Circulating endothelial progenitors are increased in COVID-19 patients and correlate with SARS-CoV-2 RNA in severe cases.

BACKGROUND: During the course of COVID-19, the disease caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thrombotic phenomena and/or diffuse vascular damage are frequent, and viral elements have been observed within endothelial cells. OBJECTIVES: CD146 + circulating endothelial cells (CD146 + CECs) and their progenitors (CEPs) are increased in cardiovascular, thrombotic, infectious, and cancer diseases. The present study was designed to investigate their kinetics in novel coronavirus (COVID-19) patients. METHODS: We used a validated flow cytometry procedure to enumerate viable and apoptotic CD146 + CECs and CEPs in COVID-19 patients during the course of the disease and in patients who recovered. RESULTS: Viable CEPs per milliliter were significantly increased in COVID-19 patients compared with healthy controls. This increase was observed in patients with mild symptoms and not further augmented in patients with severe symptoms. In patients who recovered, CEPs decreased, but were in a range still significantly higher than normal controls. Regarding mature CD146 + CECs, in COVID-19 patients, their absolute number was similar to those observed in healthy controls, but the viable/apoptotic CD146 + CEC ratio was significantly different. Both mild and severe COVID-19 patients had significantly less apoptotic CD146 + CECs compared with healthy controls. Patients who recovered had significantly less CD146 + CECs per milliliter when compared with controls as well as to mild and severe COVID-19 patients. A positive correlation was found between the copies of SARS-CoV-2 RNA in the cellular fraction and apoptotic CEPs per milliliter in severe COVID-19 patients. CONCLUSIONS: CD146 + CECs and CEPs might be investigated as candidate biomarkers of endothelial damage in COVID-19 patients.

A subpopulation of circulating endothelial cells express CD109 and is enriched in the blood of cancer patients.

BACKGROUND: The endothelium is not a homogeneous organ. Endothelial cell heterogeneity has been described at the level of cell morphology, function, gene expression, and antigen composition. As a consequence of the genetic, transcriptome and surrounding environment diversity, endothelial cells from different vascular beds have differentiated functions and phenotype. Detection of circulating endothelial cells (CECs) by flow cytometry is an approach widely used in cancer patients, and their number, viability and kinetic is a promising tool to stratify patient receiving anti-angiogenic treatment. METHODOLOGY/PRINCIPAL FINDINGS: Currently CECs are identified as positive for a nuclear binding antigen (DNA+), negative for the pan leukocyte marker CD45, and positive for CD31 and CD146. Following an approach recently validated in our laboratory, we investigated the expression of CD109 on CECs from the peripheral blood of healthy subject and cancer patients. The endothelial nature of these cells was validated by RT-PCR for the presence of m-RNA level of CDH5 (Ve-Cadherin) and CLDN5 (Claudin5), two endothelial specific transcripts. Before treatment, significantly higher levels of CD109+ CECs and viable CD109+CECs were found in breast cancer patients and glioblastoma patients compared to healthy controls, and their number significantly decreased after treatment. Higher levels of endothelial specific transcripts expressed in developing endothelial cells CLEC14a, TMEM204, ARHGEF15, GPR116, were observed in sorted CD109+CECs when compared to sorted CD146+CECs, suggesting that these genes can play an important role not only during embryogenesis but also in adult angiogenesis. Interestingly, mRNA levels of TEM8 (identified as Antrax Toxin Receptor1, Antrax1) were expressed in CD109+CECs+ but not in CD146+CECs. CONCLUSION: Taken together our results suggest that CD109 represent a rare population of circulating tumor endothelial cells, that play a potentially useful prognostic role in patients with glioblastoma. The role of CD109 expression in cancer vessel-specific endothelial cells deserves to be further investigated by gene expression studies.

Complementary populations of human adipose CD34+ progenitor cells promote growth, angiogenesis, and metastasis of breast cancer.

Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.

Circulating perivascular progenitors: a target of PDGFR inhibition.

Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells (ECs) and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumors. Similarly to what we have done with circulating ECs and progenitors, we developed a flow cytometry procedure for the enumeration of circulating PPCs and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their morphology was studied by electron microscopy, PPC specificity confirmed by reverse trascription-PCR (RT-PCR) expression of CD140b mRNA, and viability assessed by Syto16 and 7AAD. In preclinical marrow transplantation studies, 9 ± 4% of circulating PPCs were derived from the marrow donor. PPCs were increased in cancer-bearing mice and in patients affected by some types of cancer. At variance with the kinetic of circulating endothelial progenitors, high-dose cyclophosphamide reduced the number of viable PPCs. The administration of sunitinib, a drug known to inhibit PDGFR, was associated in murine models and in cancer patients with an increase of apoptotic/necrotic circulating PPC, suggesting a direct targeting of these cells. PPC enumeration might be studied as a tool for the definition of the optimal biologic dose of anti-PDGFR drugs and investigated clinically as a possible predictive/prognostic tool in patients receiving anti-PDGFR drugs.

If it is in the marrow, is it also in the blood? An analysis of 1,000 paired samples from patients with B-cell non-Hodgkin lymphoma.

BACKGROUND: Staging of B-cell non Hodgkin's lymphoma (NHL) routinely involves bone marrow (BM) examination by trephine biopsy (BM-TB). The evidence of disease in the BM-TB results in a clinical stage IV classification affecting therapeutic strategies for NHL patients. BM immunophenotyping by flow cytometry (FC) is also used, although its clinical value is still under debate. METHODS: Using FC we analyzed 1,000 paired BM aspirates and peripheral blood (PB) samples from 591 NHL patients to investigate the concordance between BM and PB. B-lymphocytes were defined monoclonal when a ratio of 0.3 < κ/l > 3 was observed. Aberrant immunophenotypes present in the B-cell subpopulation were also investigated. BM-TB was also performed in 84.1% of samples (841/1000), and concordance between BM-TB and BM-FC was evaluated. Concordance was defined as the presence of a positive (in terms of disease detection) or negative result in both BM-FC and PB-FC or BM-TB and BM-FC. RESULTS: Using FC, the overall concordance between BM and PB was 95%. Among the discordant cases (ie presence of neoplastic B-lymphocyte in the BM but under the sensibility of the technique in the PB) the most frequent diagnosis was Waldenstrom's macroglobulinemia (WM, accounting for 20.8% of all discordant cases). The expression of CXCR4, a receptor involved in B-cell trafficking and homing, was found to be down regulated in WM compared to other NHL types, thus suggesting a possible role of CXCR4 in WM cell homing in the BM. WM excluded, FC investigation of BM and PB in NHL patients gives overlapping information.BM involvement was observed by FC in 38% of samples, and concordance between BM-FC and BM-TB was 85%. CONCLUSIONS: The finding that FC data from BM and PB samples overlap in NHL might have major implications for the design of future clinical studies and for patients' follow-up.

A single dose of Pegfilgrastim versus daily Filgrastim to evaluate the mobilization and the engraftment of autologous peripheral hematopoietic progenitors in malignant lymphoma patients candidate for high-dose chemotherapy.

Pegfilgrastim has equivalent efficacy to daily G-CSF in enhancing neutrophil recovery after chemotherapy, but conclusive data concerning its use for peripheral blood stem cell (PBSC) mobilization are lacking. From 2003 to 2008 we used high-dose chemotherapy in 64 lymphoma patients. At mobilization chemotherapy (ESHAP) the first 26 patients used unconjugated G-CSF, while the remaining 38 patients received Pegfilgrastim. At the time of harvest 25 patients collected stem cells after the use of G-CSF and 36 in the Peg group. No statistical by significant differences were observed in median peripheral CD34+ cells mobilized (77 µL versus 71 µL) and in collected PBSC (12.3 × 10(6)/kg versus 9.4 × 10(6)/kg p = 0.76). In the PEG group all patients collected the target PBSC with a single apheresis with a greater proportion of "optimal" mobilizers (83% versus 64%; p = 0.05). In conclusion a single dose of Pegfilgrastim could be a valid alternative to unconjugated G-CSF to mobilize PBSC in lymphoma patients.

Angiogenic cells, macroparticles and RNA transcripts in laparoscopic vs open surgery for colorectal cancer.

BACKGROUND: Angiogenesis is crucial for tissue repair and cancer progression. We investigated a panel of angiogenic cells, macroparticles and RNA transcripts before, during and after laparoscopic colectomy or open colectomy for colorectal cancer. RESULTS: Viable and apoptotic circulating endothelial cells were significantly increased after open but not after laparoscopic colectomy (p < 0.01). A significant decrease of circulating mRNA coding for VEGFR-C and D and PDGFR-ß was found after laparoscopic but not after open colectomy. METHODS: A total of 24 patients were enrolled. Viable and apoptotic circulating endothelial cells, progenitors and macroparticles were evaluated by flow cytometry. The number of copies of angiogenesis-related RNA transcripts we reevaluated by quantitative PCR. CONCLUSION: Open, but not laparoscopic colectomy, was associated with a significant post-operative increase in circulating endothelial cells, either apoptotic (likely due to surgery-related vascular damage) and viable (likely representing vascular remodeling). Circulating RNA copies coding for some angiogenic genes were significantly decreased after laparoscopic colectomy likely because of the removal of the tumor lesion. This decrease was not observed after open colectomy,were a more pronounced wave of angiogenesis related to wound healing was expected. These results indicate a relevant wave of angiogenesis-related cells and transcripts after open but not after laparoscopic colectomy.

Predictive Potential of Angiogenic Growth Factors and Circulating Endothelial Cells in Breast Cancer Patients Receiving Metronomic Chemotherapy Plus Bevacizumab.

PURPOSE: The association of chemotherapy and antiangiogenic drugs has shown efficacy in clinical oncology. However, there is a need for biomarkers that allow selection of patients who are likely to benefit from such treatment and are useful for indicating best drug combination and schedule. EXPERIMENTAL DESIGN: We investigated the predictive potential of six angiogenic molecules/transcripts and nine subpopulations of circulating endothelial cells (CEC) and progenitors (CEP) in 46 patients with advanced breast cancer treated with metronomic cyclophosphamide and capecitabine plus bevacizumab. RESULTS: Median time to progression was 281 days. Baseline CECs higher than the first quartile were associated with an increased time to progression (P = 0.021). At progression, CECs were markedly reduced (P = 0.0002). In the cohort of 15 long-term responders, who progressed later than 1 year after beginning of therapy, circulating vascular endothelial growth factor (VEGF)-A levels measured after 2 months of therapy were significantly reduced, and there were significant trends toward lower levels of PDGF-BB, CEPs, and CECs. At the time of progression, angiogenic growth factors VEGF-A and basic fibroblast growth factor were significantly increased. CONCLUSIONS: Baseline CECs (likely reflecting an active vascular turnover) predicted a prolonged clinical benefit. At the time of relapse, a pattern of decreased CECs and increased angiogenic growth factors suggested a switch toward a different type of cancer vascularization. VEGF-A and basic fibroblast growth factor levels after 2 months of therapy were also useful to identify patients whose disease was likely to progress. These biomarkers are likely to be useful for treatment selection and might be incorporated in design of future studies. (Clin Cancer Res 2009;15(24):7652-7).

Lymphocytic toxicity in patients after peptide-receptor radionuclide therapy (PRRT) with 177Lu-DOTATATE and 90Y-DOTATOC.

PURPOSE: Peptide-receptor radionuclide therapy (PRRT) with somatostatin analogs is an efficient new tool in patients with neuroendocrine tumors, with low risk of toxicity. Since lymphocytes express somatostatin receptors, the aim of this study was to evaluate lymphocytic toxicity after PRRT. METHODS: From May 2005 to May 2007, 16 patients affected by neuroendocrine tumors received PRRT with (90)Y-DOTATOC (9), (177)Lu-DOTATATE (5), or both (2). Absolute count, percentage of leukocytes and lymphocytes, and lymphoid subsets (B, T, and NK) were tested at baseline and until 90 days after treatment. RESULTS: A significant lymphoid toxicity (G2-3), mainly affecting B-cells, was observed. It was particularly evident after (90)Y-DOTATOC. Toxicity resulted in being transient and resolved completely at the end of the follow-up (90 days). CONCLUSION: Lymphocyte toxicity in PRRT is mainly due to the selective targeting on B-cells. The relative sparing of T-lymphocytes could explain the absence of clinical side-effects in these patients, such as increased risk of infections. These findings open interesting perspectives in the treatment of B-cell lymphoproliferative disorders.

Validation of a standardized method for enumerating circulating endothelial cells and progenitors: flow cytometry and molecular and ultrastructural analyses.

PURPOSE: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. EXPERIMENTAL DESIGN: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. RESULTS: Sorted DNA/Syto16(+)CD45(-)CD31(+)CD146(+) CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 +/- 171/mL in healthy subjects (n = 37) and 951 +/- 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 +/- 14% in healthy subjects and 43 +/- 23% in cancer patients (P < 0.0001). CEPs were 181 +/- 167/mL in healthy donors and 429 +/- 507/mL in patients (P = 0.00019). Coefficients of variation were 4 +/- 4% (intrareader), 17 +/- 4% (interreader), and 17 +/- 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 +/- 8% (intrareader), 16 +/- 10% (interreader), and 26 +/- 16% (variability over 0-14 days of frozen storage), respectively. CONCLUSIONS: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

ABL1 amplification in T-cell acute lymphoblastic leukemia.

ABL1 amplification, due to a cryptic episomal translocation NUP214/ABL1, is a novel finding in T-cell acute lymphoblastic leukemia (ALL). Here we report on the incidence and clinical features of this genetic defect in a series of 30 consecutive adult T-cell ALL patients. Multiple copies of the ABL1 gene were detected in two patients (6.6%), one with the karyotype 46,XY,t(1;3)(p36;p21),del(6)(q23)/46,XY and the other without analyzable metaphases. Metaphase/interphase fluorescence in situ hybridization (FISH) detected multiple uncountable signals corresponding to ABL1 in mitotic cells and nuclei from both patients. In one patient, no signals corresponded with the 9p21 chromosomal region, which contains the p16INK4a gene, and in the other one signal was observed. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) demonstrated that in these patients ABL1 gene expression was 14- and 18-fold greater than in normal controls, and returned to normal levels only when complete remission was achieved. We reached the following conclusions: (1) FISH is the only technique that promptly identifies T-cell ALL patients with ABL1 amplification, (2) quick identification with FISH is fundamental in the clinic because this T-cell ALL subset is imatinib sensitive but may become resistant due to development of additional mutations, and (3) ABL1 quantitative RT-PCR may be easily applied to monitor minimal residual disease.

Molecularly targeted therapy in acute myeloid leukemia.

Meaningful progress has been made toward clarifying the molecular steps in the pathogenesis of acute myeloid leukemia (AML). Chromosome studies have established that translocations/inversions are the most common cytogenetic defects in AML. Cloning of chromosome breakpoints has shown that genes involved in the chromosome abnormalities are transcription factors, functional loss of which alters chromatin configuration and results in the disruption of myeloid differentiation. However, transgenic animal models have demonstrated that AML-specific translocations/inversions alone are insufficient to cause overt leukemia, which occurs only when point mutations affecting receptor tyrosine kinases (RTKs) develop. Therefore, development of AML is now considered a two-step process in which RTK mutations provide a proliferative and a survival advantage to a clonal cell population already marked by impaired differentiation. In addition, more accurate definition of such genetic lesions has led to a more precise insight as to how such lesions interact with cellular signaling pathways that are aberrantly regulated in AML. All these new data have profound clinical and therapeutic implications and will surely translate into the development of molecules that target specific mutations or signal transduction pathways.