Several studies have demonstrated that pregnancy in women with beta transfusion-dependent thalassemia (TDT) is possible with positive outcome for both the mother and the fetus if appropriately planned and followed by a multidisciplinary team. The aim of this multicenter study (AIFA Study id. 1817) is to highlight whether and how improvements in the management of the disease have influenced pregnancy in various aspects. The Italian Society for Thalassemia and Hemoglobinopathies (SITE) collected data on 341 pregnancies from 247 women with TDT in Italy. Seventy-four women had two pregnancies, 18 women had three pregnancies, and 2 women had four pregnancies. The age at first pregnancy has significantly increased over time (Person's correlation coefficient Rp = 0.26, p < 0.001). Ovulation was induced by gonadotropins in 131 out of 311 cases (42.1%) and occurred spontaneously in 180 (57.9%). Conception was spontaneous in 225 out of 307 cases (73.3%), through intrauterine insemination in 30 (9.8%), and via in vitro fertilization in 45 (14.7%). The rate of spontaneous conception following spontaneous ovulation significantly increased over the years (Rp = 0.54, p = 0.011), and the risk of non-spontaneous conception correlated with higher age at conception (OR = 1.19, 95% CI = 1.12-1.27, p < 0.001). Preimplantation diagnosis was performed in 12 out of 307 cases (3.9%) for partners carrying beta-thalassemia, and heterologous gametes were used in 4.3% (13 out of 303 cases). The rate of spontaneous abortions, excluding ongoing pregnancies (9), was 9.0% (25 out of 278), and 8 patients (2.9%) opted for voluntary pregnancy termination, one due to maternal toxoplasmosis. The percentage of complication during pregnancy was not significantly increased compared to the non-thalassemic population (p>0.05). Cardiac T2* measurements were available within 6 months from the start of pregnancy in 104 cases, indicating moderate iron overload in 8 and severe in 4. These values increased significantly over the years, and after 2015, all pregnancies began with a myocardial T2* >20 ms. Both pre-pregnancy and post-pregnancy T2* values increased significantly over time (Rp = 0.27, p = 0.007, and Rp = 0.33, p < 0.001, respectively). However, of the 72 women with pre-pregnancy heart T2* ≥ 20 ms for whom postpartum values were available, 11 showed moderate accumulation, and one showed severe accumulation. Of these 12 patients, 4 had pre-pregnancy liver iron concentration (LIC) <2 mg/g d.w., 6 had LIC between 2 and 7 mg/g d.w., and 1 had LIC >7 mg/g d.w. (one patient's data not reported). Overall, pre-pregnancy LIC did not change over the years (Rp = -0.12, p = 0.27), and 19 women with known pre-pregnancy values had moderate (11 out of 88, 12.5%) or severe (8 out of 88, 9.1%) hepatic iron accumulation. Two patients experienced heart failure immediately postpartum, one in 1990 and the other recently after a twin cesarean section following years of non-adherence to iron chelation therapy. There were two stillbirths, and, of the 245 pregnancies resulting in one or more live births, 26 were twins, and 3 were triplets (in one case, an embryo was reabsorbed in the first trimester). Only 17 deliveries (7.2%) were vaginal, 181 (77.0%) were planned cesarean sections, mainly for precautionary reasons, and 37 (15.7%) were emergency cesarean sections. The number of cesarean section did not diminish overtime. Ninety-seven babies (42%) were born preterm, 132 (57.1%) at term, and in 16 cases the gestational age was unknown. Preterm births decreased over time (Rp = -0.59, p = 0.0048), as did twin births (Rp = -0.65, p = 0.0015), and birth weight increased (Rp = 0.27, p < 0.001). Thirty-six neonates had complications at birth, and 47 were admitted to the neonatal intensive care unit. Three twins, two born preterm and one at term, died in the neonatal period. Eight children had homozygous beta-thalassemia. The rate of malformations (4 out of 237, 1.7%, 95% CI: 0-5%) was not higher than that of the general population, and no correlation was found with the duration of exposure to iron chelators during pregnancy. Two patients received two doses of luspatercept during pregnancy without complications. Exclusive or mixed breastfeeding was chosen for 66 children, with an increasing trend over time (Rp = 0.40, p = 0.07). This analysis confirms that many aspects of pregnancy in women with TDT, as well as neonatal characteristics, have changed over time along with the underlying disease.

IntroductionNonhemopoietic neoplasms (NHNs) may be encountered during routine flow cytometry (FC) immunophenotyping. The clue of their presence mainly relies on detection of CD45-negative (CD45-) cells with altered scatter parameters. MethodsIn this study, we evaluated a monoclonal antibody combination conceived to characterize the CD45- population by FC, suspected of belonging to NHNs, when present. The panel included CD45 for leucocytes identification, CD326 (clones BerEP4 and HEA-125) to mark epithelial cells, CD33 to identify myeloid cells, CD138 to trace plasma cells and CD56 useful in the identification of neuroendocrine tumours. 7AAD vital dye was used to gate out dead cells. Results were correlated with cytomorphology and confirmed by histological data, if available. ResultsAmong 9422 specimens submitted for routine FC investigation, 47 samples that included fine-needle aspirates, bone marrow aspirates, tissue biopsies and body fluids had a detectable CD45- population and a sufficient cell amount to be further investigated. FC revealed the presence of CD326-positive epithelial cells in 38 specimens; altered scatter parameters and variable reactivity to the other antigens tested allowed to suspect NHNs in the remaining nine samples. The presence of NHNs was confirmed in all cases by morphology. ConclusionsThe current results show that when CD45- cells with altered scatter parameters were detected, cytometrists involved in leukaemia/lymphoma diagnosis may require further FC investigations to rapidly identify NHNs in different specimens, thus reducing the time of the immunohistochemical diagnostic workup to reach a final diagnosis.

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) is still under investigation. The aim of the present retrospective study was to assess the role of Wilms tumor gene 1 (WT1) overexpression in a large monocentric cohort of AML patients. Among 255 enrolled patients, MRD was investigated in those in complete remission (CR) with an available WT1 at baseline (>250 copies) and at two further time-points: after induction (n = 117) and prior allogeneic hematopoietic cell transplantation (allo-HCT), n = 65. Baseline BM WT1 overexpression was not associated with response to induction (p = 0.244). Median overall survival (OS) and disease-free survival (DFS) were significantly shorter in patients with > 350 WT1 copies after induction compared to those with ≤350 (HR for mortality 2.13; 95% CI 1.14–3.97, p = 0.018 and HR for relapse 2.81; 95% CI 1.14–6.93, p = 0.025). Patients with WT1 > 150 copies pre allo-HCT had a significantly higher 2-year cumulative incidence of relapse (CIR) compared to those with WT1 ≤ 150 (HR 4.61; 95% CI 1.72–12.31, p = 0.002). The prognostic role of WT1 overexpression resulted independent from other well-established risk factors. According to these results, WT1 overexpression might represent an additional MRD tool for risk stratification in patients classified nowadays in CR.

To the editor: Although the cause of short stature in children with thalassemia major is not well understood, it is believed to be multifactorial.[1][1] Many different factors including iron overload, intensive use of iron chelators, and gonadal damage may interact, making it difficult to

Objective: Fine-needle aspiration (FNA) cytology is a safe and cost-effective technique for the diagnosis of lymphoproliferative processes, especially when correlated with clinical and imaging studies. However, cytology alone may be unable to detect a lymphoid neoplastic process, as architectural features are less obvious than in histologic preparations and, in certain cases, reactive processes may mimic lymphoma. Flow cytometry (FC) has been recognized as an important ancillary technique in the diagnosis of lymphoid neoplasms and it can be used in conjunction with FNA in the evaluation of lymphoproliferative processes. Study Design: We performed a review of the published literature concerning FC applied to the detection of salivary glands and thyroid lymphoproliferative processes, which are frequently related to autoimmune diseases and difficult to diagnose by cytomorphology alone. Results: FC is able to detect and subtype non-Hodgkin lymphomas and may contribute to the exclusion of a neoplastic process in cytologically unclear cases. Conclusions: FC can be successfully applied in the differential diagnosis of lymphoproliferative processes in the head and neck region. The FNA-FC combined approach can reduce time to therapy and may prevent unnecessary surgical biopsies.

Objectives: Flow cytometry (FC) has become a useful support for cytomorphologic evaluation (CM) of fine-needle aspirates (FNA) and serous cavity effusions (SCE) in cases of suspected non-Hodgkin lymphoma (NHL). FC results may be hampered by the scarce viability and low cellularity of the specimens. Study Design: We developed a single-tube FC assay (STA) that included 10 antibodies cocktailed in 8-color labeling, a cell viability dye, and a logical gating strategy to detect NHL in hypocellular samples. The results were correlated with CM and confirmed by histologic or molecular data when available. Results: Using the STA, we detected B-type NHL in 31 out of 103 hypocellular samples (81 FNA and 22 SCE). Of these, 8 were not confirmed by CM and 2 were considered to be only suspicious. The FC-negative samples had a final diagnosis of benign/reactive process (42/72), carcinoma (27/72), or Hodgkin lymphoma (3/72). Conclusions: The STA approach allowed obtainment of maximum immunophenotyping data in specimens containing a low number of cells and a large amount of debris. The information obtained by STA can help cytomorphologists not only to recognize but also to exclude malignant lymphomas.

BackgroundTo evaluate the efficacy of the use of flow cytometry (FC) immunophenotyping together with fine‐needle aspiration cytology (FNAC) in the diagnosis of thyroid lymphoma.MethodsFC was performed in parallel with FNAC in 35 samples of suspected thyroid lymphoma over a 12 years period. Results were correlated with histological or molecular findings and follow‐up, when available.ResultsA final diagnosis of lymphoma was given in 13 of 35 (37.1%) specimens. Among the 22 cases considered negative for lymphoma by FC, 11 were diagnosed as thyroiditis by cytology, 7 as reactive, 2 were anaplastic carcinoma, and 2 cases were considered cytologically suspicious for lymphoma but were not confirmed by further investigations. Histology on core biopsy or molecular analysis was available in 12 of 13 lymphoma cases (92.3%). Data obtained by the combination cytology/FC were confirmed in all cases on histology biopsies. Correlation with histology showed a sensitivity and a specificity of 100% for the combination cytology/FC.ConclusionsFC is an important additional test that can contribute with cytology to the identification of lymphomas of the thyroid. FC can detect the presence of small neoplastic lymphocyte populations and may contribute to the diagnosis of cases in which the lymphoid infiltrate is difficult to interpret on cytology alone. © 2014 International Clinical Cytometry Society

Immunophenotyping of paucicellular samples may represent a diagnostic challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the number of tests that can be performed. Specimens such as fine needle aspirates (FNA), human body fluids (BF), cerebrospinal fluid (CSF), or ocular fluid (OF) sent for FC investigations in the case of suspicion of lymphoma, or for lymphoma monitoring, may contain very low numbers of cells. In these cases, it is mandatory to obtain the largest amount possible of useful information from a single tube. The basic protocol described in this unit provides a method that combines the use of multiple monoclonal antibodies (MAbs) with a Boolean gating strategy to identify and quantify the main lymphocyte populations, as well as to detect lymphomatous B cells or any aberrant T cell expression, if present, in paucicellular samples.

Abstract Introduction Evaluation of WT1 expression is becoming an attractive marker in acute myeloid leukaemia (AML) for minimal residual disease (MRD) detection. Recent studies correlate therapy response with WT1 copy levels in the bone marrow (BM) offering an additional tool beside multiparameter flow-cytometry (MFC). No well-known data are available regarding its impact on the outcome after hematopoietic cell transplantation (HCT). Patients/Methods One hundred and two consecutive clinically and molecularly well characterized AML patients (pts) were transplanted in a single hematology center from 2004 to 2013. Indication for allogeneic HCT included high cytogenetic and molecular risk according to WHO criteria, high leukocytosis, extramedullary manifestations or secondary AML at diagnosis. Further, primary induction failure was considered as an indication for allogeneic HCT. WT1 expression was analyzed by real time polymerase chain reaction (RT-PCR), using the standardized European LeukemiaNet method on BM samples before HCT and during each follow up control. Only pts in first CR before HCT were included in the analysis. Cumulative incidence of relapse (CIR) was estimated considering death for other causes than relapse as a competing event. Univariate and multivariate Fine & Gray Regression models were used to test the association between CIR and pre-HCT BM WT1 levels. Linearity of the relationship between CIR and the WT1 level was investigated using a mathematical transformation (restricted cubic spline). Aim of this retrospective analysis was to investigate the impact of pre-HCT BM WT1 expression in first CR pts on predicting relapse after HCT. Results A BM WT1 evaluation pre-HCT was available in 89 out of 102 pts. Among them, 62 achieved a CR after induction treatment. Relapsed pts in CR after reinduction chemotherapy (n=16) or with disease persistence pre-HCT (n=2) and pts refractory to treatment (n=9) were excluded from analysis. The patient subgroup displaying a first CR before HCT had a median age at diagnosis of 49 years (range: 20-65). Patient/donor relationship involved 26 (42%) sibling, 32 (52%) matched unrelated and 4 (6%) haplo-identical donors. On the basis of standard cytogenetics, molecular biology and clinical criteria (global disease risk) pts were classified according to the following risk groups: 54 (87%) high, 5 (8%) intermediate and 3 (5%) low risk. Acute GVHD occurred in 24 (39%) whereas chronic GVHD was documented in 18 (28%) pts. Twenty-six (42%) deaths were documented after HCT, 21 (34%) due to relapse and 5 (8%) because of treatment related mortality (TRM). Pre-HCT BM WT1 expression was correlated with CIR. A cut point of 150 WT1 copies was applied according to the slope change of relapse hazard, and subsequently used for CIR analysis. Pts displaying a pre-HCT BM WT1 level > 150 copies (n=18) had a higher CIR (2-year CIR 52.5%, 95% CI: 27.1-77.8) compared to pts with a BM WT1 level ≤ 150 copies (n=44, 2-year CIR 27.7% (95% CI: 13.4-42.1). WT1 copy level > 150 showed a significantly higher risk of relapse in univariate analysis (HR 2.9, 95% CI 1.2-6.7, p=0.014). In multivariate analysis pre-HCT BM WT1 expression was confirmed as a significant independent risk factor for CIR when adjusted for patient/donor relationship, presence of GVHD, global disease risk and competitive risk of mortality due to TRM (HR 3.4, 95% CI 1.3-8.5, p=0,010), Figure. Conclusions In the present study, pre-HCT BM WT1 levels discriminated significantly for CIR in a cohort of AML in first CR. A cut off level of 150 BM WT1 copies pre-HCT had a powerful statistically significant discriminating value to predict the risk of relapse, independently from already established risk factors. The prognostic value of WT1 was confirmed also when TRM as competitive risk for mortality was added in the multivariable model. Based on these results, WT1 is a promising candidate as MRD tool. Further prospective studies are required to confirm these results. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Most follicular lymphomas (FLs) are genetically defined by the t(14;18)(q32;q21) translocation that juxtaposes the BCL2 gene to the immunoglobulin heavy chain (IgH) 3' regulatory regions (IgH-3'RRs). Despite this recurrent translocation, FL cases are heterogeneous in terms of intratumoral clonal diversity for acquired mutations and variations in the tumor microenvironment. Here we describe an additional mechanism that contributes to inter- and intratumoral heterogeneity in FLs. By applying a novel single-molecule RNA fluorescence-based in situ hybridization (FISH) technique to detect mRNA molecules of BCL2 and IgH in single cells, we found marked heterogeneity in the number of BCL2 mRNA transcripts within individual lymphoma cells. Moreover, BCL2 mRNA molecules correlated with IgH mRNA molecules in individual cells both in t(14;18) lymphoma cell lines and in patient samples. Consistently, a strong correlation between BCL2 and IgH protein levels was found in a series of 205 primary FL cases by flow cytometry and immunohistochemistry. Inter- and intratumoral heterogeneity of BCL2 expression determined resistance to drugs commonly used in FL treatment and affected overall survival of FL patients. These data demonstrate that BCL2 and IgH expressions are heterogeneous and coregulated in t(14;18)-translocated cells, and determine the response to therapy in FL patients.

A retrospective analysis of 1,792 solid tissues suggestive of lymphoma, submitted over a 12-year period, was carried out and flow cytometry (FC) results were compared with histologic findings. The final histologic diagnosis of cases documented in this report is as follows: 1,270 non-Hodgkin's lymphomas (NHL); 17 composite lymphomas; four NHL plus carcinomas; five post-transplant lymphoproliferative disorders; 105 Hodgkin's lymphomas (HL); eight acute leukemias; 42 tissue cancers; and 341 non-neoplastic diseases. A strong correlation between morphology and FC data was observed among hematological malignancies (1,268/1,304, 97.2%) with the exception of HL. Among B-NHL, FC detection of clonally restricted B-cell allowed the identification of lymphomas that were not histologically clear and the differential diagnosis between follicular lymphoma and reactive hyperplasia. A high correlation level (r = 0.83; P < 0.0001) was obtained in comparing proliferation results obtained by FC and immunohistochemistry. Among T-NHL, FC detection of an aberrant phenotype direct histologic diagnosis in cases having less than 20% of neoplastic cells. In nine cases, FC suggested the need to evaluate a neoplastic population, not morphologically evident. Results show that FC routinely performed on tissue samples suspected of lymphomas is a fundamental adjunct to morphology in the diagnosis of NHL and may enhance the performance of the histologic evaluation so as to achieve the final diagnosis. To the best of our knowledge, this is the first report in the literature of a wide series of tissues also studied by FC.

OBJECTIVE:Lymphoid proliferations of the salivary glands can be either reactive or malignant. Diagnosis based solely on fine needle aspiration (FNA) cytology may be troublesome in view of the difficulty in distinguishing low-grade B-cell and mucosa-associated lymphoid tissue (MALT) lymphomas from reactive lymphoid proliferations. We report our experience with FNA cytology combined with flow cytometry (FC) immunophenotyping for the diagnosis of lymphoproliferative processes affecting the salivary glands.METHODS:Sixty-one FNA specimens, obtained from salivary glands over a 10-year period, were analysed by cytology and FC. The results were correlated with histological follow-up if available.RESULTS:A diagnosis of lymphoma was given in 37 of 61 (61%) specimens; 22 of 61 (36%) specimens were considered as benign/reactive or non-lymphomatous processes; two of 61 (3%) specimens were considered as suspicious for lymphoma on cytological analysis and negative on FC. Histological control was available in 23 malignant, four non-lymphomatous and one cytologically suspicious case. Data obtained by the combination of cytology and FC were confirmed in all but one case: the case suspicious on cytology received a histological diagnosis of carcinoma. Four of seven cases with small populations of clonal cells (less than 15%) were histologically confirmed as lymphoma, whereas two remain under surveillance and one was reactive. Correlation with histological data showed a sensitivity of 100% and a specificity of 83% for the combination of cytology and FC.CONCLUSIONS:FC is fundamental for the diagnosis of lymphoproliferative lesions of the salivary glands. It may solve cytologically suspicious cases and detect the presence of neoplastic B or T cells. This combined approach reduces the time to therapy and may prevent unnecessary surgical biopsies.

To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL).CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases.CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.

Aim: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). Methods and results: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. Conclusions: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.

Flow cytometry (FC) is considered a sensitive and specific technique for the detection of occult lymphoma cells in cerebrospinal fluid (CSF).

Objective: Although endoscopic ultrasound combined with fine needle aspiration (EUS-FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS-guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. Methods: Of 1560 patients having EUS-guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS-FNA. There was adequate material to perform FC analysis for all but one case. Results: EUS-FNA-FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. Conclusions: Our results show that a combination of EUS-FNA-FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep-seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non-lymphoma cases.

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.

We report two cases of diffuse large B cell lymphoma (DLBCL), both occurring in the small bowel, which coexpress PAX5, weak or no CD20 and the CD4 antigen. The CD4 was initially identified by flow cytometry and then confirmed by immunohistochemistry. CD4 is a representative marker for helper T-lymphocytes and is present on a subset of thymocytes, peripheral T cells and monocytes or macrophages. Unlike CD2 and CD5, no B cell fractions are known to express CD4. It might be hypothesized that the deregulated control of gene expression in malignant B cells, in particular PAX5, leads to the activation of some silent or repressed genes of T cell differentiation. Although lineage infidelity is described in some B lymphomas, it remains as an uncommon phenomenon; to our knowledge, cases reported here are the first two cases of DLBCL of the gastrointestinal tract coexpressing the CD4 antigen to be described to date.