Patients In Continuous Remission Research Articles

Extended long-term follow-up of metastatic melanoma patients treated with immunotherapy: late relapses and second primary melanomas.

Immunotherapy has revolutionized the treatment of patients with advanced melanoma as well as other cancers. Most studies, whether of interleukin-2 or checkpoint inhibitor therapies, have limited follow-up after 5 years, making the incidence of late relapses uncertain. In addition, the incidence of second primary melanomas in patients with stage IV melanoma treated with immunotherapy has rarely been reported. We performed a single-institution retrospective study of stage IV melanoma patients treated with interleukin-2 or checkpoint inhibitors over the period from 1992 to 2013. We found 59 patients alive and in remission 5 years after the beginning of immunotherapy and reviewed their subsequent clinical course. This 59-patient cohort had a median follow-up of 13.1 years, with 36 patients followed up for at least 10 years. Four patients (6.8%) had relapses of their metastatic melanoma at 5, 8, 15, and 17 years after starting the successful immunotherapy. Three of the four are still alive. Only one patient in 690 patient-years of observation had a second primary invasive melanoma. Although late relapses after immunotherapy for melanoma do occur, we can conclude that the prognosis of stage IV melanoma patients in continuous remission 5 years after starting immunotherapy is excellent, with a progression-free survival of approximately 85% and a melanoma-specific survival of approximately 95% at 20 years in our series. Our incidence of second primary melanomas is lower than usually reported. These results have important implications regarding the follow-up of stage IV melanoma patients successfully treated with immunotherapy.

Long-Term Mixed Chimerism After Ex Vivo/In Vivo T Cell-Depleted Allogeneic Hematopoietic Cell Transplantation in Patients With Myeloid Neoplasms.

In patients who have undergone allogeneic hematopoietic cell transplantation (HCT), myeloid mixed donor chimerism (MC) is a risk factor for disease relapse. In contrast, several studies found favorable outcome in patients with lymphoid MC. Thus far, most studies evaluating MC focused on a short-term follow-up period. Here, we report the first case series of long-term survivors with MC. We screened 1,346 patients having undergone HCT for myeloid neoplasms at our center from 1996 to 2016; 443 patients with data on total peripheral blood mononuclear cells (PBMC)/CD4+/CD34+ short tandem repeat (STR) donor chimerism (DC) and follow-up ≥24 months post-HCT were included. We identified 10 patients with long-term MC (PBMC DC <95% at ≥12 months post-HCT). Median follow-up was 11 years. All patients had received combined ex vivo/in vivo T cell-depleted (TCD) peripheral blood stem cells; none experienced ≥grade 2 acute graft-versus-host disease (GVHD). The mean total PBMC, CD4+, and CD34+ DC of all patients were 95.88%, 85.84%, and 90.15%, respectively. Reduced-intensity conditioning (RIC) was associated with a trend to lower mean total DC. Of note, two patients who experienced relapse had lower CD34+ DC but higher CD4+ DC as compared with patients in continuous remission. Bone marrow evaluation revealed increased CD4+/FOXP3+ cells in patients with MC, which might indicate expansion of regulatory T cells (Tregs). Our results support known predictive factors associated with MC such as RIC and TCD, promote the value of CD34+ MC as a potential predictor of relapse, highlight the potential association of CD4+ MC with reduced risk of GVHD, and indicate a possible role of Tregs in the maintenance of immune tolerance post-HCT.

Bone Marrow Infiltrating T-Cell Signatures Following Allo-HCT for Adult Acute Lymphoblastic Leukaemia Differentiate between Patients in Continuous CR and Those with Persistent Minimal Residual Disease or Relapse

Allogeneic stem cell transplantation (allo-HCT) has a proven survival benefit in adult acute lymphoblastic leukaemia (ALL) and remains the most commonly applied immunotherapy. The mechanism of graft-versus-leukaemia (GVL) in allo-HCT relies upon donor T cell immunity. After contributing to initial control of ALL, allo-reactive anti-tumour T cells may either persist, thus providing continuing immune surveillance, or undergo deletion/loss of function as a result of both extrinsic regulation and/or exhaustion. Therefore, there is a clear need to identify and track T cells with potential anti-ALL reactivity. UKALL14 (ISRCTN66541317) is an ongoing clinical trial for adults with de novo ALL in the UK in which all patients over 40 years are regarded as ‘high risk’ and are assigned to reduced intensity-conditioned allo-HCT. Minimal residual disease (MRD) monitoring - by immunoglobulin heavy chain/ T cell receptor quantification - of bone marrow and peripheral blood multilineage chimerism are assessed at regular intervals for 2 years post allo-HCT.We hypothesised that it would be possible to differentiate bone marrow infiltrating T-cell (BiL-T) signatures in patients in complete molecular remission versus those with MRD or frank clinical relapse. We have evaluated the feasibility of performing gene expression profiling on BiL-T cells from cryopreserved bone marrow samples of patients following allo-HCT and correlated this information with clinical outcome data.BiL-T were flow-sorted using the BD FACSAria ™ Fusion into CD3+,TCRab+, CD4+ and CD3+,TCRab+, CD8+. After extraction of total mRNA, samples with an RNA integrity index (RIN) >7 were subjected to RNA sequencing using the Illumina Hiseq. Data were analysed using differential expression gene analysis (DeSeq 2) and gene set enrichment analysis (GSEA). The Human Immunology V2 panel on the NanoString nCounter® SPRINT Profiler was used for gene expression profiling of samples with adequate RNA but a RIN <7. Gene expression data was correlated with molecular MRD data and other clinical parameters, including T cell chimerism where available.Both CD4+ and CD8+ subsets of BiL-T were successfully sorted from all 15 cryopreserved samples. DeSeq2 and GSEA of the RNAseq data was performed for CD4+TCRab+ BiL (n=5; 3 MRD- and 2 MRD+/relapse) (Figure 1) and CD8+TCRab+ (n=6; 4 MRD- and 2 MRD+/relapse). Despite the modest number of samples in this feasibility study, CD4+ T cells from patients with relapsing or MRD+ ALL showed significantly increased expression of genes associated with terminal differentiation (e.g. LAG3, GZMK); by contrast, there was lower expression of genes associated with memory differentiation and proliferative fitness (e.g. MYC, CCR7), a finding linked to increased pathway enrichment for PD-1 and telomere maintenance. CD8+ T cells from patients with relapsing or MRD+ ALL also demonstrated a terminally differentiated signature (e.g. increased expression of KLRC1 encoding NKG2A and lower expression of CCR7). GSEA showed enrichment for pathways linked to oxidative phosphorylation and interaction with non-haematopoetic stroma). We also analysed n=9 CD8+TCRab+ samples (n=4 MRD- and n=5 MRD+ or relapse) by Nanostring. These groups could also be segregated on the basis of genes representing pathways related to T cell survival, death receptor signalling and TCR signalling; in each case, CD8+ T cells from MRD+ or relapsing patients were characterised by a hypo-functional and apoptosis-prone signature.Our data show features suggestive of T cell exhaustion and altered metabolomes in patients with residual ALL post-allo-HCT, whether they are MRD+ or in frank clinical relapse; these features were not present in patients in continuous remission. To further test the hypothesis that T cell dysfunction significantly compromises GVL efficacy, we intend to perform transcriptional profiling of BiL-T in a wider cohort of UKALL14 patients who underwent allo-HCT. Since clinical strategies for reversal of T cell exhaustion including donor lymphocyte infusion (DLI) and potential PD-1 blockade already exist, we hope the impact of this project will be to determine precisely when and in whom to use these strategies during therapy for adult ALL. We expect that our findings will have relevance to the wider field of immunotherapy for ALL including blinatumomab and CAR-T approaches. [Display omitted] DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Body Composition Change, Unhealthy Lifestyles and Steroid Treatment as Predictor of Metabolic Risk in Non-Hodgkin's Lymphoma Survivors.

Unhealthy lifestyle, as sedentary, unbalanced diet, smoking, and body composition change are often observed in non-Hodgkin’s lymphoma (NHL) survivors, and could be determinant for the onset of cancer treatment-induced metabolic syndrome (CTIMetS), including abdominal obesity, sarcopenia, and insulin resistance. The aim of this study was to assess whether changes in body composition, unhealthy lifestyles and types of anti-cancer treatment could increase the risk of metabolic syndrome (MetSyn) and sarcopenia in long-term NHL survivors. We enrolled 60 consecutive NHL patients in continuous remission for at least 3 years. Nutritional status was assessed by anthropometry-plicometry, and a questionnaire concerning lifestyles and eating habits was administered. More than 60% of survivors exhibited weight gain and a change in body composition, with an increased risk of MetSyn. Univariate analysis showed a significantly higher risk of metabolic disorder in patients treated with steroids, and in patients with unhealthy lifestyles. These data suggest that a nutritional intervention, associated with adequate physical activity and a healthier lifestyle, should be indicated early during the follow-up of lymphoma patients, in order to decrease the risk of MetSyn’s onset and correlated diseases in the long term.

Minimal Loss of Lifetime for Patients With Diffuse Large B-Cell Lymphoma in Remission and Event Free 24 Months After Treatment: A Danish Population-Based Study.

Purpose The general outlook for patients with diffuse large B-cell lymphoma (DLBCL) in first remission is important information for patients and for planning post-treatment follow-up. The purpose of this study was to evaluate the survival of patients with DLBCL in remission compared with a matched general population. Methods A total of 1,621 patients from the Danish Lymphoma Registry who were newly diagnosed with DLBCL between 2003 and 2011 were included in this study. All patients were ≥ 16 years of age at diagnosis and had achieved complete remission or complete remission unconfirmed after first-line rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) or R-CHOP-like therapy. Results The 5-year post-treatment DLBCL survival was inferior to survival in the matched general population (78%; 95% CI, 76 to 80; v 87%; standardized mortality ratio, 1.75; P < .001). Excess mortality was present but reduced for patients achieving post-treatment event-free survival for 24 months (pEFS24; standardized mortality ratio, 1.27; P < .001). In age-stratified analyses, the survival of patients < 50 years of age was normalized to the general population after achieving pEFS24 ( P = .99). During the first 8 years after pEFS24, the average loss of lifetime was 0.31 mo/y (95% CI, 0.11 to 0.50 mo/y). Excess mortality diminished when analyzing death from lymphoma as competing event to death from other causes, suggesting that early and late relapse is responsible for increased mortality in patients with DLBCL. Conclusion Although this population-based study does not support complete normalization of survival for patients with DLBCL achieving pEFS24, the estimated loss of residual lifetime was low for patients in continuous remission 2 years after ending treatment. Therefore, pEFS24 is an appealing and relevant milestone for patient counseling and could be a surrogate end point in clinical trials.

Haploidentical Natural Killer Cell Infusion Induces Remission in Non-Hodgkin Lymphoma and Overcomes Resistance to Rituximab

Patients with chemotherapy refractory non-Hodgkin lymphoma (NHL) have poor prognosis. We report the results of a phase 2 clinical trial testing the efficacy of haploidentical donor NK cell infusion with rituximab for refractory NHL. Sixteen patients were enrolled, and 14 were evaluable.Treatment included IV lymphodepleting chemotherapy (pentostatin 3.75mg x 2 days; n=6 or fludarabine 25mg/m2 x 4 days; n=10) with cyclophosphamide (60mg/kg IV day -5; n=14). To enhance NK cell function and deplete regulatory T cells(Treg), we used the IL-2 receptor-targeting agent denileukin diftitox (18 μg/kg/day IV day -1; n=5) or methylprednisolone (1 mg/kg day -2 through day +9; n=9). Donor lymphocytes were CD3- and CD19-depleted and overnight stimulated with 1000 U/ml IL-2. The cellular product at a dose of 1.5-8 x 107 NK cells/kg was infused IV on day 0 followed by subcutaneous infusion of IL-2 at 9 million IU x 6 doses. Peripheral blood (PB) donor NK cells were evaluated by short tandem repeats and/or flow cytometry analyzing donor-specific HLA alleles.Patients characteristics included median age of 52 years (range 48-67); diffuse large B cell lymphoma was the most common diagnosis (n=11); all patients were rituximab and chemotherapy refractory.Of 14 evaluable patients at 2 months post-infusion, 4 achieved remissions (2 complete, 2 partial; 28%). Three patients in continuous remission proceeded to allogeneic donor transplantation at 2,3 and 9 months after NK cell therapy.Compared to healthy controls, NK cells in patients prior to therapy had reduced function (CD107a, INFγ and TNFα production), lower expression of natural cytotoxic receptors NKp46, NKp44 and DNAM (but higher NKp30) and reduced expression of suppressor receptors TIGIT, PD1 and TIM3.In all evaluable patients (n=9), donor NK cells persisted in the PB for at least 7 days after infusion with median 18% (range 0.5-97%) of the NK cells were of donor origin. In one responding patient, donor NK cells sustained beyond day 28. Responding patients (R) had significantly lower levels of absolute number of Treg (Foxp3+CD25+CD127-) at baselineand 14 days after therapy (Figure) as compared to non-responders (NR), however neither denileukin diftitox nor methylprednisolone resulted in PB Treg depletion. Expectedly, lower PB Tregs correlated with low serum levels of IL-10 (R2=0.7; p<0.001; n=12), suggestive of attenuated immuno-suppressive milieu. In addition, frequencies of PB myeloid derived suppressor cells (MDSC) were remarkably low in responders as compared to non-responders at baseline (4.5%±0.05% vs. 11%±6%) and after therapy (day 14: 3.8%±1%; vs. 9.4%±5%).Endogenous Inteleukin-15 (IL-15) serum levels peaked at the day of NK cell infusion (range 11-33pg/dl). Responders had higher IL-15 levels compared to non-responders at day 0 (Figure). IL-15 levels also correlated with functional NK cell killing at day 14 as measured by expression of CD107a (R2=0.8; p=0.008; n=5), suggesting that higher endogenous IL-15 improved NK cell cytotoxic capacity.We observed expected, but transient hematologic toxicity. Therapy was associated with grade 3 adverse effects (tumor lysis syndrome, Pneumocystis pneumonia; Clostridium difficile colitis, neutropenic fever; thrombotic microangiopathy). One unevaluable patient developed sepsis prior to NK cell infusion, and the second died of sepsis a week after therapy.In summary, our observation supports further investigation of donor NK cellular therapies for advanced NHL as a strategy to overcome chemoresistance. NK cell expansion and therapeutic efficacy might be further improved by administration of IL-15. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Miller:Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees.

Early Stage Follicular Lymphoma. Predictive Role of Minimal Residual Disease (MRD) and Impact of MRD-Driven Treatment with Radiotherapy and Rituximab on Clinical Outcome

Background. In localized follicular lymphoma (FL, stage I-II), BCL2/IGH+ cells can be detected in the peripheral blood (PB) and/or bone marrow (BM) in 66.7% of cases (Pulsoni et al, BJH 2007). We hereby analyzed the prognostic impact of MRD in localized FL and explored the possibility of a MRD-guided therapeutic approach on a series of patients with a long follow-up.Methods. Between April 2000 and February 2015, 67 consecutive patients with a confirmed histologic diagnosis of stage I/II FL followed at our Center were enrolled in the study. PB and BM samples were collected at enrollment in all patients and investigated by qualitative PCR to identify the presence of a BCL2/IGH rearrangement. Paraffin-embedded lymph nodes (LN) were studied when available. Patients who proved positive at baseline were studied for MRD every 6 months. Real-Time Quantitative PCR (RQ-PCR) was retrospectively performed according to material availability. All patients were treated with involved field radiotherapy (RT) (24-30 Gy); from 2005, patients who were MRD+ after RT received rituximab (R) (375 mg/m2, 4 weekly administration). The median follow-up is 67 months (17-183); 21 patients (31%) have relapsed after a median of 37 months (17-165) from diagnosis.Results. At baseline, a clonal marker was found by qualitative PCR in 48/67 cases (72%): 36 were MBR+ (54%), 6 mcr+ (9%), 6 showed a minor BCL2 rearrangement (9%), while 19 (28%) were negative. Fifteen of the latter 19 were analyzed by RQ-PCR and 4 proved MBR+. Of the 13 available LNs, 11 showed the same molecular marker identified in the PB/BM; 2 cases, negative in the PB/BM, showed a rearrangement in the LN only. After RT, 40/42 MBR+/mcr+ patients were analyzed: 20 resulted MRD-, while 20 persisted MRD+. Regardless of the post-RT MRD status, an equal number of relapses was recorded in both groups (7 each). R treatment was administered to the 20 MRD+ patients after RT. Sixteen (80%) achieved a MRD- status after R: over time, 7/16 patients converted to MRD+ and 4 relapsed, whilst 9/16 patients (56.2%) remain persistently MRD- and none has relapsed so far.To evaluate the impact of R, we considered a series of 27 patients MRD+ after RT or who were MRD- and became MRD+ during the follow-up. Of the 19 patients who received R (1 could not be studied), 15 (79%) did not relapse, while of the 8 untreated patients (pre-2005), 6 (75%) relapsed (p=0.025). Progression-free survival (PFS) was significantly longer for R-treated patients (p=0.0412) (Fig. 1).To define the predictive role of MRD in the entire cohort regardless of post-RT treatment, we considered the 39 patients with molecular follow-up. Thirteen have relapsed: 10/13 (77%) were MRD+ in the follow-up, including the pre-relapse time point, while 3 resulted persistently MRD-. Contrariwise, of the 26/39 patients in continuous remission, 18 (69%) were persistently MRD- while 8 were MRD+ (p=0.015). PFS was significantly better for MRD- patients (p=0.0163) (Fig. 2).RQ-PCR was performed in 30 MBR+ patients: 17 (57%) showed a tumor burden ≥10-5 and 13 <10-5. Tumor burden at diagnosis predicted the MRD clearance following RT: 9/13 (69%) cases with low tumor burden resulted MRD- after RT compared to 2/17 (12%) cases with high tumor burden (p=0.0027). Contrariwise, tumor burden did not predict the occurrence of relapse.Conclusions. Early stage FL at diagnosis can have a heterogenous disease extension: 2 of our cases were truly localized, showing a molecular marker only in the LN. However, in most cases the use of combined qualitative approaches, including canonical MBR/mcr and minor rearrangements, together with RQ-PCR has allowed to identify circulating BCL2/IGH+ cells (52/67 cases: 77.6%), despite a negative BM biopsy. RT induced a MRD negativity in 50% of BCL2/IGH+ patients, but this did not impact on clinical outcome. The administration of R in MRD+ patients decreased significantly the risk of a subsequent relapse and improved PFS. Regardless of treatment, MRD positivity during the follow-up is a predictor of relapse and PFS. Tumor burden at diagnosis is associated with MRD clearance after RT. We support the use of a MRD-driven treatment with anti-CD20 monoclonal antibodies in patients with localized FL after RT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Seasonality and Pediatric Inflammatory Bowel Disease

Seasonal and geographic variations of inflammatory bowel disease (IBD) exacerbations have been described in adults, with inconsistent findings. We sought to determine whether disease activity in pediatric-onset IBD is associated with a seasonal pattern. We examined children with Crohn disease (CD) and ulcerative colitis (UC) using data from the ImproveCareNow Collaborative between December 2008 and November 2010. We compared the proportion of patients in continuous remission for all recorded visits in each season. We also compared the distribution of all recorded visits with a physician global assessment (PGA) of remission or active disease across seasons. A total of 1325 patients with CD (6102 visits) and 587 patients with UC (2394 visits) were included. The proportion of patients with UC in continuous remission during each season was highest in the summer (67%) and lowest in the winter (55%) (P=0.01). A similar pattern was found for CD but was not significant. Similarly, the proportion of visits in remission was highest in the summer and lowest in the winter for both UC (29%, 21%; P<0.001) and CD (28%, 23%; P<0.001); however, the distribution of visits with active disease was not significantly different across seasons. The higher proportion of patients with UC in continuous remission in the summer may be related to the higher proportion of remission visits in the summer, because the proportion of visits with active disease was similar across seasons. These findings do not support any strong associations between season of the year and disease activity in pediatric IBD.

Randomized Study of Intensified Anthracycline Doses for Induction and Recombinant Interleukin-2 for Maintenance in Patients With Acute Myeloid Leukemia Age 50 to 70 Years: Results of the ALFA-9801 Study

PURPOSE In patients with acute myeloid leukemia (AML), induction chemotherapy is based on standard doses of anthracyclines and cytarabine. High doses of cytarabine have been reported as being too toxic for patients older than age 50 years, but few studies have evaluated intensified doses of anthracyclines. PATIENTS AND METHODS In this randomized Acute Leukemia French Association 9801 (ALFA-9801) study, high doses of daunorubicin (DNR; 80 mg/m(2)/d x 3 days) or idarubicin (IDA4; 12 mg/m(2)/d x 4 days) were compared with standard doses of idarubicin (IDA3; 12 mg/m(2)/d x 3 days) for remission induction in patients age 50 to 70 years, with an event-free survival (EFS) end point. After two consolidation courses based on intermediate doses of cytarabine, patients in continuous remission were randomly assigned to receive or not receive maintenance therapy with recombinant interleukin-2 (rIL-2; 5 x 10(6) U/m(2) x 5 days each month) for a total duration of 12 months. A total of 468 patients entered the study (median age, 60 years). Results Overall complete remission rate was 77% with significant differences among the three randomization arms (83%, 78%, and 70% in the IDA3, IDA4, and DNR arms, respectively; P = .04). However, no significant differences were observed in relapse incidence, EFS, or overall survival among the three arms. In the 161 patients randomly assigned for maintenance therapy, no difference in outcome was observed between the rIL-2 and the no further treatment arms. CONCLUSION Neither intensification of anthracycline doses nor maintenance with rIL-2 showed a significant impact on AML course, at least as scheduled in this trial.

Long-term Follow-up of 14 Patients with Philadelphia Chromosome—Positive Acute Lymphoblastic Leukemia following Autologous Bone Marrow Transplantation in First Complete Remission

We describe the clinical outcome of autologous bone marrow transplantation (ABMT) for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL). Between 1985 and 2000, 14 patients in first complete remission underwent transplantation. In all cases, harvested marrow was purged with a cocktail of complement and monoclonal antibodies to common acute lymphoblastic leukemia antigen (CALLA). Eight patients died following BMT: 2 patients from interstitial pneumonia and 6 from disease recurrence. For the 6 surviving patients in continuous remission, the median follow-up period was 96 months. The probability of disease-free survival and the rate of relapse at 5 years were 41.7% and 51.4%, respectively. Minimal residual disease (MRD) was assayed quantitatively with patient-specific D-JH probes in 6 of 14 cases. A higher residual tumor burden at marrow harvest was associated with subsequent relapse. Efforts to reduce the MRD burden before harvesting stem cells should improve the clinical outcome of ABMT.

Cytochrome c–related caspase-3 activation determines treatment response and relapse in childhood precursor B-cell ALL

AbstractDeficient activation of apoptosis signaling pathways may be responsible for treatment failure in acute leukemia. Here, we address the impact of intact apoptosis signaling in 78 patients with pediatric precursor B-cell acute lymphoblastic leukemia (ALL) by analysis of 2 key apoptogenic events: caspase-3 activation and cytochrome c release in leukemia cells cultured in vitro. Both events correlated only in the group of patients who had a good response and patients in continuous remission, suggesting that intact apoptosis signaling is a characteristic for favorable outcome. By combining both parameters, we identified a novel indicator, cytochrome c–related activation of caspase-3 (CRAC). CRAC directly connects the extent of caspase-3 activation to cytochrome c release in single cells in an individual patient sample. In CRAC-positive patients, indicating proficient apoptosis signaling, the number of persisting leukemia cells on day 15 was significantly lower than in the CRAC-negative patient group (n = 27, mean 6.0% versus n = 36, mean 22.6%; P = .003). At a median follow-up of 31 months, disease-free survival was 84 months (95% CI = 76 to 91 months) and 66 months (95% CI = 52 to 80 months) for patients with positive and negative CRAC, respectively (P = .019). CRAC may serve as a functionally defined risk factor for treatment stratification.

High FLT3 Transcript Level Is Associated with Unfavorable Prognosis in Pediatric Non-Promyelocytic Acute Myeloid Leukemia.

FLT3 is a receptor tyrosine kinase involved in the survival of hematopoietic stem cells, and mutations of FLT3 have been reported to be a risk factor in acute myeloid leukemia (AML), especially for pediatric patients. Recently, FLT3 expression level is proposed to have a prognostic significance in adults, but little is known for childhood AML.FLT3 transcript levels at diagnosis were measured with real time quantitative reverse transcription polymerase chain reaction to verify the clinical significance in pediatric AML. FLT3 transcript levels were analyzed in 20 (M: 12, F: 8) pediatric patients with median 6 (0 – 16) years old received same treatment scheme (Leuk Res. 29; 617). Patients with secondary AML or RAEB-t were included, but patients with FAB M3 type were excluded. Relative gene expression levels were calculated by using standard curves made with KG-1 cell line copy and adjusted on the basis of the expression level of the GAPDH gene (106 copy). Each gene expression level was examined in triplicate, and the mean value was subjected to analysis. The copy number of FLT3 in each sample was calculated by comparing the Ct values of samples with standard curves. Mann-Whitney test was applied for comparison of means. For survival data, Kaplan-Meier life tables were constructed and the curves were compared by means of log-rank test.The median value of adjusted FLT copy number was 58 (0.06–19,302) x 103. FLT3 copy number was not associated with gender, age and other risk factor except FLT3 internal tandem duplication (FLT3/ITD) which was present in 3 patients with significantly higher mean copy number (7,130 x 103) than others (372 x 103) (P = 0.023). The mean value of FLT3 copy number of 12 refractory or relapsed patients (2,278 x 103) was higher than that of patients in continuous remission (48 x 103), significantly (P = 0.031). The overall survival rate of 10 patients with high FLT3 copy number more than 50 x 103 (25.0%) was lower than that of others (59.3%) significantly (P = 0.0457). All patients with high copy number were refractory to induction or relapsed early before post remission transplantation except 2 patients (one received bone marrow transplant from matched sibling and another received autologous peripheral stem cell transplant).High FLT3 transcript level was an unfavorable prognostic factor for early relapse and overall survival in pediatric AML and associated with FLT3/ITD. New therapeutic scheme such as stem cell transplantation with more intensive conditioning just after complete remission and administration of FLT3 inhibitor could be applied in pediatric non-promyelocytic AML patients with high FLT3 transcript level.

Treatment of Relapsed Acute Promyelocytic Leukemia by Arsenic-Based Strategies without Hematopoietic Stem Cell Transplantation in Hong Kong: A Seven-Year Experience.

Background: The optimal therapy for relapsed acute promyelocytic leukemia (APL) after arsenic trioxide (As2O3)-induced remission is unclear. Hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality. Moreover, lasting remission is observed in many patients who are not candidates for HSCT, owing to advanced age or lack of donors, implying that HSCT is not mandatory for durable remission. We evaluated our results of an As2O3-based, non-HSCT regimen for patients with relapsed APL.Materials and methods: Forty-two consecutive patients (18 men, 24 women, median age: 35 years, 12–72) with relapsed (relapse 1, R1=39, R2=3) APL were treated with an-As2O3 based, non-HSCT regimen. The time from last complete remission (CR) was 22 (6–243) months (mo). Initial treatment was As2O3 (10 mg/day) either intravenously (n=16) or orally (n=28) until CR, followed by idarubicin consolidation (6 mg/m2/day x 9). Twenty-five patients received oral-As2O3 maintenance. Post-As2O3 relapses were treated with oral As2O3 + all-trans retinoic acid (ATRA, 45 mg/m2/day) until CR, followed by maintenance (two weeks of ATRA+As2O3 every 2 mo. for 2 years). Post-As2O3/ATRA relapses were treated with oral As2O3+ATRA+ascorbic acid (1g/day) until CR, followed by consolidation/maintenance with the same regimen (2 weeks every 2 mo. for 2 years). Part of the induction and all of the maintenance therapies were given in the outpatient clinic.Results: Forty-one patients (98%) achieved CR after initial As2O3 treatment. One 72-year old man with XYY syndrome, diabetes and mental retardation died of pneumonia. Thirteen relapses occurred at a median of 15 (6–22) mo. As2O3-maintenance significantly decreased further relapses (3/24 with versus 10/17 without As2O3-maintenance, p=0.003). Two relapses died of cerebral APL before further treatment could be administered. Of eleven patients treated with As2O3+ATRA, 10 achieved CR, 8 of whom have remained in remission (median follow-up: 33 mo.). Two post-As2O3/ATRA relapses achieved CR again with As2O3+ATRA+ascorbic acid, and have remained in remission after maintenance treatment with As2O3+ATRA+ascorbic acid. All patients in continuous remission (n=38) were PML/RARa negative on polymerase chain reaction (sensitivity 10−3 to 10−4).Conclusion: Our regimen resulted in a leukemia-free-survival of 89.3%. The results suggest that an oral and mainly outpatient As2O3-based, non-HSCT strategy is efficacious for relapsed APL. In terms of survival, costs, treatment side effects and patient tolerance, the results appear to be comparable to, if not more favorable than, other treatment options based on high dose chemotherapy, graft-versus-leukemia effect, or anti-myeloid antibody therapy.

Diagnostic value of serum EBV-DNA quantification and antibody to viral capsid antigen in nasopharyngeal carcinoma patients.

We compared the amount of serum Epstein-Barr virus DNA (EBV-DNA) detected in patients with nasopharyngeal carcinoma (NPC) in a high-incidence area, represented by Taiwan, and a low-incidence area, represented by Japan, using real-time quantitative PCR. The median serum EBV-DNA value in 41 Japanese NPC cases was 5450 copies/ml, and that in in 23 Taiwanese cases was 2125 copies/ml. The median serum EBV-DNA value in all 64 NPC cases was significantly higher than in control groups. Using receiver-operating-characteristic (ROC) curves, the sensitivity and specificity of EBV-DNA quantification were determined (cut-off point, 6.87 copies/ml; sensitivity, 0.855; specificity, 0.885) and compared with those of EBV-viral-capsid-antigen (VCA) titers; the results showed that EBV-DNA was a more sensitive and specific parameter than EBV-VCA titer. Then, we analyzed 19 NPC patients in whom recurrence developed (11 Japanese and 8 Taiwanese), and 26 NPC patients in continuous remission. Although there was no significant difference in EBV-DNA values between Japanese and Taiwanese patients, the value was significantly higher in the 19 patients with recurrence than in those in remission. ROC analysis again revealed a higher diagnostic value of EBV-DNA than EBV-VCA. These results suggest EBV-DNA is a more reliable tumor marker than EBV-VCA in both high-incidence and low-incidence areas of NPC.

Pharmacogenetic determinants of outcome in acute lymphoblastic leukaemia.

Present day paediatric co-operative group acute lymphoblastic leukaemia (ALL) protocols cure approximately 80% of patients, a result achieved largely through the use of risk-stratified therapies that employ multiple chemotherapy agents. These risk-based therapies utilize host and leukaemia traits to select the most appropriate therapy. However, these risk-stratified approaches predict therapy response imperfectly and an important fraction of patients experience relapse or therapy-related toxicity. Pharmacogenetics, the study of genetic variations in drug-processing genes and individual responses to drugs, may enable the improved identification of patients at higher risk for either disease relapse or chemotherapy-associated side effects. While the impact of genetic variation in the thiopurine-S-methyltransferase gene on ALL treatment outcome and toxicity has been extensively studied, the role of other polymorphisms remains less well known. This review summarizes current research on the impact of genetic variation in drug-processing genes in paediatric ALL and reviews important methodological and statistical issues presently challenging the field of pharmacogenetics.

Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation.

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse. The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele-specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0-7.2 tumour cells/ml PB) higher than in the CD20- fractions (median 0; range 0-29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8-585; median 32 tumour cells/ml PB; CD20-: range 25-25527; median 334 tumour cells/ml PB). Enrichment with the anti-CD19 antibody as a second pan B-cell marker revealed comparable tumour cell numbers. In conclusion, an anti-CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B-cell compartment and an almost complete absence of tumour cells in the CD20- fractions.

Long-term survival of patients with acute myeloid leukemia

In 1982, the Fourth International Workshop on Chromosomes in Leukemia reviewed data prospectively collected on 716 patients with acute myeloid leukemia (AML) diagnosed between 1980 and 1982. The present study examined the extended follow-up on these patients. The analyses included cytogenetic and clinical data, with a median follow-up of 14.7 years, from 54 patients with treatment-associated AML and 628 with de novo AML. Of these patients, 291 received induction therapy that would be considered standard by today's criteria; no patient received high-dose cytarabine (HiDAC) intensification. Among the patients with treatment-associated AML, the only long-term survivor in retrospect appears to have had de novo AML. Among the patients with de novo AML, achievement of complete remission and survival varied significantly based on cytogenetic classification among all 628 patients as well as among those who did and did not receive standard induction therapy. The remission rate and survival were significantly better with standard induction therapy for patients with t(15;17) and normal cytogenetics. Multivariate analyses showed that karyotype was an independent predictor of survival for all patients and those receiving standard induction therapy. Only 8.9% of patients were alive 5 years following diagnosis, but 5 years of continuous remission was synonymous with cure. Even among 5-year survivors who had suffered a previous relapse, 41% appeared to be cured. Survival among patients in continuous remission for > or = 10 years varied significantly by cytogenetic classification. In the absence of HiDAC intensification, no complete responders with t(8;21) and only 7% with normal cytogenetics survived continuously 10 years disease free. Cure of AML following specific therapies must be evaluated in the context of cytogenetics. A meta-analysis incorporating cytogenetic data is indicated for patients with > or = 10 years of follow-up.

Treatment of metastatic breast cancer with a split-course high-dose chemotherapy regimen and autologous bone marrow transplantation.

We investigated the role of high-dose chemotherapy and autologous bone marrow transplantation (ABMT) as the initial systemic treatment in patients with hormone-unresponsive metastatic breast cancer. We studied a regimen involving a split-course schedule using sequential administration of two pairs of alkylating agents separated by 5 days of rest. The rest period was intended to provide time for recovery from the treatment-immediate adverse effects, thereby allowing further dose escalation. The treatment consisted of thiotepa 225 to 300 mg/m2/d (days - 11 to -9), cisplatin 50 to 100 mg/m2/d (days - 11 and -3), and cyclophosphamide 60 mg/kg/d (days - 3 and -2). Dose escalation was performed in the initial 15 patients before reaching dose-limiting toxicities. When feasible, responding patients received posttransplant irradiation to sites of residual or prior bulky disease. Patients with bone marrow or CNS involvement, prior pelvic irradiation, or age greater than 55 years were excluded. Thirty-nine patients with measurable or assessable tumor were enrolled: 23 with visceral metastases, 11 with only soft tissue disease, and five with skeletal involvement. Twenty-five patients had received no chemotherapy for metastatic disease before transplantation. The dose-limiting toxicities of this therapy were renal and gastrointestinal. Six patients died from complications: four of a fungal infection and two of hemorrhage. A complete response was achieved in 14 patients (36%), three of whom are free of disease at 79+, 55+, and 40+ months after transplantation. Ten of 25 patients not treated with standard-dose chemotherapy for metastatic disease achieved a complete response (40%). The three patients in continuous remission were in the untreated relapse group. This single high-dose treatment achieved a relatively high complete response rate in patients with metastatic breast cancer and may have cured some of them. On the other hand, the split-course dose schedule as tested here did not permit significant dose-intensification.

Persistence of circulating t(14;18)-positive cells in long-term remission after radiation therapy for localized-stage follicular lymphoma.

To determine the prevalence of circulating t(14;18)-positive cells in patients with long-term remission after radiation therapy for stage I and II follicular lymphoma. Peripheral-blood mononuclear cells from 21 patients in continuous remission were examined by a two-step polymerase chain reaction (PCR) assay for the detection of cells carrying a t(14;18) translocation with a breakpoint within the major breakpoint region (MBR) or minor cluster region (mcr). Follow-up duration was between 25 and 160 months, with a median of 6.5 years. Thirteen patients (62%) showed negative results on repetitive testing. Cells that were t(14;18)-positive were found in eight patients (38%), all carrying a breakpoint in the MBR. One patient relapsed in each group. Circulating t(14;18)-positive cells can persist in a high percentage of follicular lymphoma patients in long-term complete remission (CR) after radiation treatment for stage I and II disease. The significance of minimal residual t(14;18)-positive cells with regard to the risk of relapse needs to be investigated in further prospective long-term studies.

Residual disease at the end of induction therapy as a predictor of relapse during therapy in childhood B-lineage acute lymphoblastic leukemia.

More than 95% of children with B-lineage acute lymphoblastic leukemia (ALL) achieve a clinical remission after the induction phase of chemotherapy (first 28 days) as evaluated by morphologic criteria. However, relapse occurs in approximately 30% of these children. The objective of this study was to determine whether the outcome of patients in clinical remission at the end of induction therapy could be predicted using a highly sensitive method to detect residual disease. All children diagnosed with B-lineage ALL at the Children's Hospital of Philadelphia during a 2-year period were eligible. The extent of residual leukemia was quantitated in remission marrow samples obtained at the end of induction therapy in 44 children using a phage clonogenic assay in association with complementarity-determining-region 3 (CDR3)-polymerase chain reaction (PCR). Residual disease was a significant predictor of outcome independent of WBC count, age, or sex. The estimated relapse-free survival (RFS) during therapy was 50.4% (+/- 12.6%) for patients with high residual disease (> or = 0.6% leukemia cells among total marrow B cells) versus 91.9% (+/- 5.5%) for those with lower levels (P < .002). There were no significant differences in off-treatment RFS between patients with high or low residual disease who completed therapy in continuous remission (P = .82). The overall estimated RFS was 32.3% (+/- 11.6%) for patients with high residual disease versus 62.6% (+/- 10.7%) for patients with lower levels of residual leukemia cells, with a median follow-up of 5.3 years for patients in continuous remission (P < .008). PCR detection of high residual disease at the end of induction therapy identifies patients at increased risk for relapse during therapy.