B-cell chronic lymphocytic leukemia (B-CLL) follows heterogeneous clinical courses, and several biological parameters need to be added to the current clinical staging systems to predict which patients will experience an indolent or an aggressive outcome. This study analyzed CD38 expression by flow cytometry and soluble APO1/Fas (sAPO1/Fas), Bcl-2 (sBcl-2), and CD23 (sCD23) proteins by immunoenzymatic methods to evaluate their effect on the clinical course of 168 unselected B-CLL patients. Intermediate/high risk modified Rai stages were characterized by a higher CD38 ؉ B-cell number (P ‫؍‬ .0002) and higher sCD23 levels (P < .0001). Moreover, CD38 ؉ B-cell percentages were significantly and directly associated both with ␤ 2 -microglobulin and sCD23 concentrations (P < .0001 and P ‫؍‬ .002, respectively). Both a higher tumor burden (lymphadenopathy/splenomegaly) and a lymphocyte doubling time less than 12 months were significantly associated with higher CD38 ؉ percentages (P < .0001 and P ‫؍‬ .0001, respectively). With regard to clinical outcome, progression-free survival was significantly longer (75% versus 37% at 5 years; P ‫؍‬ .00006) in patients with lower CD38 ؉ B-cell percentages. Furthermore, the risk of partial or no response to fludarabine increased with increasing CD38 expression (P ‫؍‬ .003), and a shorter overall survival (50% versus 92% at 8 years; P < .00001) characterized patients with more than 30% CD38 ؉ B-cell number. The predictive value of CD38 expression was maintained among the patients within the Rai intermediate risk group and was confirmed in multivariate analysis. Thus, the percentage of CD38 ؉ B cells appears to be an accurate predictor of clinical outcome and therefore could be used to indicate when more novel chemotherapeutic approaches are needed. , and CD19 and negative for surface CD22 and FMC7. 2 These cells overexpress the Bcl-2 gene product and are resistant to apoptosis; however, examining the relative levels of this antiapoptotic protein has not been particularly helpful in predicting clinical outcome. 3,4 The clinical course of patients with B-CLL can be quite variable, with many patients surviving for prolonged periods without any therapy, whereas others succumb rapidly despite aggressive treatment. 5 Although the 2 major staging systems have provided valuable information in addressing this clinical heterogeneity, 6,7 they have been unable to predict an indolent or aggressive course within the intermediate risk category. For this reason, several parameters such as lymphocyte doubling time (LDT), 8 serum levels of ␤ 2 -microglobulin, 9 soluble CD23 (sCD23), 10 serum thymidine kinase levels, 11 bone marrow histology, 12 and cytogenetic abnormalities 13 have been added to the current staging systems to differentiate prognostic subsets.Despite having several characteristics of naive B cells, such as sequences of V H genes in germline configuration (unmutated), B-CLL cells have been shown to have somatically mutated immunoglobulin variable region genes in at least half of the case...

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of “Treg markers”. Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1729-x) contains supplementary material, which is available to authorized users.

Microglia, unique myeloid cells residing in the brain parenchyma, represent the first line of immune defense within the central nervous system. In addition to their immune functions, microglial cells play an important role in other cerebral processes, including the regulation of synaptic architecture and neurogenesis. Chronic microglial activation is regarded as detrimental, and it is considered a pathogenic mechanism common to several neurological disorders. Microglial activation and function have been extensively studied in rodent experimental models, whereas the characterization of human cells has been limited due to the restricted availability of primary sources of human microglia. To overcome this problem, human immortalized microglial cell lines have been developed. The human microglial clone 3 cell line, HMC3, was established in 1995, through SV40-dependent immortalization of human embryonic microglial cells. It has been recently authenticated by the American Type Culture Collection (ATCC®) and distributed under the name of HMC3 (ATCC®CRL-3304). The HMC3 cells have been used in six research studies, two of which also indicated by ATCC® as reference articles. However, a more accurate literature revision suggests that clone 3 was initially distributed under the name of CHME3. In this regard, several studies have been published, thus contributing to a more extensive characterization of this cell line. Remarkably, the same cell line has been used in different laboratories with other denominations, i.e., CHME-5 cells and C13-NJ cells. In view of the fact that “being now authenticated by ATCC®” may imply a wider distribution of the cells, we aimed at reviewing data obtained with the human microglia cell line clone 3, making the readers aware of this complicated nomenclature. In addition, we also included original data, generated in our laboratory with the HMC3 (ATCC®CRL-3304) cells, providing information on the current state of the culture together with supplementary details on the culturing procedures to obtain and maintain viable cells.

We used flow cytometry to quantify minimal residual disease (MRD) in 56 patients with acute myeloid leukemia (AML) expressing a leukemia-associated phenotype. Thirty-four patients aged 18 to 60 years were entered into the AML-10 protocol (induction, consolidation, and autologous stem-cell transplantation [ASCT]), whereas 22 patients older than 60 years received the AML-13 protocol (induction, consolidation, and consolidation II). After induction, the level of MRD that was best associated with treatment outcome was 4.5 × 10−4 residual leukemic cells. However, the outcome in patients with at least 4.5 × 10−4 cells (n = 26) was not significantly different from that in patients with fewer leukemic cells (n = 30); there were 15 (58%) relapses in the first group and 12 (40%) relapses in the second. After consolidation, the most predictive MRD cutoff value was 3.5 × 10−4cells: 22 patients had an MRD level of 3.5 × 10−4 cells or higher and 17 (77%) of these patients had relapse, compared with 5 of 29 patients (17%) with lower MRD levels (P < .001). An MRD level of 3.5 × 10−4 cells or higher after consolidation was significantly correlated with poor or intermediate-risk cytogenetic findings, a multidrug resistance 1 (MDR1) phenotype, short duration of overall survival, and short duration of relapse-free survival (P = .014, .031, .00022, and .00014, respectively). In multivariate analysis, this MRD status was significantly associated with a high frequency of relapse (P < .001) and a short duration of overall (P = .025) and relapse-free survival (P = .007). ASCT did not alter the prognostic effect of high MRD levels after consolidation: the relapse rate after transplantation was 70%. Thus, we found that an MRD level of 3.5 × 10−4 cells or higher at the end of consolidation strongly predicts relapse and is significantly associated with an MDR1 phenotype and intermediate or unfavorable cytogenetic findings.

Neuropilin-1 expression identifies a subset of regulatory T cells in human lymph nodes that is modulated by preoperative chemoradiation therapy in cervical cancer Introduction CD4 + T cells that constitutively express the interleukin-2 (IL-2) receptor a-chain CD25 (regulatory T cells, Treg) and the master regulator Foxp3 transcription factor play a central and prominent role in maintaining self-tolerance and in regulating responses to infectious agents, transplantation antigens and tumour antigens. SummaryWe examined the phenotype and function of CD4 + T cells expressing the semaphorin III receptor neuropilin-1 (Nrp1) in human lymph nodes and peripheral blood. In lymph nodes, Nrp1 identified a small regulatory CD4 + CD25 high T-cell subpopulation (Nrp1 + Treg) that expressed higher levels of Forkhead box P3 (Foxp3) message and protein than Nrp1 ) Treg, and various molecular markers of activated Treg, i.e. CD45RO, human leucocyte antigen (HLA)-DR and glucocorticoid-induced tumour necrosis factor receptor (GITR). Similarly to conventional Treg, Nrp1 + Treg proliferated poorly in vitro, and exerted contact-dependent in vitro suppression of T-cell proliferation and cytokine secretion. However, Nrp1 + Treg were more efficient than Nrp1 ) Treg at inducing suppression. Nrp1 was also expressed on a small subpopulation of CD25 int and CD25 ) CD4 + T cells that expressed more Foxp3, CD45RO, HLA-DR and GITR than their Nrp1 ) counterparts. In contrast, in peripheral blood Nrp1 identified a minor CD4 + T-cell subset that did not display the phenotypic features of Treg lacking Foxp3 expression and marginally expressing CD25. Hence, the function of Nrp1 + CD4 + T cells seemingly depends on their anatomical location. In a previous report, we proposed that Treg may curb the anti-tumour T-cell response in cervical cancer. We show here that Treg and Nrp1 + Treg levels dropped in the tumour-draining lymph nodes of patients with cervical cancer following preoperative chemoradiotherapy in a direct relationship with the reduction of tumour mass, suggesting that suppressor cell elimination facilitated the generation of T cells mediating the destruction of the neoplastic cells left behind after cytotoxic therapy.

Uncommon Epidermal Growth Factor Receptor (EGFR) mutations represent a distinct and highly heterogeneous subgroup of Non-Small Cell Lung Cancers (NSCLCs), that accounts for approximately 10% of all EGFR-mutated patients. The incidence of uncommon EGFR mutations is growing, due to the wider adoption of next-generation sequencing (NGS) for diagnostic purposes, which enables the identification of rare variants, usually missed with available commercial kits that only detect a limited number of EGFR mutations. However, the sensitivity of uncommon mutations to first- and second-generation EGFR Tyrosine Kinase Inhibitors (TKIs) is widely heterogeneous and less well known, compared with classic mutations (i.e., exon 19 deletions and exon 21 L858R point mutation), since most of the pivotal studies with EGFR TKIs in the first line, with few exceptions, excluded patients with rare and/or complex variants. Recently, the third generation EGFR TKI osimertinib further revolutionized the therapeutic algorithm of EGFR-mutated NSCLC, but its role in patients harboring EGFR mutations besides exon 19 deletions and/or L858R is largely unknown. Therefore, a better knowledge of the sensitivity of uncommon mutations to currently available EGFR TKIs is critical to guiding treatment decisions in clinical practice. The aim of this paper is to provide a comprehensive overview of the treatment of NSCLC patients harboring uncommon EGFR mutations with currently approved therapies and to discuss the emerging therapeutic opportunities in this peculiar subgroup of patients, including chemo-immunotherapy combinations, next-generation EGFR TKIs, and novel targeted agents.

Nivolumab is a novel therapeutic option in NSCLC, associated with a significant survival gain compared with Docetaxel. However, predictive biomarkers are lacking. The presence of systemic inflammation has been correlated with poor outcome in many cancer types. We aimed to evaluate whether there is a correlation between some indicators of inflammation and response to Nivolumab or Docetaxel in pre-treated NSCLCs. Data of 62 patients receiving Nivolumab or Docetaxel were analyzed. Baseline neutrophilia and thrombocytosis were not associated with response. High dNLR was associated with no response to Nivolumab, but not with Docetaxel, whereas high PLR correlated with low treatment response in both groups. Among refractory patients, a higher incidence of thrombocytosis, neutrophilia, high PLR, and high dNLR levels were observed compared with the overall population. This is one of the first reports in this field and suggests that indicators of inflammation might be included together with other predictive biomarkers in the baseline evaluation of patients candidate for immunotherapy.

Summary Accumulating evidence indicates an immunosuppressive role of the thymus‐derived CD4+ T‐cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age‐matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non‐neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg‐cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg‐cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg‐cell development.