ALBERT

Description: High dose therapy represents the gold standard therapy for newly diagnosed multiple myeloma (MM) patients (pts), with no definite agreement about the adoption of single or double transplant. From January 2000 to December 2004, 151 consecutive MM pts aged ≤65 years in stage II, III or I in progression according to Durie-Salmon were enrolled in a multicenter no randomised high dose program including a tandem transplant (Tx1; Tx2). The protocol was designed as follows: 2 pulse-VAD as induction, 2 DCEP to mobilise peripheral blood stem cells (PBSC), double auto-transplant 3-6 months apart each conditioned with high-dose Melphalan at the dose of 200 mg/m2. Patients characteristics at the enrolment: males 76 (51%), females 75 (49%), median age 55 (range: 35–65), stage I in progression 26 (17%), stage II 25 (16%), stage III 100 (67%). Response rates after each phase for the evaluable patients are reported in the table below VAD (151 pts) DCEP (146 pts) Tx1 (119 pts) Tx2 (63 pts) CR (%) 4 9 18 29 VGPR (%) 28 35 48 60 PR (%) 44 30 25 9 SD (%) 18 10 2 0 Progr (%) 6 16 7 0 Patients not addressed to transplant for mobilization failure were only 5%. Most of the patients (75%) collected ≥ 4x106CD34+cells/Kg after each DCEP-cycle which were considered adequate to rescue hemopoiesis after each transplant. The whole protocol was well-tolerated. In particular, no therapy related mortality was associated to pulse-VAD, or DCEP, and no difference between Tx1 and Tx2 as far the transplant related mortality was registered (1.5% after each transplant). Second transplant was not performed in 48 pts for the following reasons: 8 pts (7%) did not collect enough PBSC, 8 pts (7%) have had severe toxicity with the first transplant; 8 pts (7%) underwent allo-TMO; 7 pts (6%) had progressive disease and 15 pts (12%) refused Tx2. Finally only 76 pts (50% of the enrolled pts) completed the program with the second transplant. Analysing data on an intention-to-treat basis, median follow-up was 30 months, median Progression Free Survival (PFS) was 31 months, median overall survival (OS) was not reached. The median Event Free Survival (calculated from the completion of Tx1 to progression or any other event) was 20 months. No difference in terms of PFS and EFS was found comparing pts who finally received only Tx1, with those who completed the protocol (p=0.9; p=0.5). The EFS was not statistically different for patients receiving one or two transplant even when the analysis was performed according to the type of response achieved after Tx1. In conclusion, despite higher percentage of good quality responses (CR+VGPR) can be obtained with 2 transplants with respect to 1 (66% vs 89%) without additional toxicity, no difference in terms of PFS or EFS were observed between the patients who underwent 1 or 2 transplants. Thus, keeping into account the more complex management of patients in a tandem transplant program, it might be more advantageous to perform as initial therapeutic approach, high-dose protocol including only 1 transplant procedure.

Description: Fludarabine is a key drug in the treatment of CLL, and the oral formulation has an efficacy and safety profile similar to the intravenous formulation in previously treated patients (pts). The NCIC CTG conducted a phase II study evaluating the overall response rate (ORR) to oral fludarabine in previously untreated pts. Leukemia cells were studied for common cytogenetic abnormalities by FISH, expression of ZAP-70, and IgVH mutational status, to assess whether these parameters influence response to therapy. Eligibility criteria included diagnosis of CLL confirmed by lymphocytosis of 〉5×109/L, leukemia-cell expression of CD5, CD19, and CD23 with light chain restriction, and indication for treatment. Pts were treated with oral fludarabine 40 mg/m2/day × 5 days every 28 days for a max of 6–8 cycles. Response evaluation was based on NCI-WG criteria. Interphase FISH was performed on PBMCs, evaluating for deletion of ATM at 11q22.3, D13S319 at 13q14.3, D12Z1 at 12p11.1-q11.1, and p53 at 17p13.1. ZAP-70 expression and IgVH mutation analysis of CLL cells were assessed before therapy in all patients, and in a subset, after therapy. Results: 126 eligible pts were enrolled between Aug2002–Jan2004 at 26 institutions. Median age was 60.9 years, male 62%, female 38%. Distribution by Rai stage was I 25%, II 43%, III 14%, and IV 18%. The ORR at the completion of therapy was 64%, with 18% CR, 3% unconfirmed CR, 43% PR, 13% SD, and 13% PD. At median follow-up of 23.2 months, median progression-free survival (PFS) was 15.3 months (95% CI 13.6–16.7). Median overall survival has not been reached. 92 pts completed protocol defined therapy; 12 discontinued due to toxicity, 8 due to progressive disease and 13 for other reasons, including 1 pt withdrawn due to AIHA. Hematologic toxicity (NCI-WG) included thrombocytopenia, grade 3/4 (14/126; 11%), and neutropenia, grade 3/4 (54/126; 51%). FISH analysis revealed abnormal cytogenetics in 79% of 122 evaluable cases, with del (13) in 58%, del (11) in 23%, +12 in 14%, and del (17) in 5%, with more than 20% of CLL cells having each abnormality. All 6 pts with del (17) had a significantly poorer PFS relative to pts without a del (17) (Hazard Ratio 7.4, 95% CI 3.06–17.98). IgVH sequencing completed on 102/126 samples at the time of this analysis, and 62% of cases had unmutated IgVH (〉98% homology to known IgVH gene), whereas 39% expressed mutated IgVH genes. Forty-three (68%) of 63 cases with unmutated IgVH, but only 5 (14.6%) of 39 cases with mutated IgVH expressed ZAP-70, a concordance rate similar to that observed previously. Of total 125 cases examined, 55 were ZAP-70 positive and 70 were negative; leukemia-cell expression of ZAP-70 was unchanged after therapy in 47/52 (90%) of cases successfully examined. ORR in the pts with ZAP-70-positive/unmutated IgVH CLL cells was 63% versus 77% in the ZAP-70-negative/mutated IgVH group (P=0.22). Conclusions: Oral fludarabine as a single agent in untreated CLL is associated with response rates and toxicity profile comparable to that of intravenous fludarabine given on a similar schedule. Leukemia-cell expression of ZAP-70 generally correlated with the use of unmutated IgVH genes and appeared unaltered following treatment in most cases studied. Patients with ZAP-70-negative/mutated IgVH CLL had a better ORR although this did not reach statistical significance.

Description: Outcome of adult acute lymphoblastic leukemia (ALL) continues to be poor, and parameters to better discriminate patients with distinct prognosis are necessary. In studies comparing global gene expression differences between normal hematopoietic cells (whole bone marrow, peripheral blood, CD34+, and CD22+ sorted populations) and ALL cells using microarrays we found that the B-ALL cells showed evidence of increased expression of Connective Tissue Growth Factor (CTGF). The median log 2 transformed signal intensity of CTGF was 4.26 (range 3.95–4.76) in normal hematopoietic cells, and 6.59 (range: 3.85–10.62) in all leukemic samples; this difference in signal intensity is equivalent to a 5-fold increase in median expression of CTGF in leukemic cells. Therefore, we hypothesized that expression level of CTGF may have prognostic significance in adult ALL. Using real-time RT-PCR assays for CTGF we examined the expression of CTGF in 79 diagnostic ALL patients from SWOG protocol S9400 (28 bone marrow and 51 peripheral blood samples). Patients with L3 ALL were excluded from the study. The median age of patients was 35 (range 17–64), with the median WBC 23,400/ul (range 600–396,600), and peripheral blood blasts 56% (range: 0–98). Fifty patients had B-ALL (63%), 13 (16%) had T-ALL and lineage was unknown for 16 (20%). When treated as a continuous variable in a logistic regression model, the level of CTGF expression was significantly associated with inferior OS and DFS (p=0.007 and p=0.0012, respectively). When controlled for WBC and cell lineage, the association of CTGF with OS and DFS remained statistically significant. We then sub-grouped the ALL patients into three equal groups (tertiales) based on CTGF expression. This subgroup analysis found that the OS for patients in the highest tertile (highest CTGF expression) was approximately 11% (95% CI 0–24) at 5 years, as compared to 42% (95% CI 23–61) and 58% (95% CI 38–78) for patients with middle and low CTGF expression respectively (figure). In sub-analysis of patients with B-lineage ALL (n=50), the association of CTGF expression with OS and DFS was still statistically significant (p=0.009 and p=0.005) when treated as a continuous variable. This report is an example where a gene expression study detected a gene differentially expressed in leukemia, with clear clinical value. Moreover, this is the first report that correlates the level of expression of CTGF with outcome in ALL patients. We are actively pursuing the biological and clinical significance of CTGF in other ALL patients and model systems. Figure Figure

Description: In order to assess whether Pgp modulation with PSC833 improves disease-free (DFS) and overall (OS) survival in untreated patients (pts) with AML 〈 60 years, a phase 3 trial (CALGB 19808) was undertaken comparing two induction regimens: Ara-C (A), Daunorubicin (D), and Etoposide (E), with (ADEP) or without (ADE) PSC833. All pts received A 100 mg/m2 by CIV daily for 7 days. In ADE, D 90 mg/m2 and E 100 mg/m2 were each given daily by short IV infusion on days 1–3; the corresponding doses in ADEP were D 40 mg/m2 and E 40 mg/m2. PSC833 10 mg/m2 was given by CIV over 72 hrs after a loading dose of 2.8 mg/kg IV over 2 hrs. These doses were based on findings from parallel phase I trials that showed comparable efficacy and toxicity for the 2 regimens (JCO2004; 22:4290). Pts in complete remission (CR) received consolidation therapy according to cytogenetic risk (ASH2001, 688a) and were then randomized to a phase 3 immunotherapy sequence comparing interleukin-2 with observation. The Pgp modulation portion of the trial ended when PSC833 became unavailable after 302 of a planned 600 pts had been randomized. Prior myelodysplasia or therapy-related AML were exclusion criteria. The median age was 45 years; the arms were comparable with respect to cytogenetic risk groups (Core-Binding Factor (CBF) leukemia vs. others). Response by intent-to-treat on the part of 296 evaluable pts and toxicity data are tabulated below. The median follow-up is 2.2 years. None of the differences in CR rate, OS, or DFS are statistically significant. ADE (n=149) ADEP (n=147) CR 77% 78% CR - CBF AML 100% (21/21) 100% (25/25) CR With One Induction Course 89% 88% Induction Mortality 7% 7% Median OS (months) 21 20 Median DFS (months) 19 15 Median OS -pts ≤ 45 yrs (months) 29 Not Reached Median DFS pts ≤ 45 yrs (months) Not Reached 13 Grade 3 and 4 Toxicities Dysphagia/Esophagitis 6% 17% Stomatitis 9% 24% Bilirubin 6% 25% Rash 1% 10% Left Ventricular Function 2% 1% The high dose daunorubicin regimen was given safely, without excess induction mortality or cardiotoxicity. Hepatotoxicity due to ADEP consisted of generally reversible hyperbilirubinemia. PSC833 did not cause significant neurotoxicity. Compared with the induction regimen used in the previous group study in this pt population in which 474 pts received D 45 mg/m2 x 3 days and A 200 mg/m2 x 7 days without E or PSC833, (CALGB 9222, Blood2005; 105:3420), the CR rates in this study were higher (78% vs 72%), the number of pts achieving CR with 1 induction higher (89% vs. 77%), and the induction mortality comparable (7% vs 9%). These preliminary findings suggest that similar outcomes occur following an induction regimen using 2.25-fold higher D and 2.5-fold higher E doses (ADE) relative to a regimen employing a Pgp modulator (ADEP). PSC833 is known to delay the hepatic clearance of D and E. These data cannot distinguish between the possibilities that PSC833 augments the anti-leukemia activity of the lower dose ADE regimen or whether there is, in fact, no clinically meaningful dose-response relationship with D and E within the evaluated dose ranges. The findings of this trial to date suggest that Pgp-mediated drug efflux may not be the major or sole mechanism of drug resistance in AML or that PSC833 is an inadequate inhibitor of Pgp activity or both.

Description: The mouse is the premier model organism in human disease research because all of its life stages are accessible and there are myriad experimental tools for comparative analysis and specific manipulation of its genome. The Mouse Genome Informatics Database (MGI, http://www.informatics.jax.org) supports biological knowledge building for the laboratory mouse by integrating and providing access to a wide range of data from DNA sequence to phenotype and disease associations. The integration of complex disease phenotypes, underlying genetic causes, and gene function information can be used to confirm human disease models and provide insight into disease mechanisms. We will illustrate the utility of MGI using hemochromatosis as an example. To describe phenotypic abnormalities and similarities to human disease in the mouse, we developed and utilize a vocabulary of mouse anomalies (the Mammalian Phenotype Ontology) and utilize the human disease terms provided in the Online Mendelian Inheritance in Man (OMIM). These standard terms provide a backbone for annotation, allowing both easy access and searching for researchers via web forms and computational access for data downloads. Within MGI, more than 12,000 mouse mutant alleles have been catalogued, representing mutations in more than 6,150 genes. Of these, more than 1,000 mutant alleles in 760 genes are associated with Mammalian Phenotype terms for hematopoietic defects and approximately 150 of these have an OMIM human disease association. For example, there are 26 alleles in 13 genes associated with Hermansky-Pudlak syndrome, 6 alleles in 4 genes associated with hereditary spherocytosis, and 9 alleles in 5 genes associated with hemochromatosis. According to OMIM data, hemochromatosis in human is associated with at least 5 different genes including HFE, HFE2, HAMP, TFR2, and SLC40A1. In mouse, 12 mutant alleles in three orthologous mouse genes, Hfe, Tfr2, and Slc40a1, have been described and used as potential models for hemochromatosis. Of these mutant alleles, 6 are associated with hemochromatosis phenotypes in MGI. In addition, there are at least 6 mutant alleles in 4 additional genes (B2m, Heph, Tfrc, and Trfr2) that have been associated with the hemocromatosis phenotype in mice and may yet be discovered to influence disease in humans. Finding an appropriate model system for study of human disease is a critical step toward understanding the biological mechanism leading to disease phenotype in human and mouse. MGI provides researchers with query forms that allow simple and complex questions to be addressed. These can range from queries about a single gene or disease term to precise queries that simultaneously address phenotype, disease, gene function, expression, and genome location data. The vocabulary-based phenotype and disease annotations as well as other structured data types can assist in robust and accurate data mining when posing complex biological questions in both computational and individual formats at MGI.

Description: Background. Patients with IPI 2,3 large cell lymphoma have a poor outcome with long term survival lower than 50%. Evaluation of response only with CT scan shows often residual masses which can be tumoral or fibrotic. Gallium 67S discriminate better these two situations and therefore can help to decide further strategies. Aim. To assess the efficacy of PBSCT in patients with poor prognosis aggressive NHL according to previous early response to Mega-CHOP evaluated with CT & Ga67S Patients & methods. Inclusion criteria were: G67S positive large cell B cell lymphoma with IPI score ≥ 2 or IPI

Description: A somatic mutation in the JH2 autoinhibitory domain of the Janus kinase 2 (JAK2) tyrosine kinase was recently described in polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. The prevalence of this mutation in either “atypical” myeloproliferative disorders (MPDs) or the myelodysplastic syndromes (MDSs) is unknown. Bone marrow–derived genomic DNA from 245 patients—119 with chronic myelomonocytic leukemia (CMML), 101 with MDS, 11 with hypereosinophilic syndrome (HES), 8 with systemic mastocytosis (SM), and 6 with chronic neutrophilic leukemia (CNL)—was screened for the JAK2 V617F mutation. A mutant allele was detected in 11 patients: 3 with CMML (3%), 5 with MDS (5%), 2 with SM, and 1 with CNL. Interestingly, one of the patients with SM and the patient with CNL with JAK2 V617F had a history of lymphoma, and this patient with SM also had associated myelofibrosis and CMML. The current observation strengthens the specific association between JAK2 V617F and classic MPD, but also suggests an infrequent occurrence in other myeloid disorders.

Description: Conditioning regimens utilized in reduced intensity transplants are designed to optimize immune suppression to allow a graft vs. tumor effect. One reduced intestiy regimen reported to result in a reduced incidence of GVHD while demonstrating disease response consists of extracorporeal photopheresis, pentostatin, 4 mg/m2/day X 2, and reduced dose total body irradiation(TBI; 600 cGy given in 3 fractions) (Miller KB et al; BMT2004;34:881–9). This regimen has been utilized in our center in 45 patients who have at least 6 months of follow up. All were ineligible for a conventional conditioning regimen, and from 3/03, this regimen was utilized for all reduced intensity transplants with exception of those wherein prior radiation precluded this dose of TBI. The median age of the patients was 55 years with age range 27 through 67 years. Thirty-three of the patients were 50 years of age or older. Twenty-five patients received sibling and 20 an unrelated donor (UD) transplant. All but one sibling transplant was a 6/6 match, whereas 8/20 of the UD transplants involved mismatched loci. GVHD prophylaxis consisted of tacrolimus and short course methotrexate in 43, tacrolimus/MMF in 1, and tacrolimus/sirolimus in 1. Seventeen patients had AML, 3 MDS, 2 ALL, 2 CML, 11 CLL, and 8 NHL. One had HD, and one had Waldenstrom’s. Eight of the 45 had prior stem cell transplantation. The median number of CD34+ cells infused was 4.54 X 106/kg recipient weight. Nine patients were transplanted in CR or early disease phase. The remainder had more advanced stages of disease. Five patients died before anticipated neutrophil recovery. Two others had no neutrophil nadir, and median time to neutrophil engraftment in the remainder was 14.5 days. In those evaluable for platelet engraftment, the median time to engraftment was 18.7 days. For those surviving to approximately Day+30, the median donor chimerism as assessed by VNTRs was 94% (range 34%–100%). The overall Day 100 survival was 69% (31/45), with 80%(20/25) of sibling graft recipients alive and 55% (11/20) of UD recipients still living. Twelve patients developed regimen related toxicity. In five, this manifested as ARDS or multi-organ failure with a capillary leak syndrome, and 2 had renal failure. Ten patients had disease resistance or relapse after transplant, and all of those have died. The overall survival to date is 42%; 48% for sibling transplant recipients and 35% for UD recipients with range of follow up in survivors from 283 to 1366 days (median 535 days). Acute GVHD of grade III or IV was seen in only 3 patients. For those surviving past 100 days, 27% had extensive GVHD. No patients transplanted with this regimen for AML not in remission survived. Only one patient with NHL not in CR or a chemotherapy sensitive relapse state survived. This conditioning regimen of extracorporeal photopheresis, pentostatin, and TBI therefore appears most effective for patients with AML in first or greater remission or for CLL patients in early relapse with responsive disease. The regimen was associated with a capillary leak/organ failure syndrome in seven patients but was otherwise well-tolerated. Its long-term effects on GVHD incidence and coincident graft vs. malignancy benefit remain to be better understood as compared to other reduced intensity regimens which do not contain extracorporeal photopheresis.

Description: The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC 〉1.0x109/l and 13 d (11–24) for plt 〉50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ 〉100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT. We believe that these results are due to the rapid development of a wide T cell repertoire obtained by TK cell infusions. Immunological follow-up showed Th1/Tc1 effector memory T cells, with a wide TCR repertoire in the first 3 months after SCT in all patients. High frequencies of T cells specific for CMV (median: 35 and 93 spots/105 cells with CMV-infected donor and host fibroblasts) and EBV (median: 58 and 41 spots/105 cells with donor and host EBV-LCL) were detected by gIFN ELISpot at time of immunereconstitution, and correlated with complete control of viral infections. Normalization of the T cell repertoire was documented by spectratype, immune-phenotype for naïve and memory T cell subsets and gIFN ELIspot 6 months after treatment. A phase III randomized multicentric study will start in 2006.

Description: Background. The stomach is the most frequent site of intestinal lymphomas. However, few data are available on the clinical-endoscopic presentation of gastric lymphoma as well as on possible differences in clinical pattern and endoscopic features between low-grade (LG) and high-grade (HG) lymphomas. In this study, we evaluated such aspects on consecutive primary gastric lymphoma patients observed in the last 12 years (1993–2004) in four Italian Hospitals (1 North, 2 Centre, 2 South). Methods. Clinical, histological, and endocospic records of consecutive patients diagnosed with LG or HG gastric lymphoma were retrieved and accurately evaluated. Symptoms were categorized as “alarm” (anaemia/melaena/heamorrage, persistent vomiting, weight loss) or “no alarm” (epigastric/abdominal pain, heartburn, dyspepsia/bloating). The endoscopic findings were classified as “normal” (no macroscopic lesions) or “abnormal” (ulcer, erosions, nodular pattern, hypertrophic folds, polypoid mass). Statistical analysis was carried out by using the Chi squared test. Results. During the study period, 143 patients with primary gastric lymphoma were detected. Overall, 61 patients were observed in the first 6 years and 82 in the last 6 years. The main results of the study are summarized in the table 1. Conclusions. The incidence of primary gastric lymphoma seems to be increasing. The overall prevalence of alarm symptoms is quite low, and they may be absent in near 75% of LG lymphoma patients. Moreover, contrarily to HG, LG lymphoma may present as a normal endoscopic finding and it is more frequently associated with H. pylori infection. At diagnosis, HG lymphoma is more frequently detected in an advanced stage as compared to LG lymphoma. Overall (143 patients) LG lymphoma (73 patients) HG lymphoma (70 patients) P value Age (mean ± SD) yrs 59.5 ± 14.2 59.4 ± 13.3 59.7 ± 15.1 0.4 Sex (M /F) 83/60 44/29 39/31 0.6 Alarm symptoms 57 (40%) 19 (26%) 38 (54%) 0.0009 Normal endoscopy 15 (10%) 15 (20%) 0 (0%) 0.0004 H. pylori infection 66 (73%) 47 (86%) 27 (39%) IA) 78/65 58/15 20/50