Memory CD8+ T cell diversity and B cell responses correlate with protection against SARS-CoV-2 following mRNA vaccination.

Understanding immune responses to SARS-CoV-2 messenger RNA (mRNA) vaccines is of great interest, principally because of the poor knowledge about the mechanisms of protection. In the present study, we analyzed longitudinally B cell and T cell memory programs against the spike (S) protein derived from ancestral SARS-CoV-2 (Wuhan-1), B.1.351 (beta), B.1.617.2 (delta) and B.1.1.529 (omicron) variants of concern (VOCs) after immunization with an mRNA-based vaccine (Pfizer). According to the magnitude of humoral responses 3 months after the first dose, we identified high and low responders. Opposite to low responders, high responders were characterized by enhanced antibody-neutralizing activity, increased frequency of central memory T cells and durable S-specific CD8+ T cell responses. Reduced binding antibodies titers combined with long-term specific memory T cells that had distinct polyreactive properties were found associated with subsequent breakthrough with VOCs in low responders. These results have important implications for the design of new vaccines and new strategies for booster follow-up.

Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma.

ABSTRACT: Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor-associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Multicancer screening test based on the detection of circulating non haematological proliferating atypical cells.

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.

Dataset for reporting of the invasive carcinoma of the breast: recommendations from the International Collaboration on Cancer Reporting (ICCR).

BACKGROUND AND OBJECTIVES: Current national or regional guidelines for the pathology reporting on invasive breast cancer differ in certain aspects, resulting in divergent reporting practice and a lack of comparability of data. Here we report on a new international dataset for the pathology reporting of resection specimens with invasive cancer of the breast. The dataset was produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organizations. METHODS AND RESULTS: The established ICCR process for dataset development was followed. An international expert panel consisting of breast pathologists, a surgeon, and an oncologist prepared a draft set of core and noncore data items based on a critical review and discussion of current evidence. Commentary was provided for each data item to explain the rationale for selecting it as a core or noncore element, its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. Following international public consultation, the document was finalized and ratified, and the dataset, which includes a synoptic reporting guide, was published on the ICCR website. CONCLUSIONS: This first international dataset for invasive cancer of the breast is intended to promote high-quality, standardized pathology reporting. Its widespread adoption will improve consistency of reporting, facilitate multidisciplinary communication, and enhance comparability of data, all of which will help to improve the management of invasive breast cancer patients.

A dedicated structured data set for reporting of invasive carcinoma of the breast in the setting of neoadjuvant therapy: recommendations from the International Collaboration on Cancer Reporting (ICCR).

AIMS: The International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organisations, is an initiative aimed at providing a unified international approach to reporting cancer. ICCR recently published new data sets for the reporting of invasive breast carcinoma, surgically removed lymph nodes for breast tumours and ductal carcinoma in situ, variants of lobular carcinoma in situ and low-grade lesions. The data set in this paper addresses the neoadjuvant setting. The aim is to promote high-quality, standardised reporting of tumour response and residual disease after neoadjuvant treatment that can be used for subsequent management decisions for each patient. METHODS: The ICCR convened expert panels of breast pathologists with a representative surgeon and oncologist to critically review and discuss current evidence. Feedback from the international public consultation was critical in the development of this data set. RESULTS: The expert panel concluded that a dedicated data set was required for reporting of breast specimens post-neoadjuvant therapy with inclusion of data elements specific to the neoadjuvant setting as core or non-core elements. This data set proposes a practical approach for handling and reporting breast resection specimens following neoadjuvant therapy. The comments for each data element clarify terminology, discuss available evidence and highlight areas with limited evidence that need further study. This data set overlaps with, and should be used in conjunction with, the data sets for the reporting of invasive breast carcinoma and surgically removed lymph nodes from patients with breast tumours, as appropriate. Key issues specific to the neoadjuvant setting are included in this paper. The entire data set is freely available on the ICCR website. CONCLUSIONS: High-quality, standardised reporting of tumour response and residual disease after neoadjuvant treatment are critical for subsequent management decisions for each patient.

Unique Patterns of Heterogeneous Mismatch Repair Protein Expression in Colorectal Cancer Unveil Different Degrees of Tumor Mutational Burden and Distinct Tumor Microenvironment Features.

Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8+ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.

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.

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.

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.

Pathological examination of breast cancer samples before and after neoadjuvant therapy: recommendations from the Italian Group for the Study of Breast Pathology - Italian Society of Pathology (GIPaM-SIAPeC).

Neoadjuvant therapy (NAT) in breast cancer is administered to downstage the tumor, de-escalate surgery, and provide prognostic information that can be used to tailor subsequent adjuvant therapy. In this respect, the pathological evaluation of both pre-NAT biopsies and post-NAT surgical specimens is crucial to precisely assess the treatment response. With the increasing possibilities of NAT protocols and the rising number of eligible patients, it has become extremely important to standardize the pathological response assessment. Here, we provide an update on the recommendations of the Italian Group for the Study of Breast Pathology - the Italian Society of Pathology (GIPaM-SIAPeC) for the analysis of breast cancer samples before and after NAT.

Personalized therapeutic strategies in HER2-driven gastric cancer.

BACKGROUND: Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. METHODS: We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. RESULTS: Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. CONCLUSION: These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

The lobular neoplasia enigma: management and prognosis in a long follow-up case series.

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.

"Borderline" epithelial lesions of the breast: what have we learned in the past three decades?

Atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH) and flat epithelial atypia (FEA) are common lesions mainly detected during mammographic screening. They are considered lesions at risk for the development of breast cancer, and they have been documented as non-obligate precursors of low grade in situ carcinomas. In a monumental work in 1991 Rosai gathered them as "borderline epithelial lesions", and he described and demonstrated the subjectivity in their microscopic interpretation. Such subjectivity persists nowadays and limits considerably the diagnostic consistency. With his incredible ability to see, analyze and rationalize, Rosai introduced the concept of "mammary intraepithelial neoplasia (MIN) of either ductal or lobular type, followed by a grading system" which would have better represented the biological continuum between these lesions and benign and malignant lesions.

Survey on postmortem screening and management of COVID-19 related deaths.

The COVID-19 pandemic is associated with a high case fatality rate in some countries even thought the majority of cases are asymptomatic. Scientific studies on this novel virus is limited and there is uncertainty regarding the best practices for death investigations both in terms of detection of the disease as well as autopsy safety. An online survey was conducted to identify how different institutions responded to the screening and management of dead bodies during the early phase of the pandemic from January to May. A questionnaire was developed using Google Forms and data was collected from 14 different forensic and pathological institutions in 9 countries. None of the institutions had performed any screening prior to March. Four institutions stated that screening was done routinely. In total, 322 cases had been screened using RT-PCR, out of which 40 positive cases were detected among four institutions. The commonest types of samples obtained were nasopharyngeal and oropharyngeal swabs which also had the highest rates of positivity followed by tracheal swab. Blood, swabs from cut surfaces of lung and lung tissue also gave positive results in some cases. Majority of the positive cases were > 65 years with a history suggestive of respiratory infection and were clinically suspected to have COVID-19 before death. Except for one institution which performed limited dissections, standard autopsies were conducted on all positive cases. Disposal of bodies involved the use of sealed body bags and labelling as COVID positive. Funeral rites were restricted and none of the institutions advocated cremation. There were no reports of disease transmission to those who handled COVID positive bodies.

Real-World Data on NGS Diagnostics: a survey from the Italian Society of Pathology (SIAPeC) NGS Network.

Next Generation Sequencing (NGS) is increasingly used in diagnostic centers for the assessment of genomic alterations to select patients for precision oncology. The Italian Society of Anatomic Pathology and Diagnostic Cytopathology (SIAPEC) through the Molecular Pathology and Predictive Medicine Study Group (PMMP) has been following the progressive development of centers that have adopted NGS technology in diagnostics over time. In July 2017, a study network on massive parallel sequencing was activated in Italy and recognized as the NGS SIAPeC National Network by the SIAPeC Scientific Society Board. Since then, activities have been implemented within the network that provide for alignment of laboratories through diagnostic concordance analysis and monitoring of centers adhering to the Network. Recently, considering the growing need for extended genomic analyses, the PMMP distributed a national survey to assess activities related to the use of genomic diagnostics in oncology within the NGS SIAPEC National Network.Thirty centers participated in the survey. Eighty percent of the centers are laboratories within Pathology Departments. The distribution of laboratories in the country, the diagnostic laboratory/population ratio, the staff dedicated, the type and number of sequencing and mechatronics platforms available, the genomic panels utilized, and the type and number of diagnostic tests carried out in the last year in each center, are reported.The centers were also asked whether they participated in a multidisciplinary Molecular Tumor Board (MTB) for management of patients. Thirty percent of the centers had a MTB that was ratified by regional decree. The professionals most frequently involved in the core team of the MTB are the pathologist, oncologist, molecular biologist, geneticist, pharmacologist, and bioinformatician.The data from this survey indicate that NGS diagnostics in Italy is still heterogeneous in terms of geographical distribution and the characteristics of laboratories and diagnostic test performed. The implementation of activities that favors harmonization, the logistics and the convergence of biological material in reference centers for molecular analyses is a priority for the development of a functional laboratory network.

Integration of Ki-67 index into AJCC 2018 staging provides additional prognostic information in breast tumours candidate for genomic profiling.

BACKGROUND: The Eighth edition of the American Joint Committee on Cancer (AJCC) staging system (2018) for breast cancer (BC) introduced the prognostic stage. Moreover, multigene assessment has been indicated to tailor staging in T1/T2/N0, ER-positive/HER2-negative BC. However, many National Health Systems do not provide reimbursement for routine testing. The aim of this study was to assess whether Ki67 proliferation index is prognostically relevant for patients' candidacy for molecular testing. METHODS: A retrospective series of 686 ER+/HER2- BC were reclassified using AJCC 2018, and in the group of 521 patients for which AJCC 2018 recommends molecular evaluation, we assessed the prognostic efficacy of a prognostic stage enriched by Ki67 (Ki67-PS), considering Ki67 <20% an alternative to recurrence score <11 provided by Oncotype DX. RESULTS: We found that a group of BCs (35.6%, 58/163) assigned to IB stage by prognostic score were down classified to IA with Ki67-PS. The outcome of these 58 cases overlapped with that of lesions classified as stage IA using prognostic stage, showing a significantly better prognosis compared to IB tumours (HR = 2.79, p = 0.003). CONCLUSIONS: These data suggest that Ki67 may be a reliable marker to enrich the 2018 AJCC prognostic score in BC patients' candidacy for genomic profiling.

Post-radiotherapy vascular lesions of the breast: immunohistochemical and molecular features of 74 cases with long-term follow-up and literature review.

AIMS: A wide range of post-radiotherapy (RT) vascular lesions can occur, ranging from benign lymphangiomatous papules of the skin (BLAPs), to atypical vascular lesions (AVLs) and post-RT angiosarcomas (ASs). The relationship between benign and malignant post-RT breast lesions and their prognostic features are still controversial. The aims of this study were to investigate the relationship between benign and malignant mammary post-RT vascular lesions and to define post-RT AS prognostic features. METHODS AND RESULTS: Seventy-four post-RT vascular lesion cases were obtained and stained with antibodies against CD34, CD31, D2-40, Ki67, and c-Myc. Mutational analysis was performed by deep sequencing for the following genes: KRAS, NRAS, HRAS, BRAF, PIK3CA, TP53, NOTCH1, PTEN, CDKN2A, EGFR, AKT1, CTNNB1, hTERT, and PTPRB. Post-RT AS cases were graded according to a previously reported breast AS grading system. AVL cases showed a low number of HRAS and hTERT mutations, whereas post-RT AS cases showed a high frequency of EGFR, TP53, HRAS and hTERT mutations. On follow-up, all BLAP and AVL patients were alive with no evidence of disease. Post-RT AS 5-year overall survival declined with the increase in grade, as follows: 85.7% for grade 1, 83.3% for grade 2, and 40.4% for grade 3. CONCLUSIONS: Our findings confirm that BLAP and AVL have a good prognosis, and that post-RT AS prognosis is strongly related to histological grading. On molecular analysis, AVL and post-RT AS shared HRAS and hTERT mutations, suggesting a relationship between the two lesions.

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.

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

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