[Comparative immunohistochemistry study of different antibodies clones for detection of breast cancer markers (estrogen receptor, progesterone receptor, HER2/neu, Ki-67)]. / Sravnitel'noe issledovanie antitel razlichnykh klonov dlya vyyavleniya markerov raka molochnoi zhelezy (retseptorov estrogenov, progesterona, HER2/neu, Ki-67) immunogistokhimicheskim metodom.

OBJECTIVE: A comparative study of detection of breast cancer markers (estrogen receptors, progesterone receptors, HER2/neu, Ki-67) by immunohistochemical method with antibodies produced by PrimeBioMed (Russia) and antibodies produced by Roche Ventana (USA). MATERIAL AND METHODS: Surgical specimens and biopsies from 37 patients with invasive breast cancer were used. Sections were stained with antibodies of clones ER SP1 and GM030, PR 1E2 and PBM-5B8, HER2/neu 4B5 and PBM-46A6, Ki-67 30-9 and GM010. RESULTS: There was a high positive and significant correlation between the immunohistochemistry results and antibodies of the clones ER-SP1 and GM030, PR1E2 and PBM-5B8, HER2/neu4B5 and PBM-46A6, Ki-67 30-9 and GM010. CONCLUSION: The study showed the possibility of using antibodies of clones GM030, HER2/neu 4B5, PBM-46A6, GM010 (PrimeBioMed) on the Ventana Bench Marck Ultra automatic immunostainer using the detection system UltraView Universal DAB Detection Kit.

Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Immune-driven clonal cell selection at the intersection among cancer, infections, autoimmunity and senescence.

Immune surveillance mechanisms play a crucial role in maintaining lifelong immune homeostasis in response to pathologic stimuli and aberrant cell states. However, their persistence, especially in the context of chronic antigenic exposure, can create a fertile ground for immune evasion. These escaping cell phenotypes, harboring a variety of genomic and transcriptomic aberrances, chiefly in human leukocyte antigen (HLA) and antigen presentation machinery genes, may survive and proliferate, featuring a scenario of clonal cell expansion with immune failure characteristics. While well characterized in solid and, to some extent, hematological malignancies, little is known about their occurrence and significance in other disease contexts. Historical literature highlights the role for escaping HLA-mediated recognition as a strategy adopted by virus to evade from the immune system, hinting at the potential for immune aberrant cell expansion in the context of chronic infections. Additionally, unmasked in idiopathic aplastic anemia as a mechanism able to rescue failing hematopoiesis, HLA clonal escape may operate in autoimmune disorders, particularly in tissues targeted by aberrant immune responses. Furthermore, senescent cell status emerging as immunogenic phenotypes stimulating T cell responses, may act as a bottleneck for the selection of such immune escaping clones, blurring the boundaries between neoplastic transformation, aging and inflammation. Here we provide a fresh overview and perspective on this immune-driven clonal cell expansion, linking pathophysiological features of neoplastic, autoimmune, infectious and senescence processes exposed to immune surveillance.

A tower of babel of acronyms? The shadowlands of MGUS/MBL/CHIP/TCUS.

With the advent of outperforming and massive laboratory tools, such as multiparameter flow cytometry and next-generation sequencing, hematopoietic cell clones with putative abnormalities for a variety of blood malignancies have been appreciated in otherwise healthy individuals. These conditions do not fulfill the criteria of their presumed cancer counterparts, and thus have been recognized as their precursor states. This is the case of monoclonal gammopathy of unknown significance (MGUS), the first blood premalignancy state described, preceding multiple myeloma (MM) or Waldenström macroglobulinemia (WM). However, in the last 2 decades, an increasing list of clonopathies has been recognized, including monoclonal B cell lymphocytosis (MBL), which antecedes chronic lymphocytic leukemia (CLL), clonal hematopoiesis of indeterminate potential (CHIP) for myeloid neoplasms (MN), and T-cell clones of uncertain significance (TCUS) for T-cell large chronic lymphocytic leukemia (LGLL). While for some of these entities diagnostic boundaries are precisely set, for others these are yet to be fully defined. Moreover, despite mostly considered of "uncertain significance," they have not only appeared to predispose to malignancy, but also to be capable of provoking set of immunological and cardiovascular complications that may require specialized management. The clinical implications of the aberrant clones, together with the extensive knowledge generated on the pathogenetic events driving their evolution, raises the question whether earlier interventions may alter the natural history of the disease. Herein, we review this Tower of Babel of acronyms pinpointing diagnostic definitions, differential diagnosis, and the role of genomic profiling of these precursor states, as well as potential interventional strategies.

Treatment with the apoptosis inhibitor Asunercept reduces clone sizes in patients with lower risk Myelodysplastic Neoplasms.

In low-risk Myelodysplastic Neoplasms (MDS), increased activity of apoptosis-promoting factors such as tumor necrosis factor (TNFα) and pro-apoptotic Fas ligand (CD95L) have been described as possible pathomechanisms leading to impaired erythropoiesis. Asunercept (APG101) is a novel therapeutic fusion protein blocking CD95, which has previously shown partial efficacy in reducing transfusion requirement in a clinical phase I trial for low-risk MDS patients (NCT01736436; 2012-11-26). In the current study we aimed to evaluate the effect of Asunercept therapy on the clonal bone marrow composition to identify potential biomarkers to predict response. Bone marrow samples of n = 12 low-risk MDS patients from the above referenced clinical trial were analyzed by serial deep whole exome sequencing in a total of n = 58 time points. We could distinguish a mean of 3.5 molecularly defined subclones per patient (range 2-6). We observed a molecular response defined as reductions of dominant clone sizes by a variant allele frequency (VAF) decrease of at least 10% (mean 20%, range: 10.5-39.2%) in dependency of Asunercept treatment in 9 of 12 (75%) patients. Most of this decline in clonal populations was observed after completion of 12 weeks treatment. Particularly early and pronounced reductions of clone sizes were found in subclones driven by mutations in genes involved in regulation of methylation (n = 1 DNMT3A, n = 1 IDH2, n = 1 TET2). Our results suggest that APG101 could be efficacious in reducing clone sizes of mutated hematopoietic cells in the bone marrow of Myelodysplastic Neoplasms, which warrants further investigation.

Clonal differences underlie variable responses to sequential and prolonged treatment.

Cancer cells exhibit dramatic differences in gene expression at the single-cell level, which can predict whether they become resistant to treatment. Treatment perpetuates this heterogeneity, resulting in a diversity of cell states among resistant clones. However, it remains unclear whether these differences lead to distinct responses when another treatment is applied or the same treatment is continued. In this study, we combined single-cell RNA sequencing with barcoding to track resistant clones through prolonged and sequential treatments. We found that cells within the same clone have similar gene expression states after multiple rounds of treatment. Moreover, we demonstrated that individual clones have distinct and differing fates, including growth, survival, or death, when subjected to a second treatment or when the first treatment is continued. By identifying gene expression states that predict clone survival, this work provides a foundation for selecting optimal therapies that target the most aggressive resistant clones within a tumor. A record of this paper's transparent peer review process is included in the supplemental information.

T-cell dysfunctions in myelodysplastic syndromes.

ABSTRACT: Escape from immune surveillance is a hallmark of cancer. Immune deregulation caused by intrinsic and extrinsic cellular factors, such as altered T-cell functions, leads to immune exhaustion, loss of immune surveillance, and clonal proliferation of tumoral cells. The T-cell immune system contributes to the pathogenesis, maintenance, and progression of myelodysplastic syndrome (MDS). Here, we comprehensively reviewed our current biological knowledge of the T-cell compartment in MDS and recent advances in the development of immunotherapeutic strategies, such as immune checkpoint inhibitors and T-cell- and antibody-based adoptive therapies that hold promise to improve the outcome of patients with MDS.

Construction and characterization of a full-length infectious clone of an emerging senecavirus A strain.

Senecavirus A (SVA) is an emerging virus that causes vesicular disease in pigs. Construction of a full-length SVA cDNA clone is crucial for understanding its replication and pathogenesis. Here, we successfully constructed a CMV-promoter-driven infectious cDNA clone of the SVA isolate SVA/GX/CH/2018, which we named rSVA GX01. Sequence comparison between the pSVA GX01 and the parental isolate (SVA/GX/CH/2018) revealed three single-nucleotide differences. Four-week-old piglets were experimentally infected with either the parental virus or the cloned virus. The results showed that the cloned rSVA GX01 displayed weak pathogenicity in 4-week-old pigs compared to the parental virus SVA CH-GX-01-2018. The infectious clone of SVA will serve as a valuable tool for studying the viral replication cycle and for functional analysis of the viral genome.

O6 -methylguanine methyltransferase promoter methylation status of glioblastoma cell line clonal population.

Glioblastoma (GBM) remains a treatment-resistant malignant brain tumor in large part because of its genetic heterogeneity and epigenetic plasticity. In this study, we investigated the epigenetic heterogeneity of GBM by evaluating the methylation status of the O6 -methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. The U251 and U373 GBM cell lines, from the Brain Tumour Research Centre of the Montreal Neurological Institute, were used for the experiments. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. Moreover, mRNA and protein expression levels of MGMT in the individual GBM clones were evaluated. The HeLa cell line, which hyper-expresses MGMT, was used as control. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The methylation status by pyrosequencing was relatively high at CpG sites 3-8, 20-35, and 7-83, in both the U251 and U373 clones. Neither MGMT mRNA nor protein was detected in any clone. These findings demonstrate tumor heterogeneity among individual clones derived from a single GBM cell. MGMT expression may be regulated, not only by methylation of the MGMT promoter but by other factors as well. Further studies are needed to clarify the mechanisms underlying the epigenetic heterogeneity and plasticity of GBM.

Sarcoidosis Associated with Enlarged Mediastinal Lymph Nodes with the Detection of Streptococcus gordonii and Cutibacterium acnes Using a Clone Library Method.

A 77-year-old Japanese woman with mediastinal lymphadenopathy and uveitis was diagnosed with sarcoidosis. The bacterial flora in biopsied samples from mediastinal lymph nodes was analyzed using a clone library method with Sanger sequencing of the 16S rRNA gene, and Streptococcus gordonii (52 of 71 clones) and Cutibacterium acnes (19 of 71 clones) were detected. No previous study has conducted a bacterial floral analysis using the Sanger method for the mediastinal lymph node in sarcoidosis, making this case report the first to document the presence of S. gordonii and C. acnes in the mediastinal lymph node of a patient with sarcoidosis.

How low can you go?: Methodologic considerations in clonal hematopoiesis variant calling.

Clonal hematopoiesis (CH) is defined by the presence of an expanded clonal hematopoietic cell population due to an acquired mutation conferring a selective growth advantage and is known to predispose to hematologic malignancy. In this review, we discuss sequencing methods for CH detection in bulk sequencing data and corresponding bioinformatic approaches for variant calling, filtering, and curation. We detail practical recommendations for CH calling. Finally, we discuss how improvements in CH sequencing and bioinformatic approaches will enable the characterization of CH trajectories, its impact on human health, and therapeutic approaches to mitigate its adverse effects.

Stronger Together: Cancer Clones Cooperate to Alleviate Growth Barriers in Critical Cancer Progression Transitions.

Hershey and colleagues recently showed how clones in a triple-negative breast cancer cell line cooperate for their mutual fitness benefit. In this system, clones exchange soluble metabolites to increase their in vitro growth rate at low population densities, therefore mitigating the documented growth barrier that reduces individual fitness in small tumor cell populations (Allee effect). Such cooperation could aid important transitions in cancer progression in which cancer cell populations are small, like invasion or metastasis. Using orthotopic transplantation, the authors demonstrate that this cooperation is functional in one such transition in vivo, increasing the metastatic load and number of metastases, which are usually polyclonal. Together, these findings highlight the need to consider ecologic interactions to properly understand tumor growth dynamics, and how they complement the standing evolutionary model of cancer progression in our quest to understand and treat cancer.

Clonal Proliferation Within Smooth Muscle Cells in Unstable Human Atherosclerotic Lesions.

BACKGROUND: Studies in humans and mice using the expression of an X-linked gene or lineage tracing, respectively, have suggested that clones of smooth muscle cells (SMCs) exist in human atherosclerotic lesions but are limited by either spatial resolution or translatability of the model. METHODS: Phenotypic clonality can be detected by X-chromosome inactivation patterns. We investigated whether clones of SMCs exist in unstable human atheroma using RNA in situ hybridization (BaseScope) to identify a naturally occurring 24-nucleotide deletion in the 3'UTR of the X-linked BGN (biglycan) gene, a proteoglycan highly expressed by SMCs. BGN-specific BaseScope probes were designed to target the wild-type or deletion mRNA. Three different coronary artery plaque types (erosion, rupture, and adaptive intimal thickening) were selected from heterozygous females for the deletion BGN. Hybridization of target RNA-specific probes was used to visualize the spatial distribution of mutants. A clonality index was calculated from the percentage of each probe in each region of interest. Spatial transcriptomics were used to identify differentially expressed transcripts within clonal and nonclonal regions. RESULTS: Less than one-half of regions of interest in the intimal plaque were considered clonal with the mean percent regions of interest with clonality higher in the intimal plaque than in the media. This was consistent for all plaque types. The relationship of the dominant clone in the intimal plaque and media showed significant concordance. In comparison with the nonclonal lesions, the regions with SMC clonality had lower expression of genes encoding cell growth suppressors such as CD74, SERF-2 (small EDRK-rich factor 2), CTSB (cathepsin B), and HLA-DPA1 (major histocompatibility complex, class II, DP alpha 1), among others. CONCLUSIONS: Our novel approach to examine clonality suggests atherosclerosis is primarily a disease of polyclonally and to a lesser extent clonally expanded SMCs and may have implications for the development of antiatherosclerotic therapies.

Selection of cross-reactive T cells by commensal and food-derived yeasts drives cytotoxic TH1 cell responses in Crohn's disease.

Aberrant CD4+ T cell reactivity against intestinal microorganisms is considered to drive mucosal inflammation in inflammatory bowel diseases. The disease-relevant microbial species and the corresponding microorganism-specific, pathogenic T cell phenotypes remain largely unknown. In the present study, we identified common gut commensal and food-derived yeasts, as direct activators of altered CD4+ T cell reactions in patients with Crohn's disease (CD). Yeast-responsive CD4+ T cells in CD display a cytotoxic T helper cell (TH1 cell) phenotype and show selective expansion of T cell clones that are highly cross-reactive to several commensal, as well as food-derived, fungal species. This indicates cross-reactive T cell selection by repeated encounter with conserved fungal antigens in the context of chronic intestinal disease. Our results highlighted a role of yeasts as drivers of aberrant CD4+ T cell reactivity in patients with CD and suggest that both gut-resident fungal commensals and daily dietary intake of yeasts might contribute to chronic activation of inflammatory CD4+ T cell responses in patients with CD.

[Identification of transcriptional features and surface markers of plasma cell clones in POEMS syndrome by single-cell RNA sequencing].

POEMS syndrome is a rare monoclonal plasma cell disorder with unique symptoms distinct from other plasma cell neoplasms. To identify and find the transcriptional features of clonal plasma cells in POEMS syndrome (POEMS clones), single-cell RNA sequencing was performed on patient-derived bone marrow plasma cells. POEMS clones were identified in 5 out of 10 patients, and the proportions of POEMS clones in the plasma cells were markedly smaller than that of other plasma cell malignancies such as multiple myeloma and MGUS. The transcriptional features of POEMS clones differed from those of other plasma cell diseases, and representative MM-related oncogenes were not upregulated in POEMS clones. Notably, POEMS clones are negative for CD19 and express significantly lower MHC-II levels than normal plasma cells; thus, CD19- HLA-DRlo is confirmed as a useful marker to identify POEMS clones in patients. These findings unveil the unique features of POEMS clones and contribute to the understanding of the pathogenesis of POEMS syndrome.

Experimental and spontaneous metastasis assays can result in divergence in clonal architecture.

Intratumoural heterogeneity is associated with poor outcomes in breast cancer. To understand how malignant clones survive and grow in metastatic niches, in vivo models using cell lines and patient-derived xenografts (PDX) have become the gold standard. Injections of cancer cells in orthotopic sites (spontaneous metastasis assays) or into the vasculature (experimental metastasis assays) have been used interchangeably to study the metastatic cascade from early events or post-intravasation, respectively. However, less is known about how these different routes of injection impact heterogeneity. Herein we directly compared the clonality of spontaneous and experimental metastatic assays using the human cell line MDA-MB-231 and a PDX model. Genetic barcoding was used to study the fitness of the subclones in primary and metastatic sites. Using spontaneous assays, we found that intraductal injections resulted in less diverse tumours compared to other routes of injections. Using experimental metastasis assays via tail vein injection of barcoded MDA-MB-231 cells, we also observed an asymmetry in metastatic heterogeneity between lung and liver that was not observed using spontaneous metastasis assays. These results demonstrate that these assays can result in divergent clonal outputs in terms of metastatic heterogeneity and provide a better understanding of the biases inherent to each technique.

Anti-VEGF therapy selects for clones resistant to glucose starvation in ovarian cancer xenografts.

BACKGROUND: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis. METHODS: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy. RESULTS: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation. CONCLUSION: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy.

Copy number architectures define treatment-mediated selection of lethal prostate cancer clones.

Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance (p-values 3.07 × 10-8 and 6.4 × 10-4). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories.

Evolutionary histories of breast cancer and related clones.

Recent studies have documented frequent evolution of clones carrying common cancer mutations in apparently normal tissues, which are implicated in cancer development1-3. However, our knowledge is still missing with regard to what additional driver events take place in what order, before one or more of these clones in normal tissues ultimately evolve to cancer. Here, using phylogenetic analyses of multiple microdissected samples from both cancer and non-cancer lesions, we show unique evolutionary histories of breast cancers harbouring der(1;16), a common driver alteration found in roughly 20% of breast cancers. The approximate timing of early evolutionary events was estimated from the mutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative chromosome was acquired from early puberty to late adolescence, followed by the emergence of a common ancestor by the patient's early 30s, from which both cancer and non-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following years, these clones occupied a large area within the premenopausal breast tissues by the time of cancer diagnosis. Evolution of multiple independent cancer founders from the non-cancer ancestors was common, contributing to intratumour heterogeneity. The number of driver events did not correlate with histology, suggesting the role of local microenvironments and/or epigenetic driver events. A similar evolutionary pattern was also observed in another case evolving from an AKT1-mutated founder. Taken together, our findings provide new insight into how breast cancer evolves.