumab+pertuzumab, as compared to trastuzumab-resistant cells. Lower T-DM1 efficacy was associated with a marked reduction of HER2 expression on the cell membrane and its nuclear translocation. HER2 downregulation at the membrane level was confirmed in biopsies of four trastuzumab/pertuzumab-pretreated patients. Among the 371 patients treated with second-line T-DM1, median overall survival (mOS) from diagnosis of advanced disease and median progression-free survival to second-line treatment (mPFS2) were 52 and 6 months in 177 patients who received trastuzumab/pertuzumab in first-line, and 74 and 10 months in 194 pertuzumab-naïve patients (p = 0.0006 and 0.03 for OS and PFS2, respectively). Conclusions Our data support the hypothesis that the addition of pertuzumab to trastuzumab reduces the amount of available plasma membrane HER2 receptor, limiting the binding of T-DM1 in cancer cells. This may help interpret the less favorable outcomes of second-line T-DM1 in trastuzumab/pertuzumab pre-treated patients compared to their pertuzumab-naïve counterpart.

Aim: To analyse the role of repeated breast surgery (RBS) after breast conserving surgery (BCS) as a quality indicator in a consecutive series of breast cancer patients. Methods: Data from 1233 breast cancer patients submitted to BCS from 2015 to 2019 were reviewed. The influence of several variables on RBS rate (182/1232; 14.8%) was examined. Univariate and multivariate analyses were conducted to look for significant associations with the risk of RBS. Results: Surgical workload, BCS rate and clinicopathological variables were consistent over the study period, while RBS rate decreased after the introduction of shaving of cavity margins (from 17.9% to 9.5%). Tumor persistence at RBS was higher for mastectomy vs. re-excision (87.3% vs. 37.8%; p = 0.05), inconclusive vs. positive diagnostic biopsy (48.2% vs. 69.4%; p = 0.003), ductal carcinoma in situ vs. invasive carcinoma (69.0% vs. 51.3%; p = 0.046) and lower after neoadjuvant therapy (14.3% vs. 57.8%; p = 0.044). Several clinicopathological variables were associated with the risk of RBS, but only multifocality [Odds Ratio (OR): 1.8; p = 0.009], microcalcifications (OR: 2.0, p = 0.000), neoadjuvant therapy (OR: 0.4; p = 0.014), pathological intraoperative assessment (OR: 0.6; p = 0.010) and shaving of cavity margins (OR: 0.3; p = 0.000) retained independent value at multivariate analysis. Conclusions: RBS rate can be reduced by shaving of cavity margins. Current standards for RBS should not be made more stringent due to the existence of non-actionable risk factors. The value of RBS as a quality indicator should be scrutinzed.

Abstract Cancer of unknown primary (CUP) is an obscure disease characterized by multiple metastases in the absence of a primary tumor. No consensus has been reached whether CUPs are simply generated from cancers that cannot be detected or whether they are the manifestation of a still unknown nosological entity. Here, we report the complete expression and genetic analysis of multiple synchronous metastases harvested at warm autopsy of a patient with CUP. The expression profiles were remarkably similar and astonishingly singular. The whole exome analysis yielded a high number of mutations present in all metastases (fully shared), additional mutations (partially shared) accumulated one after another in a series, and few private mutations were unique to each metastasis. Surprisingly, the phylogenetic trajectory linking CUP metastases was atypical, depicting a common “stream”, sprouting a series of linear “brooks”, at variance from the extensive branched evolution observed in metastases from most cancers of known origin. The distinctive genetic and evolutionary features depicted suggest that CUP is a novel nosological entity.

This article is an update of the requirements of a specialist breast centre, produced by EUSOMA and endorsed by ECCO as part of Essential Requirements for Quality Cancer Care (ERQCC) programme, and ESMO.To meet aspirations for comprehensive cancer control, healthcare organisations must consider the requirements in this article, paying particular attention to multidisciplinarity and patient-centred pathways from diagnosis, to treatment, to survivorship. • The centrepiece of this article is the requirements section, comprising definitions; multidisciplinary structure; minimum case, procedure and staffing volumes; and detailed descriptions of the skills of, and resources needed by, members and specialisms in the multidisciplinary team in a breast centre. • These requirements are positioned within narrative on European breast cancer epidemiology, the standard of care, challenges to delivering this standard, and supporting evidence, to enable a broad audience to appreciate the importance of establishing these requirements in specialist breast centres.

Abstract Background Central nervous system (CNS) spreading from epithelial ovarian carcinoma (EOC) is an uncommon but increasing phenomenon. We previously reported in a small series of 11 patients a correlation between Androgen Receptor (AR) loss and localization to CNS. Aims of this study were: to confirm a predictive role of AR loss in an independent validation cohort; to evaluate if AR status impacts on EOC survival. Results We collected an additional 29 cases and 19 controls as validation cohort. In this independent cohort at univariate analysis, cases exhibited lower expression of AR, considered both as continuous (p

Genomics DNA library preparation from formalin-fixed paraffin-embedded tissues is challenging. Here the authors describe CUTseq that uses restriction enzymes and in vitro amplification to barcode samples for reduced representation genome sequencing.

Background: Tubular carcinoma (TC) is a low proliferative grade 1 (G1) breast cancer (BC). Despite its favorable outcome and allegedly lower aggressiveness, patients are treated like other luminal G1 BC, with radiotherapy (RT) and hormonal therapy (HT). We performed: (1) a retrospective study comparing a TC cohort and a control series of luminal G1 BC and (2) a systematic review and meta-analysis focused on TC outcome.Materials and Methods: We selected a series of 572 G1 luminal BC patients [111 TC, 350 not otherwise specified (NOS), and 111 special-type (ST) BC] with follow-up and clinico-pathological data, who underwent local excision followed by RT at Città della Salute e della Scienza Hospital, Turin. Moreover, 22 and 13 studies were included in qualitative and quantitative meta-analysis, respectively.Results: TCs were generally smaller (≤10 mm) (P < 0.001), with lower lymph node involvement (P < 0.001). TCs showed no local and/or distant recurrences, while 16 NOS and 2 ST relapsed (P = 0.036). Kaplan–Meier curves confirmed more favorable TC outcome (DFI: log-rank test P = 0.03). Meta-analysis data, including the results of our study, showed that the pooled DFI rate was 96.4 and 91.8% at 5 and 10 years, respectively. Meta-regression analyses did not show a significant influence of RT nor HT on the DFI at 10 years.Conclusions: Compared to the other G1 BCs, TCs have an excellent outcome. The meta-analysis shows that TC recurrences are infrequent, and HT and RT have limited influence on prognosis. Hence, accurate diagnosis of TC subtype is critical to ensuring a tailored treatment approach.

Background: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. Methods: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. Results: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. Conclusions: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate.

Purpose 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.Methods miR-100 expression was studied in 90 patients with oestrogen-receptor-positive/human-epidermal growth factor receptor 2-negative breast cancer enrolled in a prospective study of endocrine therapy given either preoperatively, or for the treatment of de novo metastatic disease. Response was defined as a Ki67 ≤2.7% after 21±3 days of treatment. The prognostic role of miR-100 expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) breast cancer datasets. Additionally, we explored the correlation between miR-100 and the expression its targets reported as being associated with endocrine resistance. Finally, we evaluated whether a signature based on miR-100 and its target genes could predict the luminal A molecular subtype.Results Baseline miR-100 was significantly anticorrelated with baseline and post-treatment Ki67 (p

Background HER2 is a therapeutic target for metastatic colorectal cancer (mCRC), as demonstrated in the pivotal HERACLES-A (HER2 Amplification for Colo-rectaL cancer Enhanced Stratification) trial with trastuzumab and lapatinib. The aim of HERACLES-B trial is to assess the efficacy of the combination of pertuzumab and trastuzumab-emtansine (T-DM1) in this setting.Methods HERACLES-B was a single-arm, phase II trial, in patients with histologically confirmed RAS/BRAF wild-type and HER2+ mCRC refractory to standard treatments. HER2 positivity was assessed by immunohistochemistry and in situ hybridisation according to HERACLES criteria. Patients were treated with pertuzumab (840 mg intravenous load followed by 420 mg intravenous every 3 weeks) and T-DM1 (3.6 mg/kg every 3 weeks) until disease progression or toxicity. Primary and secondary end points were objective response rate (ORR) and progression-free survival (PFS). With a Fleming/Hern design (H0=ORR 10%; α=0.05; power=0.85), 7/30 responses were required to demonstrate an ORR ≥30% (H1).Results Thirty-one patients, 48% with ≥4 lines of previous therapies, were treated and evaluable. ORR was 9.7% (95% CI: 0 to 28) and stable disease (SD) 67.7% (95% CI: 50 to 85). OR/SD ≥4 months was associated with higher HER2 immunohistochemistry score (3+ vs 2+) (p = 0.03). Median PFS was 4.1 months (95% CI: 3.6 to 5.9). Drug-related grade (G) 3 adverse events were observed in two patients (thrombocytopaenia); G≤2 AE in 84% of cycles (n = 296), mainly nausea and fatigue.Conclusions HERACLES-B trial did not reach its primary end point of ORR; however, based on high disease control, PFS similar to other anti-HER2 regimens, and low toxicity, pertuzumab in combination with T-DM1 can be considered for HER2+mCRC as a potential therapeutic resource.Trial registration number 2012-002128-33 and NCT03225937.

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.

Cancer of unknown primary (CUP) is an umbrella term used to classify a heterogeneous group of metastatic cancers based on the absence of an identifiable primary tumor. Clinically, CUPs are characterized by a set of distinct features comprising early metastatic dissemination in an atypical pattern, an aggressive clinical course, poor response to empiric chemotherapy and, consequently, a short life expectancy. Two opposing strategies to change the dismal prognosis for the better are pursued. On the one hand, following the traditional tissue-gnostic approach, more and more sophisticated tissue-of-origin (TOO) classifier assays are employed to push identification of the putative primary to its limits with the clear intent of allowing tumor-site specific treatment. However, robust evidence supporting its routine clinical use is still lacking, notably with two recent randomized clinical trials failing to show a patient benefit of TOO-prediction based site-specific treatment over empiric chemotherapy in CUP. On the other hand, with regards to a tissue-agnostic strategy, precision medicine approaches targeting actionable genomic alterations have already transformed the treatment for many known tumor types. Yet, an unmet need remains for well-designed clinical trials to scrutinize its potential role in CUP beyond anecdotal case reports. In the absence of practice changing results, we believe that the emphasis on finding the presumed unknown primary tumor at all costs, implicit in the term CUP, has biased recent research in the field. Focusing on the distinct clinical features shared by all CUPs, we advocate adopting the term primary metastatic cancer (PMC) to denominate a distinct cancer entity instead. In our view, PMC should be considered the archetype of metastatic disease and as such, despite accounting for a mere 2–3% of malignancies, unraveling the mechanisms at play goes beyond improving the prognosis of patients with PMC and promises to greatly enhance our understanding of the metastatic process and carcinogenesis across all cancer types.

Abstract The onset of secondary resistance represents a major limitation to long‐term efficacy of target therapies in cancer patients. Thus, the identification of mechanisms mediating secondary resistance is the key to the rational design of therapeutic strategies for resistant patients. MiRNA profiling combined with RNA‐Seq in MET‐addicted cancer cell lines led us to identify the miR‐205/ERRFI1 (ERBB receptor feedback inhibitor‐1) axis as a novel mediator of resistance to MET tyrosine kinase inhibitors (TKIs). In cells resistant to MET‐TKIs, epigenetically induced miR‐205 expression determined the downregulation of ERRFI1 which, in turn, caused EGFR activation, sustaining resistance to MET‐TKIs. Anti‐miR‐205 transduction reverted crizotinib resistance in vivo, while miR‐205 over‐expression rendered wt cells refractory to TKI treatment. Importantly, in the absence of EGFR genetic alterations, miR‐205/ERRFI1‐driven EGFR activation rendered MET‐TKI‐resistant cells sensitive to combined MET/EGFR inhibition. As a proof of concept of the clinical relevance of this new mechanism of adaptive resistance, we report that a patient with a MET‐amplified lung adenocarcinoma displayed deregulation of the miR‐205/ERRFI1 axis in concomitance with onset of clinical resistance to anti‐MET therapy.

Abstract Background The role of forkhead-box A1 (FOXA1) and Androgen receptor (AR) in breast cancer (BC) has been extensively studied. However, the prognostic role of their co-expression in Estrogen receptor positive (ER+) BC has not been investigated so far. The aim of the present study was thus to assess the co-expression (protein and mRNA) of FOXA1 and AR in BC patients, in order to evaluate their prognostic impact according to ER status. Methods Immunohistochemical expression of AR and FOXA1 was evaluated on 479 consecutive BC, with complete clinical-pathological and follow up data. Fresh-frozen tissues from 65 cases were available. The expression of AR and FOXA1 with ER was validated using mRNA analyses. Survival and Cox proportional hazard analyses were used to evaluate the relationship between FOXA1, AR and prognosis. Results Expression of ER, AR and FOXA1 was observed in 78, 60 and 85% of cases respectively. Most AR+ cases (97%) were also FOXA1+. The level of FOXA1 mRNA positively correlated with level of both AR mRNA (r = 0.8975; P

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer.More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted.Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment.Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

Abstract Background This is a retrospective cross sectional study aimed to verify whether Multiplex Ligation-dependent Probe Amplification (MLPA), a quantitative molecular assay, may represent a valuable reflex test in breast cancer with equivocal HER2 expression by immunohistochemistry and HER2 gene signals/nucleus (s/n) ranging between 4.0 and 5.9 by in situ hybridization. Methods A series of 170 breast carcinomas scored as 2+ for HER2 expression by immunohistochemistry, were selected from our files and analyzed in parallel by silver in situ hybridization and by MLPA. According to ASCO-CAP 2013 guidelines, 54/170 tumors, displaying 4.0–5.9 HER2 gene s/n, were defined as low amplified (ratio ≥ 2) or equivocal (ratio 2.0. These data were further confirmed in the external validation set. Interestingly, the 54 low amplified/equivocal breast carcinomas presented a frequency of hormonal receptor positivity significantly higher than that observed in the amplified tumors and similar to the non-amplified one (p = 0.016 for estrogen receptor and p = 0.001 for progesterone receptor). Conclusions To avoid to offer patients an ineffective therapy, HER2 status should be studied more thoroughly in low amplified and equivocal cases which can have lower response rates and shorter time to progression to trastuzumab. In this context, our data indicate that MLPA may be a reliable, objective supporting test in selecting HER2 positive breast cancer patients.

Abstract Background Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. Patients and methods We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. Results The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. Significance qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.

p140Cap adaptor proteins interfere with adhesion and growth factor-dependent signalling in cancer cells but the mechanisms are unclear. Here the authors show that p140Cap interferes with ERBB2-dependent activation of Rac GTPase-controlled circuitries reducing metastasis and cancer progression.

The co-expression of androgen (AR) and estrogen (ER) receptors, in terms of higher AR/ER ratio, has been recently associated with poor outcome in ER-positive (ER+) breast cancer (BC) patients. The aim of this study was to analyze if the biological aggressiveness, underlined in ER+ BC tumors with higher AR/ER ratio, could be due to higher expression of genes related to cell proliferation. On a cohort of 47 ER+ BC patients, the AR/ER ratio was assessed by immunohistochemistry and by mRNA analysis. The expression level of five gene proliferation markers was defined through TaqMan®-qPCR assays. Results were validated using 979 BC cases obtained from gene expression public databases. ER+ BC tumors with ratios of AR/ER ≥ 2 have higher expression levels of cellular proliferation genes than tumors with ratios of AR/ER < 2, in both the 47 ER+ BC patients (P < 0.001) and in the validation cohort (P = 0.005). Moreover, BC cases with ratios of AR/ER ≥ 2 of the validation cohort were mainly assigned to luminal B and HER2-enriched molecular subtypes, typically characterized by higher proliferation and poorer prognosis. These data suggest that joint routine evaluation of AR and ER expression may identify a unique subset of tumors, which show higher levels of cellular proliferation and therefore a more aggressive behavior.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

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.We retrospectively analyzed a case series of 139 patients with NDc who underwent breast surgery. The clinical and histological findings, types of surgery with emphasis on nipple-areola complex amputation, immunohistochemical phenotypes of the carcinomas and measurements of the tumor-nipple distance were recorded. Additionally, in patients who showed HER2-positive lesions on definitive surgery, we evaluated the HER2 immunocytochemistry of the NDc smears.Thirty-two malignant and 107 benign/borderline NDc diagnoses were identified. All 32 malignant-NDc cases were histologically confirmed as malignant. Thirty borderline/benign-NDc cases were histologically diagnosed as malignant (sensitivity 58%). The majority of the patients with malignant NDc were treated with nipple-areola complex amputations in both the mastectomy and conservative surgery groups (P

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.

In Italy, 5200 new ovarian cancers were diagnosed in 2018, highlighting an increasing need to test women for BRCA1/2. The number of labs offering this test is continuously increasing. The aim of this study was to show the results coming from the intersociety survey coordinated by four different Clinical and Laboratory Italian Scientific Societies (AIOM, SIAPEC-IAP, SIBIOC, and SIGU). A multidisciplinary team belonging to the four scientific societies drew up two different questionnaires: One was targeted toward all Italian Departments of Medical Oncology, and the second toward laboratories of clinical molecular biology. This survey was implemented from September 2017 to March 2018. Seventy-seven out of 305 (25%) Departments of Medical Oncology filled our survey form. Indeed, 59 molecular laboratories were invited. A total of 41 laboratories (70%) filled in the questionnaire. From 2014 to 2017, 16 new molecular laboratories were activated. A total of 12,559 tests were performed in the year 2016, with a mean of 339 tests and a median of 254 tests per laboratory, showing a glimpse of an extreme low number of tests performed per year by some laboratories. In terms of the type and number of professionals involved in the pre- and post-test counseling, results among the onco-genetic team were heterogeneous. Our data show that the number of laboratories providing BRCA1/2 germline assays is significantly increased with further implementation of the somatic test coming soon. The harmonization of the complete laboratory diagnostic path should be encouraged, particularly in order to reduce the gap between laboratories with high and low throughput.

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco's Modified Eagle's Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.

INTRODUCTION:Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs). METHODS:One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted. RESULTS:RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively). CONCLUSION:RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

Nature Communications 8: Article number: 14797 (2017); Published online 16 March 2017; Updated 30 March 2018 In the original version of this Article, the affiliation details for Anna Sapino were incorrectly given as Department of Medical Sciences, University of Torino, 10126 Torino, Italy instead ofCandiolo Cancer Institute-FPO, IRCCS, Str.

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.

BACKGROUND: Recent studies have demonstrated that axillary lymph node dissection (ALND) does not affect patient survival, even in those with one or two positive sentinel lymph nodes (SLNs). On the other hand, patients with 3 or more metastatic lymph nodes are eligible for chemotherapy. Therefore, it is crucial to identify a priori patients at risk of having a high number of metastatic axillary lymph nodes for their surgical and/or clinical management. Ultrasound (US) guided Fine-Needle Aspiration (FNA) has been proven to be a useful and highly specific method for detecting metastatic axillary lymph nodes. However, only one recent study has evaluated the efficiency of this method in identifying patients with high metastatic nodal involvement. Our aim was to validate US-guided FNA as a reliable method to discriminate a priori patients with >3 metastatic lymph nodes. METHODS: A retrospective series of 1287 breast cancer patients who underwent a simultaneous preoperative breast and axillary US to stage their axilla was collected. A total of 365 patients, with either positive SLNs (278) or positive axillary lymph nodes detected via US-guided FNA (87), underwent ALND. In these two subgroups, we compared the number of metastatic lymph nodes in the axilla. RESULTS: The number of metastatic axillary lymph nodes in patients who underwent US-guided FNA was significantly higher (63% had >3 metastatic lymph nodes) than that in patients with SLNs positive for micro- or macrometastases (3% and 27%, respectively) (P

Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84-100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.