Alexander Kiani

Down-Regulation of IL-4 Gene Transcription and Control of Th2 Cell Differentiation by a Mechanism Involving NFAT1

Transcription factors of the NFAT family play a critical role in the immune response by activating the expression of cytokines and other inducible genes in antigen-stimulated cells. Here we show that a member of this family, NFAT1, is involved in down-regulating the late phase of IL-4 gene transcription, thus inhibiting T helper 2 responses. Whereas stimulated T cells from wild-type mice show a transient increase and then a rapid decline in the steady-state levels of IL-4 mRNA in vitro, the levels of IL-4 gene transcripts in NFAT1-deficient T cells are maintained at high levels under the same conditions. Consistent with this observation, NFAT1-/- mice are more susceptible to infection with Leishmania major. This report provides evidence that NFAT proteins regulate not only the initiation but also the termination of gene transcription.

Randomized comparison of FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment of KRAS wild-type metastatic colorectal cancer: German AIO study KRK-0306 (FIRE-3).

LBA3506 Background: In patients (pts) with KRAS, wild-type metastatic colorectal cancer (mCRC) a head to head comparison of anti-EGFR- and anti-VEGF-directed first-line therapy has not been reported with regard to the FOLFIRI backbone. The AIO KRK-0306 study was therefore designed as a randomized multicenter trial to compare the efficacy of FOLFIRI plus cetuximab to FOLFIRI plus bevacizumab in mCRC pts not pretreated for metastatic disease. METHODS Pts were randomized to FOLFIRI (Tournigand regimen) every two wks plus cetuximab (400 mg/m² day 1, followed by 250 mg/m² wkly = arm A) or bevacizumab (5 mg/kg every two wks = arm B). The intent-to-treat (ITT) population comprised all pts who had at least completed one application of therapy. While recruitment initially was independent of KRAS status, an amendment confined inclusion to KRAS wildtype (WT) tumors. Recruitment was completed in October 2012. The primary study endpoint was objective response rate (ORR, investigators read). RESULTS Among 735 pts of the ITT-population, KRAS-WT was identified in 592. Of these, 297 pts were randomized to arm A and 295 to arm B. Median age was 64 years, 66% of pts were male, and ECOG PS 0-1 was observed in 98% of pts. Median duration of treatment was 4.7 mo vs 5.3 mo, respectively. While in the ITT analysis, ORR was comparable in arms A vs B (62% vs 57%, odds ratio 1.249), a significant superiority was found for assessable pts in arm A. Median PFS of the ITT population was nearly identical (10.3 vs 10.4 mo, HR 1.04, p=0.69), however, overall survival (OS) showed a significantly better outcome in arm A vs arm B (28.8 vs 25.0 mo, HR 0.77, p=0.0164, 95% CI: 0.620-0.953). Sixty-day mortality was low in both arms (1.01% vs 2.71%). CONCLUSIONS ORR was comparable between arms in the ITT analysis, but favored arm A in assessable pts. Significantly superior OS was observed in KRAS-WT patients receiving cetuximab plus FOLFIRI as first-line treatment. CLINICAL TRIAL INFORMATION NCT00433927.

FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab for metastatic colorectal cancer (FIRE-3): a post-hoc analysis of tumour dynamics in the final RAS wild-type subgroup of this randomised open-label phase 3 trial.

BACKGROUND FIRE-3 compared first-line 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) plus cetuximab with FOLFIRI plus bevacizumab in patients with KRAS exon 2 wild-type metastatic colorectal cancer. The same study also reported an exploratory analysis of a subgroup of patients with tumours that were wild-type at other RAS genes (KRAS and NRAS exons 2-4). We report here efficacy results for the FIRE-3 final RAS (KRAS/NRAS, exons 2-4) wild-type subgroup. Moreover, new metrics of tumour dynamics were explored during a centralised radiological review to investigate how FOLFIRI plus cetuximab conferred overall survival benefit in the absence of differences in investigator-assessed objective responses and progression-free survival. METHODS FIRE-3 was a randomised phase 3 trial comparing FOLFIRI plus cetuximab with FOLFIRI plus bevacizumab in the first-line treatment of patients with KRAS exon 2 wild-type metastatic colorectal cancer. The primary endpoint of the FIRE-3 study was the proportion of patients achieving an objective response according to Response Evaluation Criteria In Solid Tumors (RECIST) 1.0 in the intention-to-treat population. A centralised radiological review of CT scans was done in a post-hoc analysis to assess objective response according to RECIST 1.1, early tumour shrinkage, depth of response, duration of response, and time to response in the final RAS wild-type subgroup. Comparisons between treatment groups with respect to objective response rate and early tumour shrinkage were made using Fishers exact test (two-sided), while differences in depth of response were investigated with a two-sided Wilcoxon test. This trial is registered at ClinicalTrials.gov, number NCT00433927. FINDINGS In the final RAS wild-type population (n=400), median overall survival was better in the FOLFIRI plus cetuximab group than the FOLFIRI plus bevacizumab group (33·1 months [95% CI 24·5-39·4] vs 25·0 months [23·0-28·1]; hazard ratio 0·70 [0·54-0·90]; p=0·0059), although investigator-assessed objective response and progression-free survival were comparable between treatment groups. Centralised radiological review of CT-assessable patients (n=330) showed that the proportion of patients achieving an objective response (113 of 157, 72·0% [95% CI 64·3-78·8] vs 97 of 173, 56·1% [48·3-63·6]; p=0·0029), frequency of early tumour shrinkage (107 of 157, 68·2% [60·3-75·4] vs 85 of 173, 49·1% [41·5-56·8]; p=0·0005), and median depth of response (-48·9% [-54·3 to -42·0] vs -32·3% [-38·2 to -29·2]; p<0·0001) were significantly better in extended RAS wild-type patients receiving FOLFIRI plus cetuximab versus those receiving FOLFIRI plus bevacizumab. No differences in duration of response and time to response were observed between treatment groups. INTERPRETATION This analysis provides a new framework that connects alternative metrics of response to overall survival. Superior response-related outcome parameters, such as early tumour shrinkage and depth of response, obtained by centralised radiological review correlated with the overall survival benefit conferred by FOLFIRI plus cetuximab compared with FOLFIRI plus bevacizumab in the extended RAS wild-type subgroup. FUNDING Merck KGaA and Pfizer.

Monitoring of donor chimerism in sorted CD34+ peripheral blood cells allows the sensitive detection of imminent relapse after allogeneic stem cell transplantation

Detection of impending relapse following allogeneic stem cell transplantation in cases of acute leukemia or myelodysplastic syndrome is desirable for planning treatment intervention. Genetic changes harbored by tumor cells are ideal for monitoring, but are not always available. Interestingly, analysis of serial donor chimerism in CD34+ cells sorted from blood may provide an alternative. Analysis of donor chimerism is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. We prospectively investigated the predictive value of donor chimerism analyses in sorted CD34+ peripheral blood cells in 90 patients with acute leukemia and myelodysplastic syndrome. The cumulative incidence of relapse after four years was significantly increased in cases with decreasing or incomplete CD34+ donor chimerism (57% vs. 18%, p=0.0001). Multivariate analysis confirmed decreasing CD34+ donor chimerism as an independent predictor of relapse and inferior survival. The interval between a decrease of CD34+ chimerism of less than 80% and hematologic relapse was 61 days (range 0–567). Monitoring of CD34+ donor chimerism in the peripheral blood allows prediction of imminent relapse after allogeneic stem cell transplantation even when a disease-specific marker is lacking.

Impact of Subsequent Therapies on Outcome of the FIRE-3/AIO KRK0306 Trial: First-Line Therapy With FOLFIRI Plus Cetuximab or Bevacizumab in Patients With KRAS Wild-Type Tumors in Metastatic Colorectal Cancer

PURPOSE We investigated choice and efficacy of subsequent treatment, with special focus on second-line therapy, in the FIRE-3 trial (FOLFIRI plus cetuximab [arm A] or bevacizumab [arm B]) for patients with KRAS wild-type metastatic colorectal cancer. PATIENTS AND METHODS Start of subsequent-line (second or third) therapy was defined as use of an antitumor drug that was not part of the previous regimen. We evaluated choice, duration, and efficacy of subsequent therapy and determined the impact of subsequent-line treatment on outcome of patients in FIRE-3. RESULTS Of 592 patients in the intent-to-treat population, 414 (69.9%) received second-line and 256 (43.2%) received third-line therapy. In subsequent treatment lines, 47.1% of patients originally assigned to arm A received bevacizumab, and 52.2% originally assigned to arm B received either cetuximab or panitumumab. Oxaliplatin was subsequently used in 55.9% (arm A) and 53.2% (arm B) of patients. Second-line therapy was administered for a median duration of 5.0 versus 3.2 months (P < .001) in study arm A versus B. Progression-free (6.5 v 4.7 months; hazard ratio, 0.68; 95% CI, 0.54 to 0.85; P < .001) and overall survival (16.3 v 13.2 months; hazard ratio, 0.70; 95% CI, 0.55 to 0.88; P = .0021) from start of second-line therapy were longer in patients in arm A compared with arm B. CONCLUSION Our data suggest that the sequence of drug application might be more important than exposure to single agents. In patients with RAS wild-type tumors, first-line application of anti-epidermal growth factor receptor-directed therapy may represent a favorable condition for promoting effective subsequent therapy including antiangiogenic agents.

Graft-versus-Host disease Prophylaxis with Everolimus and Tacrolimus Is Associated with a High Incidence of Sinusoidal Obstruction Syndrome and Microangiopathy: Results of the EVTAC Trial

A calcineurin inhibitor combined with methotrexate is the standard prophylaxis for graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Everolimus, a derivative of sirolimus, seems to mediate antileukemia effects. We report on a combination of everolimus and tacrolimus in 24 patients (median age, 62 years) with either myelodysplastic syndrome (MDS; n = 17) or acute myeloid leukemia (AML; n = 7) undergoing intensive conditioning followed by HSCT from related (n = 4) or unrelated (n = 20) donors. All patients engrafted, and only 1 patient experienced grade IV mucositis. Nine patients (37%) developed acute grade II-IV GVHD, and 11 of 17 evaluable patients (64%) developed chronic extensive GVHD. Transplantation-associated microangiopathy (TMA) occurred in 7 patients (29%), with 2 cases of acute renal failure. The study was terminated prematurely because an additional 6 patients (25%) developed sinusoidal obstruction syndrome (SOS), which was fatal in 2 cases. With a median follow-up of 26 months, the 2-year overall survival rate was 47%. Although this new combination appears to be effective as a prophylactic regimen for acute GVHD, the incidence of TMA and SOS is considerably higher than seen with other regimens.

Cidofovir and Foscarnet for Treatment of Human Herpesvirus 6 Encephalitis in a Neutropenic Stem Cell Transplant Recipient

We report a stem cell transplant recipient who developed human herpesvirus 6 encephalitis. Human herpesvirus 6 load indicated massive intrathecal viral replication. Administration of cidofovir followed by foscarnet was associated with total clearance of human herpesvirus 6 infection. Cidofovir and foscarnet combination therapy may be beneficial for reducing mortality of (neutropenic) stem cell transplant recipients with human herpesvirus 6 encephalitis.

Gemtuzumab Ozogamicin as Part of Reduced-Intensity Conditioning for Allogeneic Hematopoietic Cell Transplantation in Patients with Relapsed Acute Myeloid Leukemia

Purpose: Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within 3 months of allogeneic hematopoietic cell transplantation (HCT). We hypothesized that GO might be safe and effective as part of a reduced-intensity conditioning regimen as salvage therapy of CD33+ acute myeloid leukemia. Experimental Design: Thirty-one patients with acute myeloid leukemia which relapsed following conventional therapy (n = 15), autologous (n = 3), or allogeneic (n = 13) HCT were included in a prospective phase I/II trial. The preparative regimen contained 6 and 3 mg/m2 of GO on days −21 and −14 before transplantation, leading to a reduction of marrow blasts in 18 patients (58%). Eight patients received further cytoreductive chemotherapy before conditioning therapy was initiated. Fludarabine-based reduced-intensity (n = 11) or nonmyelablative (n = 16) conditioning and peripheral blood stem cell infusion from related (n = 6) or unrelated (n = 21) donors could be done in 27 patients during cytopenia. Results: Primary engraftment occurred in all evaluable patients. Only one case of reversible hepatic sinusoidal obstruction syndrome was documented. Non–relapse mortality until day 100 was 22% (n = 6). The probabilities of overall and disease-free survival at 24 months were 39% and 35%, respectively. Relapse of leukemia occurring between 2 and 24 months after transplantation (median, 8 months) was the major reason for treatment failure and death. Conclusion: These data suggest that GO can be combined with reduced-intensity conditioning even after previous autologous or allogeneic HCT.

188Re anti‐CD66 radioimmunotherapy combined with reduced‐intensity conditioning and in‐vivo T cell depletion in elderly patients undergoing allogeneic haematopoietic cell transplantation

The addition of radioimmunotherapy to conventional and reduced‐intensity conditioning has been shown to be feasible and effective. Within an ongoing prospective phase II trial, 22 patients with advanced myeloid malignancies and a median age of 65 years (range 54–76) received anti‐CD66 Rhenium radioimmunotherapy followed by fludarabine (150 mg/m2), busulfan (8 mg/kg) and alemtuzumab (75 mg) before allogeneic haematopoietic stem cell transplantation from matched sibling (n = 7) and unrelated donors (n = 15). The extramedullary toxicity in the first 100 d post‐transplantation was limited and all patients engrafted with complete donor chimaerism. The incidence of non‐relapse mortality at day 100 and after 2 years was 4·5% and 23%, respectively. The probability of overall survival at 2 years was 40%. A comparison with a younger historical cohort (median age 57 years) having received the same dose of fludarabine and busulfan but neither radioimmunotherapy nor alemtuzumab showed no difference in outcome. Although the use of alemtuzumab reduced the incidence of acute graft‐versus‐host‐disease, it was associated with a relapse incidence of 40% despite the incorporation of radioimmmunotherapy. In summary, we confirmed the feasibility of combined radioimmunotherapy and reduced‐intensity conditioning in elderly patients. Further optimisation, probably involving less T cell depletion, is necessary before a randomized comparison with standard conditioning can be planned.

Osteomyelosclerosis, anemia and extramedullary hematopoiesis in mice lacking the transcription factor NFATc2

Background Nuclear factors of activated T cells (NFAT) are transcription factors that are central to cytokine production in activated T cells and regulate the development and differentiation of various tissues. NFATc2 is expressed in hematopoietic stem cells and regulated during myeloid commitment in a lineage-specific manner. The biological role of NFATc2 in hematopoiesis is, however, unclear. Design and Methods In the present study, we analyzed steady-state hematopoiesis in young (<3 months) and old (>12 months) mice lacking NFATc2. Complete blood counts were performed in the peripheral blood, bone marrow and spleen. Using cytological and histological analyses, the blood cell differential was determined. Colony-formation assays were used to determine the differentiation potential of hematopoietic cells. Bone cell cultures were derived from the bone marrow, and bone remodeling markers were determined in the serum. Results NFATc2−/− mice older than 12 months were anemic and thrombocytopenic. The bone marrows of these mice showed a markedly reduced number of hematopoietic cells, of which megakaryocytic and erythroid lineages were most affected. While the number of hematopoietic progenitor cells in NFATc2-deficent bone marrow was reduced, the myeloid differentiation potential of these cells remained intact. Aged NFATc2−/− mice showed ossification of their bone marrow space and developed extramedullary hematopoiesis in the spleen. Ex vivo differentiation assays revealed an intrinsic defect of NFATc2-deficient stromal cells, in which NFATc2−/− osteoblasts differentiated more efficiently than wild-type cells, whereas osteoclast differentiation was impaired. Conclusions Our data suggest that NFATc2 may play a role in the maintenance of steady-state hematopoiesis and bone remodeling in adult organisms.