CD3+ graft cell count influence on chronic GVHD in haploidentical allogeneic transplantation using post-transplant cyclophosphamide.

The effects of graft or donor characteristics in haploidentical hematopoietic cell transplantation (HCT) using post-transplant cyclophosphamide (PT-Cy) are largely unknown. In this multicenter retrospective study we analyzed the correlations between graft cell composition (CD34+, CD3+) and donor features on transplant outcomes in 234 patients who underwent HCT between 2010 and 2016. On multivariate analysis, the use of peripheral blood stem cells (PBSC) was associated with an increased incidence of grade 2-4 acute GVHD [HR 1.94, 95% confidence Interval (CI) = 1.01-3.98, p = 0.05]. An elevated CD3+ graft content was associated with an increased incidence of all-grade chronic GVHD [HR 1.36 (95% CI = 1.06-1.74), p = 0.01]. This effect was confirmed only for the PBSC graft group. A higher CD34+ graft content had a protective role on non-relapse mortality [HR 0.78 (95% CI = 0.62-0.96), p = 0.02] but this was confirmed only for the bone marrow (BM)-derived graft cohort. Donor characteristics did not influence any outcomes. GVHD prophylaxis should be modulated accordingly to CD3+ graft content, especially when a PBSC graft is used. These results need further validation in prospective trials.

Impact of HLA-G polymorphism on the outcome of allogeneic hematopoietic stem cell transplantation for metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) is particularly sensitive to immune intervention. HLA-G, a non-classical HLA class I molecule with immunomodulatory properties, has been studied with regard to outcome after hematopoietic stem cell transplantation (HSCT), in particular the 14 bp insertion/deletion polymorphism in the 3' untranslated region. Here we analyzed n=56 patients affected by metastatic RCC who received an allogeneic HSCT between 1998 and 2006 in Milano, Marseille, Clermont-Ferrand and Stockholm. The 14 bp polymorphism was analyzed in correlation with overall survival (OS), PFS, acute and chronic GvHD. With a median follow-up of 13 years, a trend towards better outcome was observed when homozygosity for the 14bp-del allele was present: multivariate hazard ratio was 0.50 (95% confidence interval (CI): 0.23-1.13; P=0.10) and 0.57 (95% CI: 0.26-1.26; P=0.17) for OS and PFS, respectively, when 14bp-del/del was compared with 14bp-ins/X. Further exploratory analysis revealed a significant association between T/C at p3003 and improved OS (P=0.05) and PFS (P=0.006) compared with T/T. To our knowledge this is the first study on HLA-G and outcome after HSCT for a solid malignancy. After a coordinated multicenter study, we found that the more tolerogenic polymorphisms (14bp-del/del) is associated with better PFS and OS. The finding on p3003 deserves further investigation.

Control of infectious mortality due to carbapenemase-producing Klebsiella pneumoniae in hematopoietic stem cell transplantation.

Carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections are an emerging cause of death after hematopoietic stem cell transplantation (HSCT). In allogeneic transplants, mortality rate may rise up to 60%. We retrospectively evaluated 540 patients receiving a transplant from an auto- or an allogeneic source between January 2011 and October 2015. After an Institutional increase in the prevalence of KPC-Kp bloodstream infections (BSI) in June 2012, from July 2012, 366 consecutive patients received the following preventive measures: (i) weekly rectal swabs for surveillance; (ii) contact precautions in carriers (iii) early-targeted therapy in neutropenic febrile carriers. Molecular typing identified KPC-Kp clone ST512 as the main clone responsible for colonization, BSI and outbreaks. After the introduction of these preventive measures, the cumulative incidence of KPC-Kp BSI (P=0.01) and septic shocks (P=0.01) at 1 year after HSCT was significantly reduced. KPC-Kp infection-mortality dropped from 62.5% (pre-intervention) to 16.6% (post-intervention). Day 100 transplant-related mortality and KPC-Kp infection-related mortality after allogeneic HSCT were reduced from 22% to 10% (P=0.001) and from 4% to 1% (P=0.04), respectively. None of the pre-HSCT carriers was excluded from transplant. These results suggest that active surveillance, contact precautions and early-targeted therapies, may efficiently control KPC-Kp spread and related mortality even after allogeneic HSCT.

Autologous Islet Transplantation in Patients Requiring Pancreatectomy: A Broader Spectrum of Indications Beyond Chronic Pancreatitis.

Islet autotransplantation (IAT) is usually performed in patients undergoing pancreatic surgery for chronic pancreatitis. In the present series, IAT was offered also to patients undergoing pancreatic surgery for both nonmalignant and malignant diseases, having either completion pancreatectomy as treatment for severe pancreatic fistulas (n = 21) or extensive distal pancreatectomy for neoplasms of the pancreatic neck (n = 19) or pancreatoduodenectomy because of the high risk of pancreatic fistula (n = 32). Fifty-eight of 72 patients who were eligible to this broader spectrum of indication actually received IAT. There was no evidence of a higher-than-expected rate of major complications for pancreatectomy. Forty-five patients receiving IAT were still alive at the time of the last scheduled follow-up (1375 ± 365 days). Eighteen (95%) of 19 and 11 (28%) of 39 patients reached insulin independence after partial or total pancreatectomy, respectively. The metabolic results were dependent on the transplanted islet mass. Thirty-one of 58 patients had malignant diseases of the pancreas or periampullary region, and only three patients developed ex novo liver metastases after IAT (median follow-up 914 ± 382 days). Our data demonstrate the feasibility, efficacy, and safety of IAT for a broader spectrum of clinical indications beyond chronic pancreatitis.

Haploidentical HSCT: a 15-year experience at San Raffaele.

Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular-based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed.

Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation.

Genomic loss of the mismatched human leukocyte antigen (HLA) is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their 'classical' counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (hazard ratio (HR): 10.16; confidence interval (CI): 2.65-38.92; P=0.001), whereas older patient ages had a protective role (HR: 0.16; CI: 0.05-0.46; P=0.001). A weaker association with HLA loss was observed for graft T-cell dose and occurrence of chronic graft-versus-host disease. Outcome after 'classical' and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T-cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Early molecular diagnosis of aspergillosis in a patient with acute myeloid leukaemia.

Diagnosis of invasive fungal infection remains challenging. Here we report a case of early diagnosis of invasive aspergillosis in a neutropenic patient affected by acute myeloid leukaemia, achieved through the detection of Aspergillus fumigatus species-specific ribonucleic acid sequences by a sensitive multiplex real-time polymerase chain reaction-based molecular assay. Thanks to the early diagnosis, targeted therapy was promptly established and the severe fungal infection controlled, allowing the patient to subsequently receive allogeneic hematopoietic stem cell transplantation from a haploidentical donor, her only curative option. Also in this instance, targeted secondary antifungal prophylaxis with voriconazole avoided any other fungal infection afterwards. This report suggests how the implementation of molecular assays in combination with routine diagnostic procedures, can improve microbiological diagnosis in sepsis, particularly in case of fungal infection, difficult to detect with standard microbiological culture methods.

Stem cell mobilization in patients with newly diagnosed multiple myeloma after lenalidomide induction therapy.

Lenalidomide has raised concerns regarding its potential impact on the ability to collect stem cells for autologous stem cell transplantation, especially after prolonged exposure. The use of cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) to mobilize peripheral blood stem cells may overcome this concern. In newly diagnosed multiple myeloma (MM) patients, we investigated the influence of lenalidomide on stem cell collection. In a prospective study, 346 patients received four cycles of lenalidomide-dexamethasone (Rd). Stem cells were mobilized with cyclophosphamide and G-CSF. Patients failing to collect a minimum of 4 × 10(6) CD34(+)/kg cells received a second mobilization course. After mobilization, a median yield of 8.7 × 10(6) CD34(+)/kg was obtained from patients receiving Rd induction. After first mobilization, inadequate yield was observed in 21% of patients, whereas only 9% of patients failed to collect the target yield after the second mobilization attempt. In conclusion, we confirm that a short induction with lenalidomide allowed sufficient stem cells collection to perform autologous transplantation in 91% of newly diagnosed patients.

Pre-emptive treatment of acute GVHD: a randomized multicenter trial of rabbit anti-thymocyte globulin, given on day+7 after alternative donor transplants.

We have previously shown that hemopoietic stem cell transplant (HSCT) recipients can be stratified on day+7 as having low, intermediate or a high risk of transplant-related mortality (TRM). With the aim of reducing TRM and GVHD, intermediate and high-risk patients (n=170) were randomized to receive anti-thymocyte globulin (ATG, thymoglobuline) on day+7 (n=84) or no treatment (n=86) (controls). There was a reduction of TRM from 35% in controls to 29% in ATG patients (P=0.3), of acute GVHD III-IV from 15 to 5% (P=0.02) and of chronic GVHD from 26 to 11% (P=0.03); survival was comparable. The predictive value of the day+7 score on TRM was confirmed for controls (19 vs 42% for intermediate vs high risk, respectively, P=0.03), whereas ATG abrogated this predictive effect (29 vs 29%). ATG reduced GVHD (P=0.006) in high-risk patients, but not in patients with an intermediate risk. In conclusion, we confirm that TRM can be predicted on the basis of day+7 laboratory values, after alternative donor HSCT; in high-, but not intermediate-risk patients, the administration of ATG on day+7 reduces GVHD. These results may represent a platform for risk-adapted post transplant immune modulation.

Long-term follow-up of metastatic renal cancer patients undergoing reduced-intensity allografting.

SCT from an HLA-compatible sibling donor is an adoptive immunotherapy for cytokine-refractory, metastatic clear-cell renal cell cancer (RCC). However, the recent introduction of targeted therapy compounds has reduced the interest in this therapeutic strategy. We have reanalyzed our series with the aim to assess long-term benefit from allografting. Twenty-five RCC patients received a reduced-intensity allograft from an HLA-identical sibling donor. All patients received a thiotepa, fludarabine and CY conditioning regimen, and a cyclosporine-based GVHD prophylaxis. Best response to allograft was evaluable in 24 patients: 1 CR, 4 PR, 12 minor response/stable disease, 7 progressive disease. One-year survival was 48%, and five-year survival was 20%. At a median observation time of 65 months, five patients are alive, one in CR, one in PR and three with stable disease. By multivariate analysis, C-reactive protein value before transplant, the number of CD34 + infused cells and disease status at day +90 significantly correlated with survival. Survival of patients at favorable/intermediate-risk according to the MSKCC score that underwent allografting was better in comparison to the survival predicted by historical controls. We conclude that 20% of cytokine-refractory RCC patients are alive long-term after allografting. Transplantation is able to induce long-term disease control in a fraction of relapsed RCC patients.

Allogeneic haematopoietic stem cell transplantation for metastatic renal carcinoma in Europe.

BACKGROUND: An allogeneic antitumour effect has been reported for various cancers. We evaluated the experience of allogeneic haematopoietic stem cell transplantation (HSCT) for renal cell carcinoma (RCC) in 124 patients from 21 European centres. PATIENTS AND METHODS: Reduced intensity conditioning and peripheral blood stem cells from an HLA-identical sibling (n = 106), a mismatched related (n = 5), or an unrelated (n = 13) donor were used. Immunosuppression was cyclosporine alone, or combined with methotrexate or mycophenolate mofetil. Donor lymphocyte infusions (DLI) were given to 42 patients. The median follow-up was 15 (range 3-41) months. RESULTS: All but three patients engrafted. The cumulative incidence of moderate to severe, grades II-IV acute GVHD was 40% and for chronic GVHD it was 33%. Transplant-related mortality was 16% at one year. Complete (n = 4) or partial (n = 24) responses, median 150 (range 42-600) days post-transplant, were associated with time from diagnosis to HSCT, mismatched donor and acute GVHD II-IV. Factors associated with survival included chronic GVHD (hazards ratio, HR 4.12, P < 0.001), DLI (HR 3.39, P < 0.001), <3 metastatic sites (HR 2.61, P = 0.002) and a Karnofsky score >70 (HR 2.33, P = 0.03). Patients (n = 17) with chronic GVHD and given DLI had a 2-year survival of 70%. CONCLUSION: Patients with metastatic RCC, less than three metastatic locations and a Karnofsky score >70% can be considered for HSCT. Posttransplant DLI and limited chronic GVHD improved the patient survival.

The prevalence and clinical implications of c-kit expression in plasma cell myeloma.

AIM: To evaluate the clinical implications of c-kit (CD117) expression in plasma cell myeloma (PCM). METHODS AND RESULTS: We first evaluated the reliability of immunohistochemistry in assessing c-kit expression by comparing the results with those obtained by flow cytometry and gene expression arrays in 22 PCM and in 10 PCM cell lines. Immunohistochemical results showed a perfect concordance with those of flow cytometry; likewise, immunohistochemical and gene expression data were also concordant in all but one PCM and cell lines analysed. Then, we investigated the clinical implications of c-kit immunoreactivity in bone marrow biopsies of 85 PCM patients with a mean follow-up of 41 months. C-kit immunoreactivity was detected in 24 (28.2%) of the 85 cases and it was significantly associated with a high microvessel density, but not with traditional clinicopathological characteristics or with survival. CONCLUSIONS: Our findings suggest that immunohistochemistry is a reliable indicator of c-kit gene expression and reinforce the notion that approximately one-third of PCM express high levels of c-kit. The lack of association with traditional clinicopathological parameters and patient survival suggests that c-kit expression may not be an adjunct in predicting the clinical course of the disease.

Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

Incidence of human cytomegalovirus infection in patients with refractory solid tumors receiving nonmyeloablative allogeneic stem cell transplants versus recipients of standard SCT for hematologic malignancies.

Human cytomegalovirus (HCMV) infection is the most frequent infectious complication after conventional allogeneic stem cell transplantation (alloSCT). From December 1998 to December 2002, we prospectively monitored HCMV reactivation in 59 patients affected by solid tumors and undergoing nonmyeloablative alloSCT (NST). Patients were allografted from HLA-identical sibling donors after fludarabine/cyclophosphamide-based conditioning regimens. Seventeen (28.8%) of 59 patients presented with HCMV antigenemia, and 14 received ganciclovir, with successful HCMV clearance in all cases. No patient developed HCMV viremia or disease. The median time to HCMV reactivation was 54 days (range, 16-245 days) after NST. These patients were compared with a cohort of hematologic patients who were treated with conventional myeloablative alloSCT. Matching criteria included HCMV risk group, stem cell source, donor type, and age. In the myeloablative group, HCMV active infection was observed in 47 (85.4%) of 55 patients at a median time of 30 days (range, 13-64 days) after alloSCT, and HCMV infection occurred more frequently ( P < .001) and earlier ( P = .001) than in NST patients. Patients affected with solid tumors undergoing NST had a reduced and delayed incidence of HCMV active infection.