Symptomatic BK Virus Infection Is Associated With Kidney Function Decline and Poor Overall Survival in Allogeneic Hematopoietic Stem Cell Recipients.

Nephropathy due to BK virus (BKV) infection is an evolving challenge in patients undergoing hematopoietic stem cell transplantation (HSCT). We hypothesized that BKV infection was a marker of kidney function decline and a poor prognostic factor in HSCT recipients who experience this complication. In this retrospective study, we analyzed all patients who underwent their first allogeneic HSCT at our institution between 2004 and 2012. We evaluated the incidence of persistent kidney function decline, which was defined as a confirmed reduction in estimated glomerular filtration rate of at least 25% from baseline using the Chronic Kidney Disease Epidemiology equation. Cox proportional hazard regression was used to model the cause-specific hazard of kidney function decline, and the Fine-Gray method was used to account for the competing risks of death. Among 2477 recipients of a first allogeneic HSCT, BK viruria was detected in 25% (n = 629) and kidney function decline in 944 (38.1%). On multivariate analysis, after adjusting for age, sex, acute graft-versus-host disease (GVHD), chronic GVHD, preparative conditioning regimen, and graft source, BK viruria remained a significant risk factor for kidney function decline (p < 0.001). In addition, patients with BKV infection and kidney function decline experienced worse overall survival. After allogeneic HSCT, BKV infection was strongly and independently associated with subsequent kidney function decline and worse patient survival after HSCT.

Patients with mantle cell lymphoma failing ibrutinib are unlikely to respond to salvage chemotherapy and have poor outcomes.

BACKGROUND: Although ibrutinib is highly effective in patients with relapsed/refractory mantle cell lymphoma (MCL), a substantial proportion of patients have resistant disease. The subsequent outcomes of such patients are unknown. PATIENTS AND METHODS: We carried out a retrospective review of all patients with MCL treated with ibrutinib at MD Anderson Cancer Center between January 2011 and January 2014 using pharmacy and clinical databases. Patients who had discontinued ibrutinib for any reason were included in the study. RESULTS: We identified 42 patients with MCL who discontinued therapy due to disease progression on treatment (n = 28), toxicity (n = 6), elective stem-cell transplant in remission (n = 4) or withdrawn consent (n = 4). The median age was 69 years, 35 (83%) were male; the median number of prior treatments was 2 (range 1-8) and the median time from initial diagnosis of MCL to commencing ibrutinib was 3.0 (range 0.5-15.5) years. Patients had received a median of 6.5 (range 1-43) cycles of ibrutinib. Among 31 patients who experienced disease progression following ibrutinib and underwent salvage therapy, the overall and complete response rates were 32% and 19%, respectively. After a median follow-up of 10.7 (range 2.4-38.9) months from discontinuation of ibrutinib, the median overall survival (OS) among patients with disease progression was 8.4 months. By univariate analysis, elevated serum lactate dehydrogenase at progression was associated with inferior OS. CONCLUSION: The outcome of patients with MCL who experience disease progression following ibrutinib therapy is poor, with both low response rates to salvage therapy and short duration of responses. Further studies to better understand and overcome ibrutinib resistance are urgently needed.

Vaccination guidelines after hematopoietic stem cell transplantation: practitioners' knowledge, attitudes, and gap between guidelines and clinical practice.

BACKGROUND: Hematopoietic stem cell transplant (HCT) recipients are more susceptible to infections from vaccine-preventable diseases than the general population. Despite the development of international consensus guidelines addressing immunization after HCT, studies have shown that deviations from recommended immunization practices commonly occur. METHODS: An anonymous survey aimed at determining awareness of the guidelines and attitudes toward vaccination was distributed to our HCT clinicians. In parallel, we retrospectively evaluated patients' characteristics and post-HCT vaccine administration practices from 2010 to 2013. RESULTS: The majority of survey respondents (96%) were familiar with post-HCT vaccination protocols. Seventy-four percent of respondents reported that influenza vaccines were given to >70% of their patients, and 41% stated that they prescribed live vaccines to eligible patients. However, our pharmacy database review revealed that 38% of patients received the first series of vaccinations by the recommended 6 months post HCT, and 60% received them by 1 year after HCT. Most patients who had their vaccines withheld had relapsed disease or were undergoing treatment for graft-versus-host disease. Furthermore, we identified lower immunization rates in non-English speaking individuals, African-Americans, and Hispanic patients. CONCLUSIONS: Survey respondents reported being aware of current guidelines; however, adherence to the recommendations varied, likely connected to conflicting data on vaccine effectiveness and a lack of clear recommendations in complex clinical scenarios. Similar to the general population, patient barriers also could have contributed to lower vaccination rates in some cases. To decrease the large gap between the post-HCT vaccination guidelines and clinical practice, further studies on vaccine effectiveness and specific populations are warranted.

Clofarabine combined with busulfan provides excellent disease control in adult patients with acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation.

We investigated the safety and early disease control data for i.v. busulfan (Bu) in combination with clofarabine (Clo) in patients with acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation (SCT). Fifty-one patients (median age, 36 years; range, 20-64 years) received a matched sibling (n = 24), syngeneic (n = 2), or matched unrelated donor transplant (n = 25) for acute lymphoblastic leukemia in first complete remission (n = 30), second complete remission (n = 13), or active disease (n = 8). More than one-half of the patients had a high-risk cytogenetic profile, as defined by the presence of t(9;22) (n = 17), t(4;11) (n = 3), or complex cytogenetics (n = 7). Clo 40 mg/m(2) was given once daily, with each dose followed by pharmacokinetically dosed Bu infused over 3 hours daily for 4 days, followed by hematopoietic SCT 2 days later. The Bu dose was based on drug clearance, as determined by the patient's response to a 32-mg/m(2) Bu test dose given 48 hours before the high-dose regimen. The target daily area under the receiver-operating characteristic curve was 5500 µM/min for patients age <60 years and 4000 µM/min for those age ≥60 years. The regimen was well tolerated, with a 100-day nonrelapse mortality rate of 6%. With a median follow-up of 14 months among surviving patients (range, 6-28 months), the 1-year overall survival, disease-free survival, and nonrelapse mortality rates were 67% (95% confidence interval [CI], 55%-83%), 54% (95% CI, 41%-71%), and 32% (95% CI, 16%-45%), respectively. For patients undergoing SCT in first remission, these respective rates were 74%, 64%, and 25%. Our data indicate that the combination of Clo and Bu provides effective disease control while maintaining a favorable safety profile.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Stem-cell transplantation in T-cell non-Hodgkin's lymphomas.

BACKGROUND: T-cell lymphomas are a heterogeneous group of non-Hodgkin's lymphomas (NHLs). With the exception of anaplastic lymphoma kinase protein-positive large-cell lymphoma, standard chemotherapy provides dismal long-term outcomes when compared with NHLs with B-cell immunophenotype. DESIGN: We review the literature on the role of high-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) in T-cell NHLs both as up-front treatment and in the salvage setting. The role of allogeneic transplantation will also be reviewed. RESULTS: Results from five prospective, nonrandomized and six retrospective studies evaluating the role of HDT and ASCT in the up-front setting show that patients in first complete or partial remission especially those who present with advanced disease and high prognostic index of peripheral T-cell lymphoma score may benefit from this approach. In the relapsed and/or refractory setting, most series show results that are comparable with those seen in patients with B-cell lymphomas if transplanted with chemosensitive disease. There is limited evidence to suggest that an immune-mediated graft-versus-lymphoma effect may result in long-term disease remissions in some patients after allogeneic transplantation. CONCLUSIONS: Randomized studies comparing HDT and ASCT with conventional chemotherapy are needed in T-cell lymphomas.

Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia.

Although the immune system has long been implicated in the control of cancer, evidence for specific and efficacious immune responses in human cancer has been lacking. In the case of chronic myelogenous leukemia (CML), either allogeneic bone marrow transplant (BMT) or interferon-alpha2b (IFN-alpha2b) therapy can result in complete remission, but the mechanism for prolonged disease control is unknown and may involve immune anti-leukemic responses. We previously demonstrated that PR1, a peptide derived from proteinase 3, is a potential target for CML-specific T cells. Here we studied 38 CML patients treated with allogeneic BMT, IFN- alpha2b or chemotherapy to look for PR1-specific T cells using PR1/HLA-A*0201 tetrameric complexes. There was a strong correlation between the presence of PR1-specific T cells and clinical responses after IFN-alpha and allogeneic BMT. This provides for the first time direct evidence of a role for T-cell immunity in clearing malignant cells.

Hepatosplenic gamma-delta T-cell lymphoma: clinicopathological features and treatment.

BACKGROUND: Hepatosplenic T-cell lymphoma (HSTCL) is a rare peripheral T-cell lymphoma; treatment with standard anthracycline-containing chemotherapy regimens has been disappointing, and an optimal treatment strategy for this patient population has not yet been determined. METHODS: We identified 15 cases of pathologically confirmed HSTCL in the institution's database. Clinical characteristics and treatment results were reviewed. RESULTS: Complete responses (CRs) were achieved in 7 of 14 patients who received chemotherapy. Achievement of CR was followed by hematopoietic stem-cell transplantation in three patients. Median duration of CR was 8 months (range 2 to 32+ months) with four patients currently alive and in CR at 5, 8, 12, and 32 months, respectively. Median overall survival (OS) was 11 months (range 2 to 36+ months). Patients who achieved a CR had a median OS of 13 months, compared with 7.5 months in patients who did not achieve a CR. Risk factors associated with worse outcome included male gender, failure to achieve a CR, history of immunocompromise, and absence of a T-cell receptor gene rearrangement in the gamma chain. CONCLUSION: A better understanding of the pathophysiology of HSTCL and new therapeutic strategies are needed.

High-dose chemotherapy and autologous hematopoietic progenitor cell transplantation for non-Hodgkin's lymphoma in patients >65 years of age.

PATIENTS AND METHODS: We present a retrospective analysis of 99 consecutive patients with relapsed non-Hodgkin's lymphomas who were older than 65 years at the time of high-dose chemotherapy and autologous progenitor cell transplantation. RESULTS: Median age at transplant was 68 years (range 65-82). Thirty-six percent of patients had a hematopoietic cell transplantation comorbidity index of >2 at the time of transplantation. The cumulative nonrelapse mortality was 8% [95% confidence interval (CI) 4-17] at 26 months and the 3-year overall survival (OS) was 61% (95% CI 49-71). On multivariate analysis, disease status at transplant and lactate dehydrogenase (LDH) > normal were significant predictors for OS (P = 0.002). Comorbidity index of >2 did not impact OS but did predict for higher risk of developing grade 3-5 toxicity (P = 0.006). Eight patients developed secondary myelodysplastic syndrome/acute myelogenous leukemia after transplantation (cumulative incidence 16%). CONCLUSIONS: Patients with relapsed lymphomas who are >65 years of age should be considered transplant candidates, particularly if they have chemosensitive disease and normal LDH levels at the time of transplantation. Patients with comorbidity index of >2 can also undergo transplantation with acceptable outcomes but may be at higher risk for developing toxicity.

Allogeneic hematopoietic cell transplantation for metastatic breast cancer.

We reviewed 66 women with poor-risk metastatic breast cancer from 15 centers to describe the efficacy of allogeneic hematopoietic cell transplantation (HCT). Median follow-up for survivors was 40 months (range, 3-64). A total of 39 patients (59%) received myeloablative and 27 (41%) reduced-intensity conditioning (RIC) regimens. More patients in the RIC group had poor pretransplant performance status (63 vs 26%, P=0.002). RIC group developed less chronic GVHD (8 vs 36% at 1 year, P=0.003). Treatment-related mortality rates were lower with RIC (7 vs 29% at 100 days, P=0.03). A total of 9 of 33 patients (27%) who underwent immune manipulation for persistent or progressive disease had disease control, suggesting a graft-vs-tumor (GVT) effect. Progression-free survival (PFS) at 1 year was 23% with myeloablative conditioning and 8% with RIC (P=0.09). Women who developed acute GVHD after an RIC regimen had lower risks of relapse or progression than those who did not (relative risk, 3.05: P=0.03), consistent with a GVT effect, but this did not affect PFS. These findings support the need for preclinical and clinical studies that facilitate targeted adoptive immunotherapy for breast cancer to explore the benefit of a GVT effect in breast cancer.

Designed transfer of specific immune responses with bone marrow transplantation.

Bone marrow transplant donors were immunized with tetanus/diphtheria toxoids 6-7 d before bone marrow donation to investigate the role of B cell subpopulations in reconstitution of humoral immunity. Lymphoblastoid B cells spontaneously producing IgG antitetanus and/or antidiphtheria toxoid were detected in the donor marrows at the time of transplantation. Recipients rapidly demonstrated 3-90-fold increases in serum IgG antitetanus and antidiphtheria toxoid levels. Antidiphtheria fragment A antibody in three donor/recipient pairs demonstrated spectrotypic identity indicating transfer of the donors' response. Reimmunization of three recipients 64-154 d after transplant revealed an IgG antibody response associated with reappearance of spontaneous antibody-producing B cells and an antidiphtheria fragment A response characteristics of the donor's immune response. These observations extend the understanding of the role of B cell subpopulations and provide a basis for specific modulation of immunity in the setting of bone marrow transplantation.

Treatment of donor graft failure with nonmyeloablative conditioning of fludarabine, antithymocyte globulin and a second allogeneic hematopoietic transplantation.

Graft failure is a life-threatening complication of allogeneic stem cell transplantation (SCT). We assessed the feasibility of performing a second SCT after such failure when fludarabine and antithymocyte globulin (ATG) are used for non-myeloablative conditioning and tacrolimus for graft-versus-host disease (GVHD) prophylaxis. Nine patients with SCTs for various hematologic malignancies were enrolled, eight with primary and one with secondary graft failure. The median time between the first and second SCT was 53 days. Eight patients had the same donor for their second SCT, and one had a cord blood transplant. Three patients were not evaluable because of early death; the other six had evidence of donor cell engraftment. Six of the nine patients developed acute grade II-IV GVHD, the main cause of death. Overall, we found that fludarabine and ATG conditioning before a second SCT allows engraftment of donor hematopoiesis. Future studies should include more intense GVHD prophylaxis.

High-dose caspofungin combination antifungal therapy in patients with hematologic malignancies and hematopoietic stem cell transplantation.

Pneumocandins have concentration-dependent antifungal activity and higher dose of caspofungin (HD-CAP) in combination with other licensed antifungal therapy (OLAT) may improve response. Thirty-four patients who received HD-CAP were compared with 63 patients who received standard dose (SD)-CAP. There were no differences between the groups in either patient or disease characteristics. Significantly more patients in the HD-CAP arm had extrapulmonary infections (29 vs 8% in SD group; P=0.0053), and non-Aspergillus species infection (21 vs 6%; P=0.05) and had received prior antifungal therapy (71 vs 33%; P=0.0004). No serious adverse reactions were noted in patients receiving HD- or SD-CAP therapy. Twelve weeks after treatment commenced 44% had a complete or partial response compared with 29% in SD-CAP group (P=0.1). Logistic regression analysis showed a significant probability of a favorable outcome at 12 weeks in patients who received HD-CAP (OR 3.066, 95% CI, 1.092-8.61; P=0.033). This may in part reflect higher number of patients in HD group had received granulocyte-macrophage colony-stimulating factor (41 vs 14% in SD group; P=0.04) and/or interferon gamma (26 vs 5% in SD group; P=0.003) immune enhancement. Further studies are needed to evaluate efficacy of HD-CAP in severely immunosuppressed cancer patients with invasive fungal infections.

Risk factors associated with late cytomegalovirus reactivation after allogeneic stem cell transplantation for hematological malignancies.

We analyzed the clinical factors associated with late cytomegalovirus (CMV) reactivation in a group of 269 consecutive recipients of allogeneic stem cell transplant (SCT) for hematological malignancies. Eighty-four subjects (31%) experienced late CMV reactivation, including 64 with prior early reactivation and 20 with isolated late reactivation. Multivariate analyses were conducted in patients with early CMV reactivation to identify factors associated with late recurrence. Important risk factors included lymphoid diagnosis, occurrence of graft-versus-host disease (GVHD), greater number of episodes of early reactivation, persistent day 100 lymphopenia and the use of a CMV-seronegative donor graft. We combined these risk factors in a predictive model to identify those at relatively low, intermediate and high risk. The low-risk group (15% cumulative incidence, CI) encompassed patients without early CMV reactivation, and subjects transplanted for a myeloid malignancy from a matched-related (MR) donor without subsequent acute GVHD. The high-risk patients (73% CI) met all of the following criteria: (1) received an MR graft but developed GVHD, or received a non-MR graft irrespective of GVHD; (2) had more than two episodes of early reactivation; and (3) received a CMV-seronegative graft and/or remained persistently lymphopenic at day 100 after SCT. The remaining patients had an intermediate incidence of 32%.

Autologous stem cell transplantation is safe and feasible in elderly patients with multiple myeloma.

Several clinical trials have shown the superiority of autologous stem cell transplantation over conventional dose therapy for patients with multiple myeloma. This treatment, however, is limited to younger patients (<65 years) owing to concerns about toxicity and treatment-related mortality (TRM) in older patients. We treated 26 elderly myeloma patients (>70 years), who received a preparative regimen of melphalan 200 mg/m2 (19 patients), melphalan 180 mg/m2 (six patients) or melphalan 140 mg/m2 (one patient). Twenty-two of the 26 patients were alive after a median follow-up of 25 months (range=8-74). Responses (complete+partial response) were seen in 20 patients (77%), five (19%) of which were complete responses. Median PFS was 24 months, whereas median OS has not been reached. Cumulative incidence of 100-day TRM was 0%. Three-year PFS and OS were 39% (range=16-61) and 65% (range=35-83), respectively. A low serum albumin (<3.5 g/dl) was associated with a shorter PFS (P=0.02). Patients with relapsed disease at transplant, and an interval of >12 months between diagnosis and autotransplant, had a shorter OS (P=0.0004 and 0.04). HDT and autologous transplant is safe and feasible in elderly myeloma patients.

Multi-center analysis of the effect of T-cell acute lymphoblastic leukemia subtype and minimal residual disease on allogeneic stem cell transplantation outcomes.

This study aims to provide a detailed analysis of allogeneic stem cell transplantation (allo-SCT) outcomes in a large T-cell acute lymphoblastic leukemia (T-ALL) cohort with a specific emphasis on the effects of pre-transplant minimal residual disease (MRD) and disease subtype, including the aggressive early-thymic precursor (ETP) subtype. Data from 102 allo-SCT patients with a diagnosis of T-ALL from three centers were retrospectively analyzed. Patients were grouped into four T-ALL subtypes: ETP, early, cortical and mature. At 3 years, overall survival (OS), PFS, non-relapse mortality and cumulative incidence (CI) progression were 35, 33, 11 and 55%, respectively. Patients transplanted in first complete remission (CR1) had a 3-year OS of 62% versus those transplanted in CR2 or greater (24%) (hazards ratio 1.6, P=0.2). Patients with MRD positivity at the time of transplant had significantly higher rates of progression compared with those with MRD negativity (76 vs 34%, hazards ratio 2.8, P=0.006). There was no difference in OS, PFS or cumulative incidence (CI) progression between disease subtypes, including ETP (n=16). ETP patients transplanted in CR1 (n=10) had OS of 47%, comparable to other disease subtypes, suggesting that allo-SCT can overcome the poor prognosis associated with ETP. MRD status at transplant was highly predictive of disease relapse, suggesting novel therapies are necessary to improve transplant outcomes.

PR1 peptide vaccine induces specific immunity with clinical responses in myeloid malignancies.

PR1, an HLA-A2-restricted peptide derived from both proteinase 3 and neutrophil elastase, is recognized on myeloid leukemia cells by cytotoxic T lymphocytes (CTLs) that preferentially kill leukemia and contribute to cytogenetic remission. To evaluate safety, immunogenicity and clinical activity of PR1 vaccination, a phase I/II trial was conducted. Sixty-six HLA-A2+ patients with acute myeloid leukemia (AML: 42), chronic myeloid leukemia (CML: 13) or myelodysplastic syndrome (MDS: 11) received three to six PR1 peptide vaccinations, administered subcutaneously every 3 weeks at dose levels of 0.25, 0.5 or 1.0 mg. Patients were randomized to the three dose levels after establishing the safety of the highest dose level. Primary end points were safety and immune response, assessed by doubling of PR1/HLA-A2 tetramer-specific CTL, and the secondary end point was clinical response. Immune responses were noted in 35 of 66 (53%) patients. Of the 53 evaluable patients with active disease, 12 (24%) had objective clinical responses (complete: 8; partial: 1 and hematological improvement: 3). PR1-specific immune response was seen in 9 of 25 clinical responders versus 3 of 28 clinical non-responders (P=0.03). In conclusion, PR1 peptide vaccine induces specific immunity that correlates with clinical responses, including molecular remission, in AML, CML and MDS patients.

Fludarabine with pharmacokinetically guided IV busulfan is superior to fixed-dose delivery in pretransplant conditioning of AML/MDS patients.

We hypothesized that IV busulfan (Bu) dosing could be safely intensified through pharmacokinetic (PK-) dose guidance to minimize the inter-patient variability in systemic exposure (SE) associated with body-sized dosing, and that this should improve outcome of AML/MDS patients undergoing allogeneic stem cell transplantation. To test this hypothesis, we treated 218 patients (median age 50.7 years, male/female 50/50%) with fludarabine 40 mg/m2 once daily x4, each dose followed by IV Bu, randomized to 130 mg/m2 (N=107) or PK-guided to average daily SE, AUC of 6000 µM min (N=111), stratified for remission status and allo-grafting from HLA-matched donors. Toxicity and GvHD rates in the groups were similar; the risk of relapse or treatment-related mortality remained higher in the fixed-dose group throughout the 80-month observation period. Further, PK-guidance yielded safer disease control, leading to improved overall and PFS, most prominently in MDS patients and in AML patients not in remission at allogeneic stem cell transplantation. We conclude that AML/MDS patients receiving pretransplant conditioning treatment with our 4-day regimen may benefit significantly from PK-guided Bu dosing. This could be considered an alternative to fixed-dose delivery since it provides the benefit of precise dose delivery to a predetermined SE without increasing risk(s) of serious toxicity and/or GvHD.

Phase II study of unrelated cord blood transplantation for adults with high-risk hematologic malignancies.

Cell dose is a critical determinant of outcomes in unrelated cord blood (CB) transplantation. We investigated a strategy in which CB units should contain at least 2 x 10(7) total nucleated cells/kg of recipient weight, otherwise a second unit had to be added. We report the results of a study that was prematurely closed owing to toxicity. Patients with advanced hematologic malignancies without a human leukocyte antigen-matched sibling or unrelated donor were eligible. Conditioning regimen consisted of fludarabine and 12 Gy of total body irradiation (n=11), or melphalan (n=4), with antithymocyte globulin. Graft-versus-host disease prophylaxis was tacrolimus and methotrexate. Fifteen patients with acute leukemia (n=9), chronic myelogenous leukemia (n=2), multiple myeloma (n=2) and lymphoma (n=2) were treated; 60% had relapsed disease at transplantation. Three patients received double CB transplants. The 100-day and 1-year treatment-related mortality rates were 40 and 53%, respectively. Median time to neutrophil and platelet engraftment was 22 days (n=10) and 37 days (n=10), respectively. One patient had secondary graft failure and five patients failed to engraft. Two patients are alive and disease free; 4-year actuarial survival is 33 versus 0% for patients transplanted in remission versus in relapse. We concluded that disease status was the main determinant of treatment failure in this study.