Roberta Della Pepa

Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal disorders characterised by ineffective haematopoiesis with high risk of leukaemia progression. The relevance of immune‐dysregulation for emergence, dominance and progression of dysplastic clones has been suggested, but valuable criteria to obtain insight into these connections are lacking. This study showed significant increase of CD8 lymphocytes and mature B cells in the bone marrow (BM) compared to peripheral blood (PB) of low risk MDS patients. Different BM levels of Regulatory T cells (Treg) identified two sub‐groups in these patients; only the sub‐group with lower Treg percentage showed BM recruitment of CD8 lymphocytes. Different levels of CD54 on BM CD8 cells revealed two sub‐groups of Intermediate‐1 (Int‐1) patients. The sub‐group with higher CD54 expression on BM CD8 showed high levels of this molecule also on CD4 cells. BM recruitment of CD8 lymphocytes in the low risk group and/or the presence of high CD54 expression on BM CD8 in Int‐1 patients were associated with more pronounced dyserythropoiesis and erythropoietin treatment. Our data shed light on the involvement of immune‐mediated mechanisms in Low and Int‐1 risk MDS patients and suggest that BM versus PB levels of immune effectors could represent useful criteria for a more homogeneous grouping of MDS patients.

Eculizumab treatment modifies the immune profile of PNH patients

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced. Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged.

Intravenous versus subcutaneous immunoglobulin replacement in secondary hypogammaglobulinemia.

In this study, we compared intravenous immunoglobulins (IVIG) and subcutaneous immunoglobulins (SCIG) in terms of serum IgG concentration and incidence of infections in patients with hypogammaglobulinemia secondary to chemo-immunotherapy regimens including the anti-CD20 monoclonal antibody rituximab. Fourteen patients with a B-cell lymphoproliferative disease treated for at least six months with a rituximab-including chemo-immunotherapy regimen were recruited. Mean serum levels of IgG were higher during replacement therapy than at the end of rituximab treatment (p<0.001). Moreover, serum IgG level was higher during replacement therapy with SCIG than with IVIG (p<0.001). No differences in the incidence of infections were observed. Although the non-randomized design and the small number of patients do not allow definitive conclusions to be drawn, study results suggest that higher mean serum IgG levels are reached when using the subcutaneous route after a switch from the intravenous regimen, and that IVIG and SCIG offer comparable protection against infections.

HLA-E and HLA class I molecules on bone marrow and peripheral blood polymorphonuclear cells of myelodysplatic patients

Relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones in myelodysplastic syndromes (MDS) was suggested, but valuable or predictive criteria on this involvement are lacking. We previously reported that reduced T-regulatory cells (Treg) and high CD54 expression on T cell identify a sub-group of patients in whom an immune-pathogenesis might be inferred. Here, we suggest the occurrence of immune-selection of dysplastic clones in a subgroup of MDS patients, with reduced HLA-I and HLA-E on PMN, and propose that an altered immune profile might represent a valuable criterion to classify Low/Int-1 patients on the basis of immune-pathogenesis of MDS.

Pegfilgrastim in primary prophylaxis of febrile neutropenia following frontline bendamustine plus rituximab treatment in patients with indolent non-Hodgkin lymphoma: a single center, real-life experience

BackgroundIn this prospective study, the impact of granulocyte colony-stimulating factors (G-2 CSF) administered during induction treatment with bendamustine plus rituximab for indolent non- Hodgkin Llymphoma (NHL) was evaluated by comparing patients who received secondary prophylaxis with filgrastim (control group) versus. patients who received pegfilgrastim as primary prophylaxis (peg-group). The primary endpoint was the incidence rate of febrile neutropenia (FN)- related chemotherapy disruptions (regarding dose-dense and/or dose-intensity of schedule). The Ssecondary endpoint included days of hospitalization due to FN, and G-CSF-related side effects (grade ≥3 WHO toxicity criteria) in each group.MethodsOne hundred twenty-two: 122 consecutive patients, with untreated indolent NHL, were referred to our outpatient unit for remission induction immuno-chemotherapy with bendamustine-rituximab. During the first period, 61 patients received secondary prophylaxis with filgrastim, given “on demand” if ANC was <1000/mm3. During the second period, 61 patients received primary prophylaxis with pegfilgrastim in a single administration.ResultsPegfilgrastim was significantly associated with fewer incidence rate of FN-related chemotherapy disruptions (11.4% in the control group vs. 1.6% in the peg-group, p = 0.04) and fewer days of hospitalization due to FN (median number 18 days in the control group vs. 6 in the peg-group, p = 0.04). In terms of G-CSF-related extra-hematological grade III side effects, no significant difference has been found in the two groups (9.8% in the control group vs. 11.5% in the peg-group, p = 0.77). Only one patient stopped the treatment in the peg-group due to intolerance.ConclusionsIn patients with indolent NHL, in front-line treatment with bendamustine plus rituximab, primary prophylaxis with pegfilgrastim seems to reduce the incidence of chemotherapy disruptions due to FN, and the days of hospitalization. Moreover, it is well- tolerated and may increase the opportunity to maintain the planned schedule of treatment. These results make pegfilgrastim an advantageous option in most cases both in terms of cost-effectiveness and quality of life. These preliminary observations need to be validated by controlled clinical trials.

Diagnostic-driven antifungal approach in neutropenic patients at high risk for chronic disseminated candidiasis: preliminary observations on the role of 1,3-β-D-glucan antigenemia and multiphasic contrast-enhanced computed tomography

Dear Editor, Chronic disseminated candidiasis (CDC) is a critical form of invasive fungal infection (IFI) that affects mainly the liver, spleen, and, occasionally, kidneys [1]. Typical clinical, microbiological, and/or radiological manifestations have late onset, leading frequently to misdiagnosis [1, 2]. A late diagnosis leads to a delay in starting an effective antifungal therapy against Candida infection resulting in a severe morbidity and high mortality [3]. Recent studies have shown effective alternatives to traditional microbiological and radiological procedures for the diagnosis of CDC, in particular, 1,3-β-Dglucan (BDG) antigenemia and contrast-enhanced computed tomography (CE-CT) [4, 5]. The preemptive approach, based on the routine surveillance with serum BDG and hepatosplenic CE-CT, has been proposed for obtaining a reliable and early diagnosis of CDC, and for establishing a proper antifungal treatment [6]. However, guidelines give moderate evidence to support recommendation for the use of such approach in clinical practice [7]. In our institution, patients with acute leukemia at high-risk for CDC underwent diagnosticdriven approach, which was based on the identification of the clinical settings requiring intensive diagnostic efforts. Between January 2013 and December 2014, 20 of 24 consecutive patients older than 18 years with several risk factors for Candida infection (and on fluconazole prophylaxis), who underwent intensive chemotherapy or autologous stem cell transplantation (SCT), developed febrile neutropenia (FN). In the event of FN, a standard diagnostic work-up (SDWU) was performed as already reported [8]. Patients with persisting fever after 4–6 days of broad-spectrum antibiotics or patients with fever relapsing after 48 h of defervescence underwent a baseline diagnostic work-up (BDWU) including serum BDG antigenemia (Fungitell, manufacturer; Associated of Cape Cod, Inc., East Falmouth, MA). An intensive diagnostic work-up (IDWU) was performed in patients with a positive BDG test (≥ 80 pg/mL). It includedmultiphasic CE helical CT of the liver and spleen, as already described [9]. Among this series of 24 patients, we report a patient suffering from CDC, which was definitively proven by ultrasonography (US)-guided core needle cutting biopsy (CNCB) of the liver. The most important aspect, revealed by the clinical case herein described, is the crucial role of the serum BDG test and the hepatosplenic multiphasic CE helical CT for the early diagnosis of deep-seated Candida infection.

Rituximab in a risk-adapted treatment strategy gives excellent therapeutic results in nodular lymphocyte-predominant Hodgkin lymphoma

Nodular lymphocyte-predominant Hodgkin lymphoma (NLP-HL) is a rare variant of Hodgkin lymphoma, histologically characterized by prominent lymphocytic proliferation mixed with histiocytes that form at least a partially nodular pattern. Due to its rarity, this type of lymphoma still lacks consolidated and widely accepted treatment guidelines. Current first-line therapy approaches are often based on radiotherapy (RT) alone for non-bulky early-stage NLP-HL, while chemotherapy regimens [ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) or CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone)] plus RT have been widely used for the most advanced stages (Eichenauer et al, 2014; Hoppe et al, 2015). The observation that malignant lymphocytes of NLP-HL, although negative for CD15 and CD30, consistently express CD20 (Hartmann et al, 2014), provides the rationale for the inclusion of anti-CD20 antibody rituximab in the treatment of this disease as a low toxic component for replacing chemotherapy and/or RT. Therefore, the outcome and side effects of 12 consecutive patients with newly diagnosed NLP-HL, who were treated in our Institution using a risk-adapted, rituximab-based protocol, were compared with those of a historical cohort of NLPHL patients, previously treated with a conventional modality approach, i.e. chemotherapy and/or RT (Table I). The clinical features of these two groups of patients were similar(see Supplementary data), except that the rituximab group presented more sub-diaphragmatic disease (P = 0 01) and the historical cohort included more patients with bulky disease (P = 0 03), thus balancing adverse prognostic factors (see Table SI). All 24 patients showed the typical CD20 positive stain in at least 30% of tumour cells without either CD15 or CD30 reactivity (Swerdlow et al, 2008). Comparing the treatments in the two series, 9 historical patients, and no patients of the rituximab group, underwent involved-field (IF)RT; cytotoxic agent administration was spared in 42% of the patients of the rituximab group (Table I). At final restaging, 23 of the 24 patients (95 8%) were in complete remission [CR; (Cheson et al, 2007)], while one historical patient showed refractory disease and was underwent rescue therapy. Patients who received rituximab-based risk-adjusted therapy showed excellent response to the treatment and had better progression-free survival (PFS, P = 0 04) but not overall survival (OS) than those of the historical cohort (see Figure S1). Indeed, during follow-up (median 4 3 years; range, 0 5–8 2 in the whole cohort), four patients, all belonging to the historical cohort and treated with ABVD IFRT, showed refractory (n = 1) or recurrent disease (n = 3). These results confirm that NLP-HL, despite a more indolent clinical behaviour than classical HL, may show late multiple relapses after conventional therapy particularly in advanced stage patients. Therefore, there is a need for novel treatment approaches that, along with low toxicity, have an increased efficacy to improve the overall prognosis. In our more recent series of NLP-HL patients, the use of rituximab as single agent in the induction phase was restricted only to stage I and II patients without risk factors. However, in order to minimize the possibility of relapses, we prolonged the anti-CD20 antibody treatment, with eight further infusions (once every 3 months) distributed over a 2year maintenance therapy in these patients with early favourable disease. Therefore, as seen in indolent non-Hodgkin lymphoma, the use of rituximab-maintenance in early favourable NLP-HL seems to delay or avoid relapse. In the remaining patients, according to a risk-adapted treatment approach, rituximab therapy was combined with polychemotherapy, giving a total of four cycles of ABVD to patients with stages I or II disease but with ≥1 risk factors (early unfavourable) and six cycles of ABVD to those who presented with advanced stages (Fig 1). Importantly, the inclusion of rituximab in this therapeutic scheme appears to provide long-term protection from relapse when compared with the classical chemo-radiotherapy. Notably, we observed a higher rate of grade 3–4 neutropenia in the historical cohort (54%) than the rituximab group (10%; P = 0 009); non-haematological toxicity was recorded only in the historical cohort and was prevalently RT-related (4/8 thyroid disease, 2/8 lung fibrosis and 2/8 osteoarthritic degeneration). Over the study period, one patient died due to bleomycin-induced pneumonitis following the last cycle of R-ABVD. These results underscore the role of anti-CD20 antibody in NLP-HL, which provides substantial benefit to many patients, avoiding the need for RT and cytotoxic agent treatment or, at least, potentially delaying the need for both, in those with early favourable stage disease. In the recent study by Advani et al (2014), rituximab was given as single agent once per week for 4 weeks to early-stage NLPHL patients, giving an overall response rate (ORR) of 100% with a 67% CR rate. The last 16 patients also had received a maintenance phase with rituximab (MR) once per week for

Reply to the letter to the editor “chronic disseminated candidiasis” by Kenneth Rolston

Dear Editor, We would like to thank Kenneth Rolston for his comments regarding our recent Supportive Care in Cancer article on chronic disseminated candidiasis (CDC) in patients with hematological malignancies on the behalf of SEIFEM (Sorveglianza Epidemiologica Infezioni Fungine in Ematologia) group [1]. We acknowledge the small sample size (N = 20) and the retrospective nature of the study, which is probably not enough capable to lead to significant modifications of the CDC treatment recommendations. However, we would like to underline some aspects. First, the guidelines of the Infectious Diseases Society of America (IDSA) strongly recommend the first line therapy of CDC with lipid formulation amphotericin B (AmB) 3–5 mg/kg daily [2]. Our data suggest that high-dose (HD) liposomal AmB (5 mg/kg daily) is the better choice for the treatment of CDC. This is likely due to the fungicide action of HD liposomal AmB in the liver and spleen derived from better tissue concentrations (target of liposomal formulation: reticuloendothelial system) than that of triazoles and echinocandins [3]. In addition, the 5 mg/kg daily dosage for liposomal AmB may be useful for less susceptible species, such as Candida glabrata and Candida krusei [2]. On the other hand, in our series, the majority of patients were receiving triazoles prophylaxis and thus had an increased risk of developing infection with a fluconazoleresistant organism [2]. Moreover, according to the IDSA guidelines, fluconazole (6 mg/kg daily) should be administered only for maintenance therapy [2]. Second, 13/20 (65%) patients received diagnosis of probable CDC according to standard criteria, i.e., an alkaline phosphatase increase, hepatic and/or splenic nodules with typical bull’s eye aspect (seen at imaging tools), and blood cultures positive for Candida spp. (no polymicrobic sepsis occurred in our series) [4]. Such patients had negative serum galactomannan monitoring and negative thorax radiological assessments; three cases had a serum βD-glucan assay >80 pg/ml (270, 520, and 370 pg/ml, respectively). Altogether, it is very unlikely that these findings may represent infections due to other organisms, particularly molds. According to the policy of the SEIFEM group, when clinically indicated, we performed liver biopsy using a Menghini-type automatic fine-cutting needle (1.2 mm, 18G) under color ultrasound guidance, as already reported [5, 6]. In fact, the remaining seven patients underwent a mini-invasive procedure that was well tolerated with no discomfort and provided reliable information regarding liver histology, leading to the definitive diagnosis of CDC. Third, both cases no. 11 and no. 20 died early as a result of CDC (before the definitive microbiological results from blood samples); they were receiving empirical antifungal treatment, respectively, with fluconazole and itraconazole. Finally, no liposomal AmB-related toxicity of grade ≥3, according to the Common Terminology Criteria for Adverse Events (CTCAE), occurred in our series [7]. * Roberta Della Pepa roberta.dellapepa@unina.it

Ultrasonography-driven combination antibiotic therapy with tigecycline significantly increases survival among patients with neutropenic enterocolitis following cytarabine-containing chemotherapy for the remission induction of acute myeloid leukemia

Neutropenic enterocolitis (NEC) is an abdominal infection reported primarily in patients with acute myeloid leukemia (AML) following chemotherapy, especially cytarabine, a notable efficacious cytotoxic agent for AML remission. Specific data regarding the impact of different cytarabine schedules and/or antibacterial regimens for NEC are sparse. The aim of the study was to identify the predictors of outcome within 30 days of NEC onset. NEC episodes were retrospectively pinpointed among 440 patients with newly diagnosed AML hospitalized in our Institution, over a 10‐year period, for receiving chemotherapy protocols with 100–6000 mg/m2 daily of cytarabine. Two subgroups, survivors versus nonsurvivors, were compared by using logistic regression analysis. NEC was documented in 100 of 420 (23.8%) analyzed patients: 42.5% had received high‐dose cytarabine, whereas 19% and 15% intermediate‐dose and standard‐dose cytarabine, respectively (P < 0.001). The 30‐day NEC attributable mortality rate was 23%. In univariate analysis, antileukemic protocols containing robust dosages of cytarabine were significantly associated with high mortality (P < 0.001); whereas, standard‐dose cytarabine and prompt initiation (at the ultrasonographic appearance of intestinal mural thickening) of NEC therapy with antibiotic combinations including tigecycline were significantly associated with low mortality. In multivariate analysis, high‐dose cytarabine‐containing chemotherapy was the independent predictor of poor outcome (odds ratio [OR]: 0.109; 95% confidence interval [CI]: 0.032–0.364; P < 0.001), whereas ultrasonography‐driven NEC therapy with antibiotic regimens including tigecycline was associated with a favorable outcome (OR: 13.161; 95% CI: 1.587–109.17; P = 0.017). Chemotherapy schedules with robust dosages of cytarabine for AML remission are associated with a high rate of NEC incidence and attributable. Vigorous antibacterial therapy, triggered off pathologic ultrasonographic findings, with drug combinations which have broad antimicrobial coverage and good gut penetration, specifically those also including tigecycline, may be effective in improving 30‐day survival rate after NEC onset.