Andreas K. Klein

Prospective study of extracorporeal photopheresis in steroid-refractory or steroid-resistant extensive chronic graft-versus-host disease: analysis of response and survival incorporating prognostic factors

Summary:We enrolled 25 patients with extensive, steroid-refractory chronic graft-versus-host disease (cGVHD) in a prospective trial evaluating the efficacy of extracorporeal photophoresis (ECP) in both skin and visceral cGVHD. The median time from transplant to initiation of ECP was 790 days. ECP was administered for 2 consecutive days every 2 weeks in 17 patients and once a week in eight patients until best response or stable disease. The median duration of therapy was 9 months (range 3–24 months). In all, 20 patients had improvement in cutaneous GVHD and six had healing of oral ulcerations. Steroid sparing or discontinuation of immunosuppressive medications was possible in 80% of patients. Response rates were similar between patients receiving treatment weekly vs every 2 weeks and in patients commencing ECP less than vs greater than 18 months from transplant (70 vs 66%). When patients were stratified based on the Akpek prognostic score, there was no difference in overall response between the favorable (Akpek score<2.5) and unfavorable risk groups, but patients with progressive onset cGVHD tended to have a higher response than those with de novo onset. In summary, we report improvement in skin and/or visceral cGVHD in 71% overall and 61% of high-risk patients.

Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia

The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.

Molecular Pathogenesis of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is unique among malignancies since it represents an accumulation of B-lymphocytes resistant to apoptosis. Several factors are thought to confer this unusual feature to a CLL B-cell. Misbalance between cytoplasmic pro-survival and pro-death molecules, such as Bcl-2, Mcl-1 and alike, appears to be one of the key factors defining B-cell longevity. Autocrine pathways, such as vascular endothelial growth factor-receptor pathway, also contribute to survival. The role of B-cell receptor (BCR) is less straightforward. In the last decade it became clear that CLL does not constitute a uniform disease, but, based on the prevalence of mutations in the BCR heavy chain (IgVH), can be classified into two distinct subgroups. Several molecular markers correlate with IgVH mutations. Some of them, like zeta-chain associated protein kinase, are also involved in BCR signaling and influence cell cycle. Yet the primary pathogenic event leading to increased proliferation and survival in CLL is difficult to ascertain. Molecules involved in BCR signaling pathways and cytoplasmic pro-survival players probably act in concert to confer resistance to apoptosis. In this respect, the role of the B-CLL environment, which includes nurse-like cells and T-cells, cannot be underestimated. Nurse-like cells provide stimuli necessary for perpetuation of life in CLL. On the other hand, abnormal T-cell function, whether it is excessive immunosuppression delivered by regulatory T-cells or insufficient anti-tumor immunity rendered by T-helpers, allows malignant CLL cells to go unnoticed by the cellular immune system.

Reduced-Intensity Allografting as First Transplantation Approach in Relapsed/Refractory Grades One and Two Follicular Lymphoma Provides Improved Outcomes in Long-Term Survivors.

This study was conducted to compare long-term outcomes in patients with refractory/relapsed grades 1 and 2 follicular lymphoma (FL) after allogeneic (allo) versus autologous (auto) hematopoietic cell transplantation (HCT) in the rituximab era. Adult patients with relapsed/refractory grades 1 and 2 FL undergoing first reduced-intensity allo-HCT or first autograft during 2000 to 2012 were evaluated. A total of 518 rituximab-treated patients were included. Allo-HCT patients were younger and more heavily pretreated, and more patients had advanced stage and chemoresistant disease. The 5-year adjusted probabilities, comparing auto-HCT versus allo-HCT groups for nonrelapse mortality (NRM) were 5% versus 26% (P < .0001); relapse/progression: 54% versus 20% (P < .0001); progression-free survival (PFS): 41% versus 58% (P < .001), and overall survival (OS): 74% versus 66% (P = .05). Auto-HCT was associated with a higher risk of relapse/progression beyond 5 months after HCT (relative risk [RR], 4.4; P < .0001) and worse PFS (RR, 2.9; P < .0001) beyond 11 months after HCT. In the first 24 months after HCT, auto-HCT was associated with improved OS (RR, .41; P < .0001), but beyond 24 months, it was associated with inferior OS (RR, 2.2; P = .006). A landmark analysis of patients alive and progression-free at 2 years after HCT confirmed these observations, showing no difference in further NRM between both groups, but there was significantly higher risk of relapse/progression (RR, 7.3; P < .0001) and inferior PFS (RR, 3.2; P < .0001) and OS (RR, 2.1; P = .04) after auto-HCT. The 10-year cumulative incidences of second hematological malignancies after allo-HCT and auto-HCT were 0% and 7%, respectively. Auto-HCT and reduced-intensity-conditioned allo-HCT as first transplantation approach can provide durable disease control in grades 1 and 2 FL patients. Continued disease relapse risk after auto-HCT translates into improved PFS and OS after allo-HCT in long-term survivors.

Reduced respiratory and skeletal muscle strength in survivors of sibling or unrelated donor hematopoietic stem cell transplantation

We performed a retrospective analysis of muscle strength testing obtained following sibling or unrelated donor hematopoietic stem cell transplant (HSCT) between 1 January 1999 and 31 December 2003 in a cohort of 44 subjects at Tufts-New England Medical Center. Maximal inspiratory pressure (PImax) was ⩽80% predicted in 52% of subjects and ⩽60% predicted in 20% of subjects; maximal expiratory pressure (PEmax) was ⩽80% predicted in 88% of subjects and ⩽60% predicted in 74% of subjects. Patients with a PImax ⩽60% predicted spent significantly longer time in hospital following HSCT compared with subjects with PImax 60% predicted. Grip strength (n=32) was reduced to ⩽80% predicted in 75% of subjects and ⩽60% predicted in 47% of subjects following HSCT. Analysis of paired measurements obtained before and after HSCT in 20 of the 44 subjects (45%) showed significant reduction in both PImax and PEmax between the two measures. Respiratory and skeletal muscle weakness is present in a significant percentage of subjects undergoing pulmonary function testing in the post-HSCT period, and may contribute to pulmonary morbidity in subjects with pulmonary complications of HSCT.

Autologous Stem Cell Transplant Recipients Tolerate Haploidentical Related-Donor Natural Killer Cell Enriched Infusions

BACKGROUND: In the setting of allogeneic stem cell transplantation (SCT), infusing natural killer (NK) cells from a major histocompatibility complex (MHC)‐mismatched donor can mediate an antileukemic effect. The graft‐versus‐tumor effect after autologous stem cell transplantation (ASCT) may result in less disease relapse.

Selection of B-cell chronic lymphocytic leukemia cell variants by therapy with anti-CD20 monoclonal antibody rituximab

OBJECTIVE Anti-CD20 chimeric monoclonal antibody rituximab (Mabthera; IDEC-C2B8) is currently tested in several clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). In the present study, we investigated whether rituximab therapy may select for CD20(-) subclones. MATERIALS AND METHODS Leukemic B-CLL cells were isolated from patients with B-CLL and sensitivity to rituximab-induced cell death was examined. Levels of CD20 protein and mRNA were determined using flow cytometry and real-time PCR, respectively. Clonality analyses of leukemic cells throughout rituximab therapy were performed by GeneScan analysis of patient clone specific rearrangements of the complementarity determining region III of the heavy chain immunoglobulin. RESULTS Cytotoxicity of rituximab in vitro did not depend on the protein levels of CD20. During therapy with rituximab CD20(+) B-CLL cells were depleted and CD20(-) leukemic cells emerged. After treatment, the initial CD20(+) B-CLL cell clone reexpanded. CD20(-) B-CLL cells retained their capacity to synthesize the CD20 molecule. CONCLUSIONS These data support the concept that in B-CLL rituximab treatment may not lead to the emergence of CD20(-) leukemic variants. Our findings support clinical studies investigating the benefit of prolonged period of rituximab therapy in B-CLL disease.

Variable contribution of monoclonal antibodies to ADCC in patients with chronic lymphocytic leukemia.

Monoclonal antibodies (mAbs) are increasingly used in treatment protocols for chronic lymphocytic leukemia (CLL). Here we determined (i) the extent of antibody-dependent cellular cytotoxicity (ADCC) of four different mAbs against primary CLL cells, (ii) whether ADCC correlates with antigen density on CLL cells, and (iii) whether allogeneic natural killer (NK) cells display superior ADCC than autologous. Effector cells for ADCC were (i) NK-92 cells not expressing FcR, (ii) NK-92 cells transfected with a high-affinity Fc receptor, (iii) autologous NK cells from patients with CLL, (iv) allogeneic NK cells. Results suggest that ADCC contributes to killing of CLL cells by anti-CD20 antibodies (rituximab and veltuzumab), whereas mAbs against CD22 (epratuzumab) and CD23 (lumiliximab) showed minimal ADCC. The magnitude of anti-CD20 mediated ADCC did not correlate with antigen density of CD20. ADCC was not influenced by the FcR genotype expressed by autologous NK cells. Allogeneic NK cells were superior to autologous NK cells in killing primary CLL cells.

Gray zone lymphoma with features intermediate between classical Hodgkin lymphoma and diffuse large B‐cell lymphoma: Characteristics, outcomes, and prognostication among a large multicenter cohort

Gray zone lymphoma (GZL) with features between classical Hodgkin lymphoma and diffuse large B‐cell lymphoma (DLBCL) is a recently recognized entity reported to present primarily with mediastinal disease (MGZL). We examined detailed clinical features, outcomes, and prognostic factors among 112 GZL patients recently treated across 19 North American centers. Forty‐three percent of patients presented with MGZL, whereas 57% had non‐MGZL (NMGZL). NMGZL patients were older (50 versus 37 years, P = 0.0001); more often had bone marrow involvement (19% versus 0%, P = 0.001); >1 extranodal site (27% versus 8%, P = 0.014); and advanced stage disease (81% versus 13%, P = 0.0001); but they had less bulk (8% versus 44%, P = 0.0001), compared with MGZL patients. Common frontline treatments were cyclophosphamide‐doxorubicin‐vincristine‐prednisone +/− rituximab (CHOP+/−R) 46%, doxorubicin‐bleomycin‐vinblastine‐dacarbazine +/− rituximab (ABVD+/−R) 30%, and dose‐adjusted etoposide‐doxorubicin‐cyclophosphamide‐vincristine‐prednisone‐rituximab (DA‐EPOCH‐R) 10%. Overall and complete response rates for all patients were 71% and 59%, respectively; 33% had primary refractory disease. At 31‐month median follow‐up, 2‐year progression‐free survival (PFS) and overall survival rates were 40% and 88%, respectively. Interestingly, outcomes in MGZL patients seemed similar compared with that of NMGZL patients. On multivariable analyses, performance status and stage were highly prognostic for survival for all patients. Additionally, patients treated with ABVD+/−R had markedly inferior 2‐year PFS (22% versus 52%, P = 0.03) compared with DLBCL‐directed therapy (CHOP+/−R and DA‐EPOCH‐R), which persisted on Cox regression (hazard ratio, 1.88; 95% confidence interval, 1.03–3.83; P = 0.04). Furthermore, rituximab was associated with improved PFS on multivariable analyses (hazard ratio, 0.35; 95% confidence interval, 0.18–0.69; P = 0.002). Collectively, GZL is a heterogeneous and likely more common entity and often with nonmediastinal presentation, whereas outcomes seem superior when treated with a rituximab‐based, DLBCL‐specific regimen. Am. J. Hematol. 90:778–783, 2015.

A randomized, double-blind, placebo-controlled trial of valacyclovir prophylaxis to prevent zoster recurrence from months 4 to 24 after BMT

Reactivation of latent VZV remains a significant cause of morbidity after SCT. Twenty-five percent or more of patients undergoing SCT will experience zoster within the first year after transplant. Short-course (<1 year) prophylaxis with acyclovir has been shown to be effective, but compliance with five times daily dosing may be problematic. We conducted a randomized, double-blind, placebo-controlled trial of valacyclovir (VACV) 1000 mg twice daily from 4 through 24 months after SCT for the prevention of VZV. Fifty-three VZV-seropositive transplant recipients (17 auto-SCT, 36 allo-SCT) were randomized at a median of 163 days after SCT. In a modified intent-to-treat analysis of 49 patients who took study drug, 0 of 22 in the VACV arm experienced zoster reactivation, compared with 6 of 26 (23%) in the placebo arm (P=0.025). Thirty-two subjects completed therapy through the second year post transplant or first episode of zoster. Adverse events resulting in discontinuation were more frequent in the placebo group (5 of 26 vs 3 of 27 for placebo and study drug, respectively). VACV at a dose of 1000 mg twice daily through 24 months after transplant is well tolerated and effective in suppressing shingles after SCT.