Flow cytometric evaluation of measurable residual disease in chronic lymphocytic leukemia: Where do we stand?

Measurable residual disease (MRD) has emerged as a relevant parameter of response to therapy in chronic lymphocytic leukemia (CLL). Although several methods have been developed, flow cytometry has emerged as the most useful and standardized approach to measure and quantify MRD. The improved sensitivity of MRD measurements has been paralleled by the development of more effective therapeutic strategies for CLL, increasing the applicability of MRD detection in this setting. Chemotherapy and chemoimmunotherapy have firstly demonstrated their ability to obtain a deep MRD. Combined targeted therapies are also demonstrating a high molecular response rate and prospective trials are exploring the role of MRD to guide the duration of treatment in this setting. In this review we briefly summarize what we have learned about MRD with emphasis on its flow cytometric detection.

Gene signature and immune cell profiling by high-dimensional, single-cell analysis in COVID-19 patients, presenting Low T3 syndrome and coexistent hematological malignancies.

BACKGROUND: Low T3 syndrome is frequent in patients admitted to intensive care units for critical illness and pneumonia. It has been reported also in patients with COVID-19, Hodgkin disease and chronic lymphocytic leukemia. We analyzed the clinical relevance of Low T3 syndrome in COVID-19 patients and, in particular, in those with associated hematological malignancies. METHODS: Sixty-two consecutive patients, hospitalized during the first wave of SARS-CoV-2 outbreak in Sant'Andrea University Hospital in Rome, were subdivided in 38 patients (Group A), showing low levels of FT3, and in 24 patients (Group B), with normal FT3 serum values. During the acute phase of the disease, we measured serum, radiologic and clinical disease severity markers and scores, in search of possible correlations with FT3 serum values. In addition, in 6 COVID-19 patients, 4 with Low T3 syndrome, including 2 with a hematological malignancy, and 2 with normal FT3 values, we performed, high-dimensional single-cell analysis by mass cytometry, multiplex cytokine assay and gene expression profiling in peripheral blood mononuclear cells (PBMC). RESULTS: Low FT3 serum values were correlated with increased Absolute Neutrophil Count, NLR and dNLR ratios and with reduced total count of CD3+, CD4+ and CD8+ T cells. Low FT3 values correlated also with increased levels of inflammation, tissue damage and coagulation serum markers as well as with SOFA, LIPI and TSS scores. The CyTOF analysis demonstrated reduction of the effector memory and terminal effector subtypes of the CD4+ T lymphocytes. Multiplex cytokine assay indicates that mainly IL-6, IP-10 and MCAF changes are associated with FT3 serum levels, particularly in patients with coexistent hematological malignancies. Gene expression analysis using Nanostring identified four genes differently expressed involved in host immune response, namely CD38, CD79B, IFIT3 and NLRP3. CONCLUSIONS: Our study demonstrates that low FT3 serum levels are associated with severe COVID-19. Our multi-omics approach suggests that T3 is involved in the immune response in COVID-19 and coexistent hematological malignancy and new possible T3 target genes in these patients have been identified.

Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

TCD4pos lymphocytosis in rheumatoid and psoriatic arthritis patients following TNFα blocking agents.

BACKGROUND: Lymphocyte expansion and true lymphocytosis are commonly observed in the everyday clinical practice. The meaning of such phenomenon is often poorly understood so that discrimination between benign and malignant lymphocytosis remains difficult to establish. This is mainly true when lymphocytosis rises in patients affected by immune-mediated chronic inflammatory diseases under immunosuppressive treatment, conditions potentially associated with lymphomagenesis. In this brief report the development of mild T CD4pos lymphocytosis in a group of patients with chronic arthritis under anti-TNF-α treatment is described. METHODS: Two hundred eight rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients have been evaluated longitudinally for at least 1-year before and 2-years after anti-TNF-α therapy introduction for the possible appearance of a lymphocyte expansion. In patients who developed lymphocyte expansion, T, B and NK cells were analysed. RESULTS: Twenty-five out of 208 (12%) subjects developed a mild T CD4pos lymphocytosis, during anti-TNF-α therapy, which reverted after its interruption. Higher lymphocyte count, more frequent use of steroids and shorter disease duration, before biological therapy start, have emerged as risk factors for lymphocytosis development. CONCLUSIONS: This is the first longitudinal cohort study evaluating the onset of lymphocytosis in RA and PsA patients under anti-TNF-α treatment and its possible clinical relevance. A mild T CD4pos lymphocytosis has been observed in 12% of RA and PsA patients probably related to anti-TNF-α treatment as previously reported by anecdotal cases. Patients with higher baseline lymphocyte count, use of steroids and shorter disease duration before the introduction of biologic therapy, seem to be prone to develop this laboratory reversible abnormality.

MicroRNA miR-24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a.

Cell cycle progression is controlled by numerous mechanisms ensuring correct cell division. The transition from one cell cycle phase to another occurs in an orderly fashion and is regulated by different cellular proteins. Therefore an alteration of the regulatory mechanisms of the cell cycle results in uncontrolled cell proliferation, which is a distinctive feature of human cancers. Recent evidences suggest that microRNAs (miRs) may also control the levels of multiple cell cycle regulators and therefore control cell proliferation. In fact miRs are a class of small non-coding RNAs, which modulate gene expression. They are involved in numerous physiological cellular processes and most importantly accumulating evidence indicates that many miRs are aberrantly expressed in human cancers. In this report we describe that miR-24 directly targets p27(Kip1) and p16(Ink4a) in primary keratinocyte and in different cancer derived cell lines promoting their proliferation, suggesting that miR-24 is involved in cyclin-dependent kinase inhibitors post-transcriptional regulation and that upregulation of miR-24 may play a role in carcinogenesis.

Consensus for Flow Cytometry Clinical Report on Multiple Myeloma: A Multicenter Harmonization Process Merging Laboratory Experience and Clinical Needs.

Flow cytometry is a highly sensitive and specific approach for discriminating between normal and clonal plasma cells in multiple myeloma. Uniform response criteria after treatment have been established by the International Myeloma Working Group and the EuroFlow Group; however, the way in which flow cytometry data are reported has suffered from no collaborative or multicentre efforts. This study, involving 8 expert laboratories and 12 clinical hematology units of the Lazio region in Italy, aims to produce a uniform and shared report among the various Centres. From the pre-analytical phase to sample processing, data acquisition, analysis, and evaluation of the potential limitations and pitfalls of the entire process, the study reaches a final conclusion shared by laboratories and clinicians according to the most updated principles and recommendations. The aim was to identify the necessary data to be included in the clinical report by using multiple-choice questionnaires at every single stage of the process. An agreement of more than 75% of the laboratories was considered mandatory for the data to be included in the report. By ensuring the operational autonomy of each laboratory, this study provides a clear report that limits subjective interpretations and highlights possible bias in the process, better supporting clinical decision-making.

Circulating mitochondrial DNA is an early indicator of severe illness and mortality from COVID-19.

BackgroundMitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether circulating cell-free MT-DNA quantitation could be used to predict the risk of poor COVID-19 outcomes remains undetermined.MethodsWe measured circulating MT-DNA levels in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at hospital presentation. Our primary outcome was mortality. Intensive care unit (ICU) admission, intubation, vasopressor, and renal replacement therapy requirements were secondary outcomes. Multivariate regression analysis determined whether MT-DNA levels were independent of other reported COVID-19 risk factors. Receiver operating characteristic and area under the curve assessments were used to compare MT-DNA levels with established and emerging inflammatory markers of COVID-19.ResultsCirculating MT-DNA levels were highly elevated in patients who eventually died or required ICU admission, intubation, vasopressor use, or renal replacement therapy. Multivariate regression revealed that high circulating MT-DNA was an independent risk factor for these outcomes after adjusting for age, sex, and comorbidities. We also found that circulating MT-DNA levels had a similar or superior area under the curve when compared against clinically established measures of inflammation and emerging markers currently of interest as investigational targets for COVID-19 therapy.ConclusionThese results show that high circulating MT-DNA levels are a potential early indicator for poor COVID-19 outcomes.FundingWashington University Institute of Clinical Translational Sciences COVID-19 Research Program and Washington University Institute of Clinical Translational Sciences (ICTS) NIH grant UL1TR002345.

Circulating Mitochondrial DNA is an Early Indicator of Severe Illness and Mortality from COVID-19.

Mitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether the appearance of cell-free MT-DNA is linked to poor COVID-19 outcomes remains undetermined. Here, we quantified circulating MT-DNA in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at the time of hospital presentation. Circulating MT-DNA were sharply elevated in patients who eventually died, required ICU admission or intubation. Multivariate regression analysis revealed that high circulating MT-DNA levels is an independent risk factor for all of these outcomes after adjusting for age, sex and comorbidities. Additionally, we found that circulating MT-DNA has a similar or superior area-under-the curve when compared to clinically established measures of systemic inflammation, as well as emerging markers currently of interest as investigational targets for COVID-19 therapy. These results show that high circulating MT-DNA levels is a potential indicator for poor COVID-19 outcomes.

Hemopoietic recovery and infectious complications in breast cancer and multiple myeloma after autologous CD34+ cell-selected peripheral blood progenitor cell transplantation.

Autografting with CD34+ cell-selected peripheral blood progenitor cells (PBPC) is often associated with a prolonged recovery time and a higher incidence of infections. The aim of our study was to evaluate whether underlying disease influences hemopoietic recovery and the infectious complications occurring after transplantation. We studied 19 breast cancer (BC) patients and 17 multiple myeloma (MM) patients entered in a high-dose chemotherapy (HDC) program of tandem autografting with CD34+ cell-selected PBPC. PBPC were collected after mobilizing chemotherapy plus granulocyte colony-stimulating factor and were processed for selection of CD34+ cells. After selection, a median of 53% CD34+ cells was recovered with a median final purity of 92% with no significant differences between the MM (52% and 92%, respectively) and BC (53% and 89%, respectively) patients. Medians of 4.5 x 10(6)/kg CD34+ cells (BC, 4.4 x 10(6)/kg; MM, 5.4 x 10(6)/kg) and 18 x 10(4)/kg colony-forming units-granulocyte-macrophage (BC, 21 x 10(4)/kg: MM, 16 x 10(4)/kg) were reinfused after each HDC. Twenty-six patients (10 MM and 16 BC) underwent tandem autografting, and 10 patients received only 1 autograft because of inadequate collection (5 patients), clinical condition (3 patients), and refusal (2 patients). In the BC patients, the HDC regimen included a high-dose melphalan course followed by an ICE (ifosfamide, carboplatin, and etoposide) course. In the MM patients, the regimen consisted of a course of high-dose melphalan therapy and a course of ICBV (idarubicin, cyclophosphamide [Cytoxan], BCNU, and etoposide) or total body irradiation, etoposide, and Cytoxan. We found a significantly prolonged time for neutrophil recovery to > 500/microL in the MM patients (13 days versus 10 days; P < .002), whereas the times for platelet recovery to > 20,000/microL in the two groups were not different (13 days versus 12 days; not significant). No late engraftment failures and no toxic deaths were observed. The incidences of extrahematologic toxicity were similar for the two patient groups. All patients received similar anti-infection prophylaxis for 3 months after transplantation. After 12 months of observation, we found a statistically significant higher incidence of bacterial infections in MM patients in both the early (77.8% versus 48.6%; P < .034) and the late (41.1% versus 0%; P < .014) posttransplantation periods, whereas the incidences of fungal infections were similar in the two groups. Viral infections consisted of herpes zoster virus infection in 2 patients of each group, and cytomegalovirus infection was observed in 3 MM patients and no BC patients. Our experience demonstrates a prolonged neutrophil recovery time and higher incidences of bacterial and viral infections in MM patients compared with BC patients. These observations, although limited by the small sample size, suggest that the underlying disease may influence the incidence of infections after CD34- cell-selected transplantation and should be considered in the planning of appropriate antimicrobial prophylaxis in the autologous transplantation setting.

Peripheral blood lymphocyte typing as a useful tool to objectify the oral mucosa patch test in the diagnosis of allergic contact mucositis to nickel.

Nickel (Ni) exposure through the intestinal mucosa may cause a hypersensitivity reaction recently defined as allergic contact mucositis (ACM). This condition is identifiable by the oral mucosa patch test (omPT), a qualitative and subjective examination that requires clinical expertise. Our aim was to evaluate if a peripheral blood lymphocyte typing performed before and after the omPT for Ni may be able to objectify this examination for diagnostic purposes. Thirty patients with symptoms referable to the ingestion of Ni-rich foods were subjected to omPT for Ni. Before and after the omPT, each patient underwent blood sampling for the typing of total lymphocytes and their subsets (T, T helper or Th, T cytotoxic or Tc, B, natural killer or NK). Statistical analysis was performed by Student t test and receiver operating characteristic (ROC) curve analysis. According to the omPT outcomes, 18 patients were defined as Ni-sensitive and the remaining 12 as controls. In Ni-sensitive patients, the number of total, T, Th, Tc, and B lymphocytes/µL whole blood increased after the omPT (p<0.0001 for the first three, p=0.0004 and p=0.0001 for the last two lymphocyte types). No omPT-dependent lymphocyte increase was observed in controls. The post/pre omPT cell ratio, especially if calculated for Th lymphocytes, appears to be an effective index for diagnostic purposes (sensitivity=100%, specificity=83.3%, Youden index=0.833, area under curve (AUC)=0.926, p<0.0001). In conclusion, the peripheral blood lymphocyte typing with calculation of post/pre omPT cell ratio has the potential to support the omPT in diagnosing ACM, with the advantage of providing quantitative and objective data.

Immunological characterization of the allergic contact mucositis related to the ingestion of nickel-rich foods.

BACKGROUND: The ingestion of nickel (Ni)-rich foods may result in allergic contact mucositis (ACM), a not yet well defined condition identifiable by oral mucosa patch test (omPT). Our aim was to characterize immunologically the ACM taking advantage from the allergen exposure that occurs during the omPT for Ni. METHODS: Thirty-seven symptomatic patients underwent to omPT for Ni. Before and after omPT, serum and urine Ni concentrations were determined by mass spectrometry, the white blood cells were counted by hemochromocytometric assay, the peripheral lymphocyte typing was carried out by flow cytometry, total IgE and cytokine serum concentrations were measured by immunoenzymatic assays. The local lymphocyte typing was performed by immunohistochemistry only after omPT. RESULTS: According to the omPT outcomes, 25 patients were defined as Ni-sensitive and the remaining 12 as controls. After omPT, serum and urine Ni concentrations increased significantly in all patients, while a significant increment of circulating lymphocytes and neutrophils was highlighted, respectively, in Ni-sensitive and control patients. Consistently, the Th and Tc circulating lymphocytes, as well as the Th/Tc ratio increased significantly in Ni-sensitive patients after omPT. No noteworthy increment in serum concentrations of total IgE and selected cytokines was observed in any patient after omPT. The presence of CD3+, CD4+, and CD8+ cells was highlighted on the oral mucosa biopsy samples taken from Ni-sensitive patients after omPT. CONCLUSIONS: In patients with ACM, a local adaptive response with increased lymphocyte trafficking appears to be the most likely mechanism of reaction to Ni administered with the omPT.

A pilot study for the integration of cytometry reports in digital cytology telemedicine applications.

Up to date, tele-pathology in the three different forms of application, "dynamic", "static" and "virtual microscopy" has been mainly based on tele-hystology remote consulting. Today the diffusion of specialized WAN connections is guiding the research of new applications of tele-pathology. A specific analysis has been conducted, focused on digital cytology, in the biomedical laboratory of Sant'Andrea Hospital to investigate the technologies potentially useful to integrate in the LAN/WAN for telemedicine applications. Among the possible tools useful to be integrated in the LAN/WAN for telemedicine applications, the cytometry equipment available in the technical unity of cytometry has been considered important. The study finally provides a proposal for a tele-consulting architecture for the integration of cytometry reports both in the hospital LAN and the WAN for possible cooperative diagnosis and second opinion support.

Inflammation and immune response in acute aortic dissection.

OBJECTIVE: The aim of our study was to evaluate the lymphocyte subpopulations and the cytokines in the peripheral blood of patients with type-A Stanford acute aortic dissection (AAD group) and to determine whether inflammatory cells are present at the site of aortic dissection. METHODS: Thirty-five consecutive patients with type-A Stanford dissection were evaluated for haemochrome and lymphocyte subpopulations (CD3+, CD4+, CD8+, CD19+, CD4+CD25+, CD16+CD56+, CD4+CD28-, CD8+CD28-) by flow cytometry. C-reactive protein (CRP), tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, interferon-gamma (IFN-γ), and monocyte chemoattractant protein (MCP)-1 were measured by ELISA. In addition, immunohistochemical staining with cell type-specific antibodies was performed to study the inflammatory cells detected inside the aortic wall. RESULTS: In the AAD group, a significant increase in natural killer (NK) (P = 0.032), B cells (P = 0.022), and CD8+CD28- (P = 0.045) subpopulations was observed, whereas there was a significant decrease in total T lymphocytes (P = 0.004) and T helper fractions (P = 0.005). Moreover, a significant increase in CRP (P < 0.0001), IL-6 (P < 0.0001), IL-8 (P < 0.0001), IL-10 (P < 0.0001), TNF-α (P < 0.0001), and MCP-1 (P < 0.001) was observed; macrophages represented the main population detected inside the media. CONCLUSIONS: Our results strongly support the hypothesis of a pivotal role of innate immunity in type-A Stanford AAD.

Low absolute lymphocyte count is a poor prognostic factor in diffuse-large-B-cell-lymphoma.

The prognostic value of absolute lymphocytic count (ALC), has been a recent matter of debate in non-Hodgkin-lymphoma (NHL). We assessed prospectively the value of ALC at diagnosis and also after the completion of immuno-chemotherapy in 101 diffuse-large-B-cell-lymphoma (DLBCL). Analysis of prognostic factors with respect to overall survival (OS), event free survival (EFS) and progression free survival (PFS) was done by two-tailed log-rank test. The ALC cut-off value was calculated as <0.84 x 10(9)/L at diagnosis: this was a strong negative prognostic factor for OS (p = 0.0004), EFS (p < 0.00001) and PFS (p < 0.00001) and in multivariate analysis was independent from the revised-international-prognostic-index (R-IPI). ALC after chemo-immunotherapy was not of prognostic value. As R-IPI and ALC < 0.84 x 10(9)/L, were the factors better discriminating poor prognosis, a new trichotomous score (ALC/R-IPI) was built up: (1) low risk: R-IPI = very good or good and ALC < 0.84 x 10(9)/L; (2) intermediate risk: patients with at least one risk factor (R-IPI = poor or ALC < 0.84 x 10(9)/L). (3) high risk: patients with both risk factors. This new prognostic score was highly significant in univariate analysis for OS (p = 0.0002), EFS (p < 0.00001) and PFS (p < 0.00001). In multivariate analysis ALC/R-IPI was the most predictive factor for OS (OR = 2.954; p = 0.002) and EFS (OR = 2.381; p < 0.00001) and the only predictive factor for PFS (OR = 4.018; p < 0.00001). Our data, show that ALC at diagnosis has a strong prognostic relevance and is independent from the R-IPI. The new score including both values proved the most powerful predictor at multivariate analysis.