Tissue Fixation with a Formic Acid-Deprived Formalin Better Preserves DNA Integrity over Time.

INTRODUCTION: Optimization of pre-analytic procedures and tissue processing is a basic requirement for reliable and reproducible data to be obtained. Tissue fixation in formalin represents the extensively favored method for surgical tissue specimen processing in diagnostic pathology; however, formalin fixation exerts a blasting effect on DNA and RNA. METHODS: A formic acid-deprived formaldehyde solution was prepared by removing acids with an ion-exchange basic resin and the concentrated, acid-deprived formaldehyde (ADF) solution was employed to prepare a 4% ADF solution in 0.1 M phosphate buffer, pH 7.2-7.4. Human (n = 27) and mouse (n = 20) tissues were fixed in parallel and similar conditions in either ADF or neutral buffered formalin (NBF). DNAs and RNAs were extracted, and fragmentation analyses were performed. RESULTS: Besides no significant differences in terms of extraction yield and absorbance ratio, ADF fixation reduced DNA fragmentation, i.e., the largest fragments (>5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (p < 0.001 in both cohorts). Moreover, we observed that DNA preservation is more stable in ADF-fixed tissue compared to NBF-fixed tissues. CONCLUSION: Although DNA fragmentation in FFPE tissues is a multifactor process, we showed that the removal of formic acid is responsible for a significant improvement in DNA preservation.

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.

Unusual Patterns of HER2 Expression in Breast Cancer: Insights and Perspectives.

The biomarker human epidermal growth factor receptor-2 (HER2) has represented the best example of successful targeted therapy in breast cancer patients. Based on the concept of "oncogene addiction," we have learnt how to identify patients likely benefitting from anti-HER2 agents. Since HER2 gene amplification leads to marked overexpression of the HER2 receptors on the cell membrane, immunohistochemistry with clinically validated antibodies and scoring system based on intensity and completeness of the membranous expression constitute the screening method to separate negative (score 0/1+) and positive (score 3+) carcinomas and to identify those tumours with complete yet only moderate HER2 expression (score 2+, equivocal carcinomas), which need to be investigated further in terms of gene status to confirm the presence of a loop of oncogene addiction. This process has demanded quality controls and led to recommendations by Scientific Societies, which pathologists routinely need to follow to guarantee reproducibility. In this review, we will span from the description of classical HER2 evaluation to the discussion of those scenarios in which HER2 expression is unusual and/or difficult to define. We will dissect HER2 heterogeneity, HER2 conversion from primary to relapsed/metastatic breast cancer, and we will introduce the new category of HER2-low breast carcinomas.

Molecular Characterization of Prostate Cancers in the Precision Medicine Era.

Prostate cancer (PCa) therapy has been recently revolutionized by the approval of new therapeutic agents in the metastatic setting. However, the optimal therapeutic strategy in such patients should be individualized in the light of prognostic and predictive molecular factors, which have been recently studied: androgen receptor (AR) alterations, PTEN-PI3K-AKT pathway deregulation, homologous recombination deficiency (HRD), mismatch repair deficiency (MMRd), and tumor microenvironment (TME) modifications. In this review, we highlighted the clinical impact of prognostic and predictive molecular factors in PCa patients' outcomes, identifying biologically distinct subtypes. We further analyzed the relevant methods to detect these factors, both on tissue, i.e., immunohistochemistry (IHC) and molecular tests, and blood, i.e., analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Moreover, we discussed the main pros and cons of such techniques, depicting their present and future roles in PCa management, throughout the precision medicine era.

Pursuit of Gene Fusions in Daily Practice: Evidence from Real-World Data in Wild-Type and Microsatellite Instable Patients.

Agnostic biomarkers such as gene fusions allow to address cancer patients to targeted therapies; however, the low prevalence of these alterations across common malignancies poses challenges and needs a feasible and sensitive diagnostic process. RNA-based targeted next generation sequencing was performed on 125 samples of patients affected either by colorectal carcinoma, melanoma, or lung adenocarcinoma lacking genetic alterations in canonical driver genes, or by a colorectal carcinoma with microsatellite instability. Gene fusion rates were compared with in silico data from MSKCC datasets. For NTRK gene fusion detection we also employed a multitarget qRT-PCR and pan-TRK immunohistochemistry. Gene fusions were detected in 7/55 microsatellite instable colorectal carcinomas (12.73%), and in 4/70 of the "gene driver free" population (5.71%: 3/28 melanomas, 10.7%, and 1/12 lung adenocarcinomas, 8.3%). Fusion rates were significantly higher compared with the microsatellite stable and "gene driver positive" MSKCC cohorts. Pan-TRK immunohistochemistry showed 100% sensitivity, 91.7% specificity, and the occurrence of heterogeneous and/or subtle staining patterns. The enrichment of gene fusions in this "real-world" cohort highlights the feasibility of a workflow applicable in clinical practice. The heterogeneous expression in NTRK fusion positive tumours unveils challenging patterns to recognize and raises questions on the effective translation of the chimeric protein.

Basic principles of biobanking: from biological samples to precision medicine for patients.

The term "biobanking" is often misapplied to any collection of human biological materials (biospecimens) regardless of requirements related to ethical and legal issues or the standardization of different processes involved in tissue collection. A proper definition of biobanks is large collections of biospecimens linked to relevant personal and health information (health records, family history, lifestyle, genetic information) that are held predominantly for use in health and medical research. In addition, the International Organization for Standardization, in illustrating the requirements for biobanking (ISO 20387:2018), stresses the concept of biobanks being legal entities driving the process of acquisition and storage together with some or all of the activities related to collection, preparation, preservation, testing, analysing and distributing defined biological material as well as related information and data. In this review article, we aim to discuss the basic principles of biobanking, spanning from definitions to classification systems, standardization processes and documents, sustainability and ethical and legal requirements. We also deal with emerging specimens that are currently being generated and shaping the so-called next-generation biobanking, and we provide pragmatic examples of cancer-associated biobanking by discussing the process behind the construction of a biobank and the infrastructures supporting the implementation of biobanking in scientific research.

Identification of TENM4 as a Novel Cancer Stem Cell-Associated Molecule and Potential Target in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is insensitive to endocrine and Her2-directed therapies, making the development of TNBC-targeted therapies an unmet medical need. Since patients with TNBC frequently show a quicker relapse and metastatic progression compared to other breast cancer subtypes, we hypothesized that cancer stem cells (CSC) could have a role in TNBC. To identify putative TNBC CSC-associated targets, we compared the gene expression profiles of CSC-enriched tumorspheres and their parental cells grown as monolayer. Among the up-regulated genes coding for cell membrane-associated proteins, we selected Teneurin 4 (TENM4), involved in cell differentiation and deregulated in tumors of different histotypes, as the object for this study. Meta-analysis of breast cancer datasets shows that TENM4 mRNA is up-regulated in invasive carcinoma specimens compared to normal breast and that high expression of TENM4 correlates with a shorter relapse-free survival in TNBC patients. TENM4 silencing in mammary cancer cells significantly impaired tumorsphere-forming ability, migratory capacity and Focal Adhesion Kinase (FAK) phosphorylation. Moreover, we found higher levels of TENM4 in plasma from tumor-bearing mice and TNBC patients compared to the healthy controls. Overall, our results indicate that TENM4 may act as a novel biomarker and target for the treatment of TNBC.

Evolving concepts in HER2 evaluation in breast cancer: Heterogeneity, HER2-low carcinomas and beyond.

The human epidermal growth factor receptor 2 (HER2) is a well-known negative prognostic factor in breast cancer and a target of the monoclonal antibody trastuzumab as well as of other anti-HER2 compounds. Pioneering works on HER2-positive breast cancer in the 90s' launched a new era in clinical research and oncology practice that has reshaped the natural history of this disease. In diagnostic pathology the HER2 status is routinely assessed by using a combination of immunohistochemistry (IHC, to evaluate HER2 protein expression levels) and in situ hybridization (ISH, to assess HER2 gene status). For this purpose, international recommendations have been developed by a consensus of experts in the field, which have changed over the years according to new experimental and clinical data. In this review article we will document the changes that have contributed to a better evaluation of the HER2 status in clinical practice, furthermore we will discuss HER2 heterogeneity defined by IHC and ISH as well as by transcriptomic analysis and we will critically describe the complexity of HER2 equivocal results. Finally, we will introduce the clinical impact of HER2 mutations and we will define the upcoming category of HER2-low breast cancer with respect to emerging clinical data on the efficacy of specific anti-HER2 agents in subgroups of breast carcinomas lacking the classical oncogene addition dictated by HER2 amplification.

The Oncocytic Variant of Poorly Differentiated Thyroid Carcinoma Shows a Specific Immune-Related Gene Expression Profile.

BACKGROUND: Poorly differentiated thyroid cancer (PDTC) is a rare, follicular cell-derived neoplasm with an unfavorable prognosis. The oncocytic variant of PDTC may be associated with even more adverse outcome than classical PDTC cases, but its specific molecular features are largely unknown. Our aim was to explore the immune-related gene expression profile of oncocytic and classical PDTC, in correlation with clinical and pathological characteristics (including programmed death ligand 1 [PD-L1] expression) and outcome, and in comparison with a control group of well-differentiated follicular carcinomas (WDFCs), including conventional follicular carcinomas (FTCs) and Hürthle cell carcinomas (HCCs). METHODS: A retrospective series of 48 PDTCs and 24 WDFCs was analyzed by means of NanoString technology employing the nCounter PanCancer Immune Profiling panel. Gene expression data were validated using quantitative real-time polymerase chain reaction. RESULTS: Oncocytic PDTCs showed a specific immune-related gene expression profile, with higher expression of LAIR2, CD274, DEFB1, IRAK1, CAMP, LCN2, LY96, and APOE, and lower expression of NOD1, as compared to conventional PDTCs. This molecular signature was associated with increased intratumoral lymphocytic infiltration, PD-L1 expression, and adverse outcome. Three of these genes, CD274, DEFB1, and IRAK1, as well as PD-L1 expression, were also the hallmarks of HCCs as compared to FTCs. By contrast, the panel of genes differentially regulated in PDTCs as compared to WDFCs was unrelated to the oncocytic phenotype. CONCLUSIONS: Our results revealed a distinctive immune-related gene expression profile of oncocytic PDTC and confirmed a more aggressive outcome in this cancer subtype. These findings may provide guidance when exploring novel immunotherapeutic options for oncocytic PDTC patients.

DSCAM-AS1-Driven Proliferation of Breast Cancer Cells Involves Regulation of Alternative Exon Splicing and 3'-End Usage.

DSCAM-AS1 is a cancer-related long noncoding RNA with higher expression levels in Luminal A, B, and HER2-positive Breast Carcinoma (BC), where its expression is strongly dependent on Estrogen Receptor Alpha (ERα). DSCAM-AS1 expression is analyzed in 30 public datasets and, additionally, by qRT-PCR in tumors from 93 BC patients, to uncover correlations with clinical data. Moreover, the effect of DSCAM-AS1 knockdown on gene expression and alternative splicing is studied by RNA-Seq in MCF-7 cells. We confirm DSCAM-AS1 overexpression in high grade Luminal A, B, and HER2+ BCs and find a significant correlation with disease relapse. In total, 908 genes are regulated by DSCAM-AS1-silencing, primarily involved in the cell cycle and inflammatory response. Noteworthily, the analysis of alternative splicing and isoform regulation reveals 2085 splicing events regulated by DSCAM-AS1, enriched in alternative polyadenylation sites, 3'UTR (untranslated region) shortening and exon skipping events. Finally, the DSCAM-AS1-interacting splicing factor heterogeneous nuclear ribonucleoprotein L (hnRNPL) is predicted as the most enriched RBP for exon skipping and 3'UTR events. The relevance of DSCAM-AS1 overexpression in BC is confirmed by clinical data and further enhanced by its possible involvement in the regulation of RNA processing, which is emerging as one of the most important dysfunctions in cancer.

Prognostic role of PD-L1 and immune-related gene expression profiles in giant cell tumors of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastatic tumor, with a relatively unpredictable clinical course. A retrospective series of 46 GCTB and a control group of 24 aneurysmal bone cysts (ABC) were selected with the aim of investigating the PD-L1 expression levels and immune-related gene expression profile, in correlation with clinicopathological features. PD-L1 and Ki67 were immunohistochemically tested in each case. Furthermore, comprehensive molecular analyses were carried out using NanoString technology and nCounter PanCancer Immune Profiling Panel, and the gene expression results were correlated with clinicopathological characteristics. PD-L1 expression was observed in 13/46 (28.3%) GCTB (and in 1/24, 4.2%, control ABC, only) and associated with a shorter disease free interval according to univariate analysis. Moreover, in PD-L1-positive lesions, three genes (CD27, CD6 and IL10) were significantly upregulated (p < 0.01), while two were downregulated (LCK and TLR8, showing borderline significance, p = 0.06). Interestingly, these genes can be related to maturation and immune tolerance of bone tissue microenvironment, suggesting a more immature/anergic phenotype of giant cell tumors. Our findings suggest that PD-L1 immunoreactivity may help to select GCTB patients with a higher risk of recurrence who could potentially benefit from immune checkpoint blockade.

AR/ER Ratio Correlates with Expression of Proliferation Markers and with Distinct Subset of Breast Tumors.

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.

The Multifaceted Nature of Tumor Microenvironment in Breast Carcinomas.

Heterogeneity in breast carcinomas can be appreciated at various levels, from morphology to molecular alterations, and there are well-known genotypic-phenotypic correlations. Clinical decision-making is strictly focused on the evaluation of tumor cells and is based on the assessment of hormone receptors and of the HER2 status, by means of a combination of immunohistochemical and in situ hybridization techniques. The tumor microenvironment (TME) also shows a multifaceted nature stemming from the different actors populating the intratumoral and the peritumoral stroma of breast carcinomas. Of note, we have now evidence that tumor-infiltrating lymphocytes (TILs) are clinically meaningful as their quantification in the intratumoral stroma strongly correlates with good prognosis, in particular in triple-negative and HER2-positive breast cancer patients. Nevertheless, TILs are just one of the many actors orchestrating the complexity of the TME, which is populated by immune and non-immune cells (cancer-associated fibroblasts, cancer-associated adipocytes), as well as non-cellular components such as chemical inflammation mediators. In this review article we will overview the main features of the distinct cell compartments by discussing (i) the potential impact the TME may have on the prognostic stratification of breast cancers and (ii) the possible predictive value of some markers in the context of immunotherapy in light of the recent results of phase III studies in advanced and early triple-negative breast cancer patients.

Cold Formalin Fixation Guarantees DNA Integrity in Formalin Fixed Paraffin Embedded Tissues: Premises for a Better Quality of Diagnostic and Experimental Pathology With a Specific Impact on Breast Cancer.

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.

PAX8-GLIS3 gene fusion is a pathognomonic genetic alteration of hyalinizing trabecular tumors of the thyroid.

The hyalinizing trabecular adenoma/tumor is a rare and poorly characterized follicular-derived thyroid neoplasm recently shown to harbor recurrent PAX8-GLIS1 or PAX8-GLIS3 gene fusions. Here we sought to define the repertoire of genetic alterations of hyalinizing trabecular tumors, and whether PAX8-GLIS3 fusions are pathognomonic for hyalinizing trabecular tumors. A discovery series of eight hyalinizing trabecular tumors was subjected to RNA-sequencing (n = 8), whole-exome sequencing (n = 3) or targeted massively parallel sequencing (n = 5). No recurrent somatic mutations or copy number alterations were identified in hyalinizing trabecular tumor, whereas RNA-sequencing revealed the presence of a recurrent genetic rearrangement involving PAX8 (2q14.1) and GLIS3 (9p24.2) genes in all cases. In this in-frame fusion gene, which comprised exons 1-2 of PAX8 and exons 3-11 of GLIS3, GLIS3 is likely placed under the regulation of PAX8. Reverse transcription RT-PCR and/or fluorescence in situ hybridization analyses of a validation series of 26 hyalinizing trabecular tumors revealed that the PAX8-GLIS3 gene fusion was present in all hyalinizing trabecular tumors (100%). No GLIS1 rearrangements were identified. Conversely, no PAX8-GLIS3 gene fusions were detected in a cohort of 237 control thyroid neoplasms, including 15 trabecular thyroid lesions highly resembling hyalinizing trabecular tumor from a morphological standpoint, as well as trabecular/solid follicular adenomas, solid/trabecular variants of papillary carcinoma, and Hurthle cell adenomas or carcinomas. Our data provide evidence to suggest that the PAX8-GLIS3 fusion is pathognomonic for hyalinizing trabecular tumors, and that the presence of the PAX8-GLIS3 fusion in thyroid neoplasms may be used as an ancillary marker for the diagnosis of hyalinizing trabecular tumor, thereby avoiding overtreatment in case of misdiagnoses with apparently similar malignant tumors.

Paclitaxel-Loaded Nanosponges Inhibit Growth and Angiogenesis in Melanoma Cell Models.

This study investigated the effects of free paclitaxel (PTX) and PTX-loaded in pyromellitic nanosponges (PTX-PNS) in reducing in vitro and in vivo melanoma cell growth and invasivity, and in inhibiting angiogenesis. To test the response of cells to the two PTX formulations, the cell viability was evaluated by MTT assay in seven continuous cell lines, in primary melanoma cells, both in 2D and 3D cultures, and in human umbilical vein endothelial cells (HUVECs) after exposure to different concentrations of PTX or PTX-PNS. Cell motility was assessed by a scratch assay or Boyden chamber assay, evaluating cell migration in presence or absence of diverse concentrations of PTX or PTX-PNS. The effect of PTX and PTX-PNS on angiogenesis was evaluated as endothelial tube formation assay, a test able to estimate the formation of three-dimensional vessels in vitro. To assess the anticancer effect of PTX and PTX-PNS in in vivo experiments, the two drug formulations were tested in a melanoma mouse model obtained by B16-BL6 cell implantation in C57/BL6 mice. Results obtained were as follows: 1) MTT analysis revealed that cell proliferation was more affected by PTX-PNS than by PTX in all tested cell lines, in both 2D and 3D cultures; 2) the analysis of the cell migration showed that PTX-PNS acted at very lower concentrations than PTX; 3) tube formation assay showed that PTX-PNS were more effective in inhibiting tube formation than free PTX; and 4) in vivo experiments demonstrated that tumor weights, volumes, and growth were significantly reduced by PTX-PNS treatment with respect to PTX; the angiogenesis and the cell proliferation, detected in the tumor samples with CD31 and Ki-67 antibodies, respectively, indicated that, in the PTX-PNS-treated tumors, the tube formation was inhibited, and a low amount of proliferating cells was present. Taken together, our data demonstrated that our new PTX nanoformulation can respond to some important issues related to PTX treatment, lowering the anti-tumor effective doses and increasing the effectiveness in inhibiting melanoma growth in vivo.

Phenotypic characterisation of immune cells associated with histological regression in cutaneous melanoma.

Histological regression and tumour infiltrating lymphocytes represent an early sign of activation of the immune system against primary melanoma. The first phenomenon has been especially discussed in the literature because of its prognostic role, but no clear agreement on its evaluation has been reached. Immunotherapy of advanced stage melanoma has recently shown promising results; an improved understanding of the initial interplay between melanoma cells and the immune system would potentially help tailor treatment for patients. Seventy consecutive melanomas with regression were analysed to identify a prognostic cut-off value of regression extension. Then, we compared the immune infiltrate between regressed and not regressed areas of these regressed melanomas, assessing CD3, CD4, CD8, CD20, CD123, PD1 and FOXP3/CD25 expression. The immune infiltrate of these cases was further compared with 28 control melanomas without regression. A regression extension of 10% represented a reliable cut-off to distinguish two different risk categories in regressed melanomas. Regressed areas were less infiltrated by CD4/CD25, FOXP3/CD4 or PD1/CD4 compared to not regressed areas of each sample. These lymphocyte subsets are associated with anergy and hamper the immune CD8+ response towards the cancer cells. Moreover, the relevance of these findings was further supported by the observation that not regressed controls were significantly more infiltrated by these anergic immune cell subsets compared to the regressed cases. These results help understand the real meaning of regression in melanoma. Moreover, the association here identified between specific immunomodulatory immune cell subsets and regression could help in developing new therapeutic strategies.

Tissues under-vacuum to overcome suboptimal preservation.

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.

Eccrine spiradenoma of the nipple: Case report, differential diagnosis and literature review.

Eccrine spiradenoma is a rare lesion originating from eccrine sweat glands, with only few cases reported in breast tissue: we here describe for the first time, an eccrine spiradenoma arising in the nipple. An 84 year-old woman with a lesion enlarging her right nipple, showing ulcerations and eczema-like changes of the covering skin, was admitted to our hospital. Surgical excision of the central quadrant with nipple-areola complex was performed, followed by histopathological evaluation which revealed an adenoma with predominantly basaloid epithelial cells. The lesion was composed of tightly packed small and large groups of cells, arranged in diffuse alveolar/pseudorosette formations. The small cells expressed p63 and calponin, while a positive expression of CK7 and CD117 was detected in large cells. After careful and detailed examination, excluding various similar entities, a diagnosis of eccrine spiradenoma has been rendered. Although extremely rare, eccrine spiradenoma should be taken into account in the differential diagnosis of subcutaneous primary breast tumors.

Thymidylate synthase maintains the de-differentiated state of triple negative breast cancers.

Cancer cells frequently boost nucleotide metabolism (NM) to support their increased proliferation, but the consequences of elevated NM on tumor de-differentiation are mostly unexplored. Here, we identified a role for thymidylate synthase (TS), a NM enzyme and established drug target, in cancer cell de-differentiation and investigated its clinical significance in breast cancer (BC). In vitro, TS knockdown increased the population of CD24+ differentiated cells, and attenuated migration and sphere-formation. RNA-seq profiling indicated repression of epithelial-to-mesenchymal transition (EMT) signature genes upon TS knockdown, and TS-deficient cells showed an increased ability to invade and metastasize in vivo, consistent with the occurrence of a partial EMT phenotype. Mechanistically, TS enzymatic activity was found essential for maintenance of the EMT/stem-like state by fueling a dihydropyrimidine dehydrogenase-dependent pyrimidine catabolism. In patient tissues, TS levels were found significantly higher in poorly differentiated and in triple negative BC, and strongly correlated with worse prognosis. The present study provides the rationale to study in-depth the role of NM at the crossroads of proliferation and differentiation, and depicts new avenues for the design of novel drug combinations for the treatment of BC.