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Background: Previous studies have provided a comprehensive picture of genomic alterations in primary and metastatic Hormone Receptor (HR)-positive, Human Epidermal growth factor Receptor 2 (HER2)-negative breast cancer (HR+ HER2- BC). However, the evolution of the genomic landscape of HR+ HER2- BC during adjuvant endocrine therapies (ETs) remains poorly investigated. Methods and findings: We performed a genomic characterization of surgically resected HR+ HER2- BC patients relapsing during or at the completion of adjuvant ET. Using a customized panel, we comprehensively evaluated gene mutations and copy number variation (CNV) in paired primary and metastatic specimens. After retrieval and quality/quantity check of tumor specimens from an original cohort of 204 cases, 74 matched tumor samples were successfully evaluated for DNA mutations and CNV analysis. Along with previously reported genomic alterations, including PIK3CA, TP53, CDH1, GATA3 and ESR1 mutations/deletions, we found that ESR1 gene amplification (confirmed by FISH) and MAP3K mutations were enriched in metastatic lesions as compared to matched primary tumors. These alterations were exclusively found in patients treated with adjuvant aromatase inhibitors or LHRH analogs plus tamoxifen, but not in patients treated with tamoxifen alone. Patients with tumors bearing MAP3K mutations in metastatic lesions had significantly worse distant relapse-free survival (hazard ratio [HR] 3.4, 95% CI 1.52–7.70, p value 0.003) and worse overall survival (HR 5.2, 95% CI 2.10–12.8, p-value < 0.001) independently of other clinically relevant patient- and tumor-related variables. Conclusions: ESR1 amplification and activating MAP3K mutations are potential drivers of acquired resistance to adjuvant ETs employing estrogen deprivation in HR+ HER2- BC. MAP3K mutations are associated with worse prognosis in patients with metastatic disease. Author summary: Breast cancer is the most frequently diagnosed cancer and represents the leading cause of cancer-related death in women. Hormone receptor positive tumors account for 70–80% of all breast cancers. They are characterized by estrogen dependent growth, and are routinely treated by endocrine therapy, aiming at blocking estrogen receptor (e.g., tamoxifen) or inhibiting the production of estrogen (aromatase inhibitors and LHRH analogues). Unfortunately, a significant proportion of patients develops endocrine resistance, ultimately leading to tumor recurrence. In this study, we analyzed a cohort of 74 hormone receptor positive breast cancer patients by performing a deep molecular characterization of treatment-naïve primary tumor samples and their matched metastatic localizations, to highlight putative mechanisms of endocrine resistance. Along with expected acquired molecular alterations, including mutation in ESR1 gene, that encodes for estrogen receptor, we found that an increase of the number of copies of the ESR1 gene (amplification) and mutations in MAP3K are significantly enriched in relapsing tumors, thus expanding the spectrum of known endocrine therapy resistance mechanisms. Interestingly, we found that patients with MAP3K mutations were associated with a worse prognosis. [ABSTRACT FROM AUTHOR]

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods: Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in selfselected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results: The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56-0.72). However, we found very high inter-observer variabilities (Kappa 0.04-0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were reexamined 4 months later (Kappa 0.01-0.04) and intra-observer agreement was likewise poor (Kappa 0.00-0.35). Conclusion: Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas. [ABSTRACT FROM AUTHOR]

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs. [ABSTRACT FROM AUTHOR]

Background: Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system. Methodology/Principal Findings: In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the ''normal'' samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease. Conclusions/Significance: The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition. [ABSTRACT FROM AUTHOR]

Donkeys (Equus asinus) have been used extensively in agriculture and transportations since their domestication, ca. 5000-7000 years ago, but the increased mechanization of the last century has largely spoiled their role as burden animals, particularly in developed countries. Consequently, donkey breeds and population sizes have been declining for decades, and the diversity contributed by autochthonous gene pools has been eroded. Here, we examined coding-region data extracted from 164 complete mitogenomes and 1392 donkey mitochondrial DNA (mtDNA) control-region sequences to (i) assess worldwide diversity, (ii) evaluate geographical patterns of variation, and (iii) provide a new nomenclature of mtDNA haplogroups. The topology of the Maximum Parsimony tree confirmed the two previously identified major clades, i.e. Clades 1 and 2, but also highlighted the occurrence of a deep-diverging lineage within Clade 2 that left a marginal trace in modern donkeys. Thanks to the identification of stable and highly diagnostic coding-region mutational motifs, the two lineages were renamed as haplogroup A and haplogroup B, respectively, to harmonize clade nomenclature with the standard currently adopted for other livestock species. Control-region diversity and population expansion metrics varied considerably between geographical areas but confirmed North-eastern Africa as the likely domestication center. The patterns of geographical distribution of variation analyzed through phylogenetic networks and AMOVA confirmed the co-occurrence of both haplogroups in all sampled populations, while differences at the regional level point to the joint effects of demography, past human migrations and trade following the spread of donkeys out of the domestication center. Despite the strong decline that donkey populations have undergone for decades in many areas of the world, the sizeable mtDNA variability we scored, and the possible identification of a new early radiating lineage further stress the need for an extensive and large-scale characterization of donkey nuclear genome diversity to identify hotspots of variation and aid the conservation of local breeds worldwide., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Migliore et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)