Outcomes of Breast Cancer Patients Treated with Chemotherapy, Biologic Therapy, Endocrine Therapy, or Active Surveillance During the COVID-19 Pandemic.

PURPOSE: Provide real-world data regarding the risk for SARS-CoV-2 infection and mortality in breast cancer (BC) patients on active cancer treatment. METHODS: Clinical data were abstracted from the 3778 BC patients seen at a multisite cancer center in New York between February 1, 2020 and May 1, 2020, including patient demographics, tumor histology, cancer treatment, and SARS-CoV-2 testing results. Incidence of SARS-CoV-2 infection by treatment type (chemotherapy [CT] vs endocrine and/or HER2 directed therapy [E/H]) was compared by Inverse Probability of Treatment Weighting. In those diagnosed with SARS-CoV-2 infection, Mann-Whitney test was used to a assess risk factors for severe disease and mortality. RESULTS: Three thousand sixty-two patients met study inclusion criteria with 641 patients tested for SARS-COV-2 by RT-PCR or serology. Overall, 64 patients (2.1%) were diagnosed with SARS-CoV-2 infection by either serology, RT-PCR, or documented clinical diagnosis. Comparing matched patients who received chemotherapy (n = 379) with those who received non-cytotoxic therapies (n = 2343) the incidence of SARS-CoV-2 did not differ between treatment groups (weighted risk; 3.5% CT vs 2.7% E/H, P = .523). Twenty-seven patients (0.9%) expired over follow-up, with 10 deaths attributed to SARS-CoV-2 infection. Chemotherapy was not associated with increased risk for death following SARS-CoV-2 infection (weighted risk; 0.7% CT vs 0.1% E/H, P = .246). Advanced disease (stage IV), age, BMI, and Charlson's Comorbidity Index score were associated with increased mortality following SARS-CoV-2 infection (P ≤ .05). CONCLUSION: BC treatment, including chemotherapy, can be safely administered in the context of enhanced infectious precautions, and should not be withheld particularly when given for curative intent.

Features of triple-negative breast cancer: Analysis of 38,813 cases from the national cancer database.

The aim of this study was to determine the features of triple-negative breast cancer (TNBC) using a large national database. TNBC is known to be an aggressive subtype, but national epidemiologic data are sparse. All patients with invasive breast cancer and known molecular subtype diagnosed in 2010 to 2011 were identified from the National Cancer Data Base (NCDB). Patients with and without TNBC were compared with respect to their sociodemographic and clinicopathologic features. TNBC was present in 38,628 of 295,801 (13%) female patients compared to 185 of 3136 (6%) male patients (P < 0.001). The incidence of TNBC varied by region from 10.8% in New England to 15.8% in the east south central US (P < 0.001), as well as by race with the highest rates in African-Americans (23.7%), and lowest in Filipino patients (8.9%). The incidence of TNBC also varied by histology, accounting for 76% of metaplastic cancers, but only 2% of infiltrating lobular carcinomas. TNBCs were significantly larger than non-TNBC (mean 2.8 cm vs 2.1 cm, P < 0.001), and more TNBC were poorly differentiated compared to other subtypes (79.7% vs 25.8%, P < 0.001). On univariate analysis, TNBC was no more likely than non-TNBC to have node-positive disease (32.0% vs 31.7%, respectively, P = 0.218) but in a multivariable analysis controlling for tumor size and grade, TNBC was associated with significantly less node-positivity (OR = 0.59; 95% confidence interval [CI]: 0.57-0.60). TNBC has distinct features regarding age, gender, geographic, and racial distribution. Compared to non-TNBC, TNBC is larger and higher grade, but less likely to have lymph node metastases.

Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21.

BACKGROUND: Tissue engineering is often limited by the time required for culture expansion of cells necessary for scaffold seeding. Cell cycle regulators control entry and exit into the cell cycle and as such regulate cellular proliferation rates. The authors hypothesized that transient alteration in cell cycle regulators can be utilized as a means to accelerate stem cell proliferation. METHODS: Mesenchymal stem cells were harvested from wild-type mice and mice deficient in the cell cycle regulator p21. Wild-type cells were treated with small interfering RNA against p21 in two- or three-dimensional cultures in vitro. Cellular proliferation and the potential for cellular differentiation into the bone or fat lineage were assessed. RESULTS: Mesenchymal stem cells treated with small interfering RNA targeting p21 demonstrated a significant decrease in p21 protein and mRNA expression 96 hours after treatment. They also proliferated significantly faster than control cells (2.5 to three times) in both two- and three-dimensional culture. Similarly, cells harvested from p21-deficient mice demonstrated a significant acceleration in cellular proliferation. Inhibition of p21 expression was not associated with significant changes in spontaneous cellular differentiation. However, transient p21 inhibition promoted both osteoblastic and adipogenic differentiation when cells were exposed to differentiation medium. CONCLUSIONS: Transient inhibition of the cell cycle regulator p21 results in significant acceleration of mesenchymal stem cell proliferation without promoting spontaneous cellular differentiation. Exposure to differentiation medium results in increased cellular differentiation toward the osteoblast and fat lineage. Manipulation of cell cycle regulators may represent a novel means by which stem cell proliferation can be accelerated, thereby decreasing the time required for scaffold synthesis in tissue engineering.

Enforced expression of NUP98-HOXA9 in human CD34(+) cells enhances stem cell proliferation.

The t(7;11)(p15;p15) translocation, observed in acute myelogenous leukemia and myelodysplastic syndrome, generates a chimeric gene where the 5' portion of the sequence encoding the human nucleoporin NUP98 protein is fused to the 3' region of HOXA9. Here, we show that retroviral-mediated enforced expression of the NUP98-HOXA9 fusion protein in cord blood-derived CD34(+) cells confers a proliferative advantage in both cytokine-stimulated suspension cultures and stromal coculture. This advantage is reflected in the selective expansion of hematopoietic stem cells as measured in vitro by cobblestone area-forming cell assays and in vivo by competitive repopulation of nonobese diabetic/severe combined immunodeficient mice. NUP98-HOXA9 expression inhibited erythroid progenitor differentiation and delayed neutrophil maturation in transduced progenitors but strongly enhanced their serial replating efficiency. Analysis of the transcriptosome of transduced cells revealed up-regulation of several homeobox genes of the A and B cluster as well as of Meis1 and Pim-1 and down-modulation of globin genes and of CAAT/enhancer binding protein alpha. The latter gene, when coexpressed with NUP98-HOXA9, reversed the enhanced proliferation of transduced CD34(+) cells. Unlike HOXA9, the NUP98-HOXA9 fusion was protected from ubiquitination mediated by Cullin-4A and subsequent proteasome-dependent degradation. The resulting protein stabilization may contribute to the leukemogenic activity of the fusion protein.

Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome.

Immune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS). Immune inhibition can result from cytotoxic T cell (CTL) attack against normal hematopoiesis or reflect immune surveillance. We used clonally unique T-cell receptor (TCR) variable beta-chain (VB) CDR3 regions as markers of pathogenic CTL responses and show that while marrow failure syndromes are characterized by polyclonal expansions, overexpanded clones exist in these diseases and can serve as investigative tools. To test the applicability of clonotypic assays, we developed rational molecular methods for the detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 AA, 37 MDS, and 21 paroxysmal nocturnal hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, respectively. In total, 81 immunodominant signature clonotypes were identified. Based on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays for monitoring corresponding clones, including clonotypic Taqman polymerase chain reaction (PCR) and clonotype-specific sequencing. No correlation was found between clonality and disease severity but in patients treated with immunosuppression, truly pathogenic clones were identified based on the decline that paralleled hematologic response. We conclude that immunodominant clonotypes associated with marrow failure may be used to monitor immunosuppressive therapy.

In-vivo dominant immune responses in aplastic anaemia: molecular tracking of putatively pathogenetic T-cell clones by TCR beta-CDR3 sequencing.

BACKGROUND: Aplastic anaemia is a bone-marrow-failure syndrome characterised by low blood-cell counts and fatty bone marrow. In most cases, no obvious aetiological factor can be identified. However, clinical responses to immunosuppression strongly suggest an immune pathophysiology. METHODS: To test the hypothesis that aplastic anaemia results from antigen-specific lymphocyte attack against haemopoietic tissue, we analysed effector immunity, seeking especially dominant specific T-cell responses. Blood samples from 54 patients with aplastic anaemia were subjected to flow cytometry to define T-cell-receptor Vbeta-chain usage and expansion of particular Vbeta subsets. We measured the size distribution of the complementarity-determining region 3 (CDR3) for expanded Vbeta subsets, then cloned and sequenced skewed, oligoclonal, or monoclonal peaks. FINDINGS: Expanded Vbeta subsets were identified in almost all the patients. Over-represented Vbeta subsets from CD8-positive cells showed oligoclonal or monoclonal CDR3 size patterns. The CDR3 sequence repertoire in aplastic anaemia showed much redundancy compared with healthy donors. We identified patient-specific putative pathogenetic clonotypes that were not detectable in controls. In selected patients who were assessed longitudinally, these clonotypes were quantitatively related to disease activity. Selective killing of autologous haemopoietic progenitors by the Vbeta-specific lymphocyte population was shown in one patient. These apparently pathogenetic CDR3 sequences showed homology between individuals, suggesting a role for a "semi-public" immune response in the pathophysiology of aplastic anaemia. INTERPRETATION: In-vivo dominant clonal immune response can be identified in many patients with aplastic anaemia, which is evidence for an underlying antigen-driven immune process. Longitudinal tracking by molecular techniques could inform individual clinical decisions and the development of new treatments in autoimmune diseases. RELEVANCE TO PRACTICE: Although the target of the aberrant immune response is the haemopoietic stem cell, the triggering antigens remain unknown. We combined cell phenotypic, molecular biology, and functional analyses to study the effector arm of immunity in an attempt to establish an immune pathophysiology. Clinical application of such a model could broadly extend to other autoimmune diseases.

Shared and individual specificities of immunodominant cytotoxic T-cell clones in paroxysmal nocturnal hemoglobinuria as determined by molecular analysis.

OBJECTIVE: Similar immune mechanisms have been suggested to operate in aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH), and the presence of PNH clones in AA may indicate that an immune reaction directed against hematopoietic stem cells may be responsible for the immune selection pressure leading to PNH evolution. We previously described expansions of selective cytotoxic T-lymphocyte (CTL) clones in AA patients. MATERIALS AND METHODS: We applied a molecular analysis of the T-cell receptor repertoire to study the characteristics of CTL response in patients with various forms of PNH. Immunodominant T-cell clones were detected using combined flow cytometric and molecular analysis of the variable beta (VB) chain and CDR3 representation, followed by determination of the frequency of individual CDR3 clonotypes. Clonotypic polymerase chain reaction (PCR) was performed to establish clonotypic utilization pattern. RESULTS: In patients with a past history of AA, and when subgrouped by current blood counts as "hypoproliferative" PNH patients (in contrast to purely hemolytic form of PNH), more pronounced skewing of VB family utilization was found, consistent with T-cell responses involving several immunodominant CTL clones. Sequences of the PNH-derived clonotypes were used to design PCR-based assays for the utilization analysis of individual clones in PNH patients. The clonotypic distribution pattern established by this PCR method indicated that immunodominant T-cell specificities were shared between some patients but also may be found at low frequencies in controls. CONCLUSION: Analysis of the CDR3 sequence pattern as a marker for expanded immunodominant clonotypes may have an application in the study of T-cell responses in PNH.

Molecular analysis of TCR clonotypes in LGL: a clonal model for polyclonal responses.

Large granular lymphocytic (LGL) leukemia is a clonal lymphoproliferative disorder of CTL associated with cytopenias resulting from an immune and cytokine attack on hemopoietic progenitor cells. Extreme clonality of CTL expansions seen in LGL leukemia makes it an ideal model to study the role of the T cell repertoire in other less-polarized immune-mediated disorders. Complementarity-determining region 3 (CDR3) of the TCR is a unique Ag-specific region that can serve as a molecular marker, or clonotype, of the disease-specific T cells. We studied the variable portion of the beta-chain spectrum in a cohort of LGL leukemia patients. The CDR3 sequences were determined for the immunodominant clones and used to design clonotype-specific primers. By direct and semi-nested amplification, clonotype amplicons were found to be shared by multiple patients and controls. Analysis of the generated sequences demonstrated that the original clonotypes are rarely encountered in normal control samples; however, high levels of homology were found in both controls and patients. Clonotypes derived from individual LGL patients can be used as tumor markers for the malignant clone. More generally, clonotypic analysis and comparison of the variable portion of the beta-chain CDR3-specific sequences from a large number of patients may lead to better subclassification of not only LGL but also other immune-mediated disorders.

Diamond blackfan anemia stem cells fail to repopulate erythropoiesis in NOD/SCID mice.

Diamond Blackfan Anemia (DBA) is a congenital disorder characterized by decreased red blood cell production and developmental abnormalities. We herein show that DBA progenitors produced lower numbers of phenotypically normal erythroid colonies in vitro, whereas nonerythroid colonies were normally abundant and developed. To determine whether DBA stem cells are capable of producing early erythroid, monocyto-granulocytic, and lymphoid progenitors in vivo we used a mouse xenotransplantation model. We demonstrate that DBA stem cells poorly repopulated erythroid progeny in NOD/SCID mice, whereas the monocyto-granulocytic and lymphoid progenies were repopulated normally. Therefore, we conclude that disordered DBA erythropoiesis may be a result of defective erythroid-lineage commitment and maintenance of early erythroid progenitors.

Application of the molecular analysis of the T-cell receptor repertoire in the study of immune-mediated hematologic diseases.

The basis for the vast recognition spectrum of the T-cell receptor (TCR) can be determined by the rearrangement and recombination of the variable, diversity and joining regions of the variable portions of beta (B) and alpha (A) chains as well as their recombination and modification. Analysis of the TCR rearrangement has been routinely used to detect clonality for the diagnosis of lymphoid malignancies. However, molecular analysis of the TCR repertoire can be a powerful tool in the study of T-cell responses to pathogens and in autoimmune diseases. The concept of the oligoclonality in the context of cellular immune responses is based on the presence of immunodominant T-cell clones within distinct T-cell subpopulations used for analysis. Under normal circumstances, a limited number of clones undergo periodic expansions in reaction to foreign antigens. Under pathologic conditions, though, the derailment of immune regulation allows expansions of specific and potentially pathogenic T-cell clones. For example, large granular lymphocyte (LGL) leukemia illustrates an extreme expansion of a single T-cell clone associated with a distinct autoimmune pathology, which suggests an exaggerated clonal response to a specific antigenic target. In immune-mediated bone marrow failure syndromes, clonal rearrangement of the TCR cannot be detected in unseparated blood or marrow. Nevertheless, individual T-cell clones can significantly expand and may allow for demonstration of oligoclonality in selected T-cell populations. These subpopulations are defined, for example, by a specific beta (B)-chain usage or other phenotypic markers. Given the diversity of the TCR recognition spectrum, the task of identifying immunodominant clonotypes derived from unique complementarity determining region-3 (CDR3) sequences is very complex. However, expanded T-cell clones likely represent immunodominant responses which can be detected on the molecular level using analysis of the individual TCR VB-chain representation, CDR3 size fragment skewing, and determination of the frequency of individual clonotypic sequences. In the future, TCR VB clonotypes may be applied as a diagnostic tool, analogous to serologic markers. As an investigative tool in hematology, molecular analysis of the TCR utilization pattern and the detection of immunodominant clonotypes represents a novel approach in the study of immune-mediated hematologic diseases, such as aplastic anemia (AA), some forms of myelodysplasia (MDS), anti-leukemic immune surveillance, graft-versus-leukemia effects and graft-versus-host disease (GvHD).

Mitochondria play a central role in apoptosis induced by alpha-tocopheryl succinate, an agent with antineoplastic activity: comparison with receptor-mediated pro-apoptotic signaling.

alpha-Tocopheryl succinate (alpha-TOS) is a semisynthetic vitamin E analogue with high pro-apoptotic and anti-neoplastic activity [Weber, T et al. (2002) Clin. Cancer Res. 8, 863-869]. Previous studies suggested that it acts through destabilization of subcellular organelles, including mitochondria, but compelling evidence is missing. Cells treated with alpha-TOS showed altered mitochondrial structure, generation of free radicals, activation of the sphingomyelin cycle, relocalization of cytochrome c and Smac/Diablo, and activation of multiple caspases. A pan-caspase inhibitor suppressed caspase-3 and -6 activation and phosphatidyl serine externalization, but not decrease of mitochondrial membrane potential or generation of radicals. For alpha-TOS, but not Fas or TRAIL, apoptosis was suppressed by caspase-9 inhibition, while TRAIL- and Fas-resistant cells overexpressing cFLIP or CrmA were susceptible to alpha-TOS. The central role of mitochondria was confirmed by resistance of mtDNA-deficient cells to alpha-TOS, by regulation of alpha-TOS apoptosis by Bcl-2 family members, and by anti-apoptotic activity of mitochondrially targeted radical scavengers. Co-treatment with alpha-TOS and anti-Fas IgM showed their cooperative effect, probably by signaling via different, convergent pathways. These data provide an insight into the molecular mechanism, by which alpha-TOS kills malignant cells, and advocate its testing as a potential anticancer agent or adjuvant.

Expression of genes regulating angiogenesis in human circulating hematopoietic cord blood CD34+/CD133+ cells.

OBJECTIVES: Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors. MATERIAL AND METHODS: We have measured the expression of angiogenesis regulating genes angiopoietin-1 (Ang-1), angiopoietin-1 (Ang-2) and their receptor Tie-2, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in sorted population of CD34+ and CD34+/CD133+ cells from human cord blood and bone marrow, and in their differentiating progeny, using real time reverse transcriptase polymerase chain reaction. The hematopoietic differentiation of CD34+ cells was induced in semisolid or liquid differentiation supporting media containing appropriate hematopoietic growth factors. RESULTS: A higher expression of Ang-1, Ang-2, and Tie-2 mRNAs was detected in CD34+/CD133+ cord blood cells as compared with CD34-/CD133- fraction, but no expression of these genes was detected in burst-forming unit erythroid (BFU-E) nor colony-forming unit granulocyte macrophage (CFU-GM) colonies. The level of Ang-1 and Tie-2 mRNAs, but not that of Ang-2 mRNA gradually decreased during a 14-d incubation of cord blood CD34+ cells in a liquid culture. A significantly higher expression of VEGF mRNA was in BFU-E as compared with CFU-GM cell colonies and CD34+/CD133+ cells. VEGFR-1 mRNA was equally expressed in CD34+/CD133+ and CD34-/CD133- cells as well as in BFU-E and CFU-GM colonies. Expression of VEGFR-2 mRNA was detected at the borderline of method sensitivity only in CD34+/CD133+ cells. CONCLUSION: CD34+/CD133+ cord blood cells express Ang-1, Ang-2 and VEGF as well as their receptor mRNAs, suggesting a role of these cells in regulation both angiopoiesis and hematopoiesis.