ALBERT

Description: Multiple Myeloma (MM) is a malignancy of monoclonal plasma cell proliferation in the bone marrow, which accounts for 10% of all hematologic cancers. This slow proliferating B-cell malignancy accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics, which target rapidly replicating cells. MM remains an incurable disease in need of new therapeutic approaches. We have previously shown that the nucleoside analog, 8-chloro-adenosine (8-Cl-Ado), has significant activity for MM and that its sister compound, 8-amino-adenosine (8-NH2-Ado), appears to be even more potent, exhibiting greater activity in pre-clinical studies. 8-NH2-Ado is cytotoxic to MM cell lines both sensitive and resistant to conventional chemotherapies. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its tri-phosphate form, 8-NH2-ATP, and accumulation of 8-NH2-ATP results in a concomitant loss of endogenous ATP levels. 8-NH2-Ado induces apoptosis in MM cells as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a subG1 DNA content. Here we demonstrate the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. MM cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including ERK1/2, p38 MAPK and Akt kinase, within 30–120 minutes. Cells depleted of ATP by antimycin A or 2-deoxyglucose treatments to mimic 8-NH2-Ado-induced ATP depletion do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may be affecting the activity of protein phosphatases involved in the negative regulation of these signaling molecules. Based on studies using the phosphatase inhibitor okadaic acid, it appears that PP2A may play a role in the 8-NH2-Ado-induced decrease in phosphorylation of p38. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analog drug and appears to be unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical pro-survival and apoptosis-regulating proteins suggest that the modulation of these proteins by de-phosphorylation at early time-points may be an important mechanistic step in 8-NH2-Ado-induced programmed cell death.

Description: Treatment of advanced mycosis fungoides (MF)/Sézary syndrome (SS) remains difficult as virtually all pts become refractory to most standard therapies. The CD52 monoclonal antibody alemtuzumab is a new therapeutic option. We report from a clinical trial as well as clinical use in E-CTCL. Alemtuzumab was IV administered at a dose of 30 mg three times per week (t.i.w.) for 4 weeks (Dose 1 and 2: 3 mg and 10 mg, respectively) followed by SQ administration of 30 mg for an additional 8 weeks t.i.w. All pts received prophylactic treatment with TMP-SMZ, acyclovir, and fluconazole until immune reconstitution. 19 pts with E-CTCL have been treated to date. 15 pts have been enrolled into the study; while 4 pts who did not meet the pre-study criteria were treated off study (including second malignancy without recurrence diagnosed within 5 years prior study entry in 2 pts and living too remote from study center in 2 pts). Median age was 63 yrs, (39 to 88 years). Clinical stages were: 8 (42%) stage III; 10 (53%) stage IVA; 1 (5%) stage IVB. All pts were heavily pretreated with a median of 5 prior treatments. The ORR was 79% (15 pts.) with CR in 47% (9 pts) and PR in 32 % (6 pts) with effective clearing of circulating Sézary cells in 100%. Four pts (21%) developed PD with the development of cutaneous tumors in one patient despite complete clearing of circulating Sézary cells. Median response duration was 7 months (1–39 months). We have analyzed minimal residual disease (MRD) by PCR of γ/δ TCR after treatment in skin and/or blood samples in 11 pts who achieved CR and PR. MRD was detected in 8 pts with subsequent relapse in 5 pts after a median time of 6 months. Only 3 pts had negative blood samples tested with histologic evidence of cutaneous residual disease in 2 pts (PR). Patients remained in remission for 17, 4, and 5 months, respectively. Another patient with CR had negative skin, but a positive blood sample tested and relapsed after 3 months. The median overall survival of all pts. was 18 months (1–50 months). Ten pts (53%) have died, 7 (37%) attributable to MF. One patient developed a second B-cell lymphoma approximately 6 months after completing alemtuzumab. One patient died of aplastic anemia. None of the pts died of infectious complications. In general, treatment was well tolerated, with the majority of toxicities being Grade 2 in severity and transient. The major hematologic side effect was T-cell depletion (lymphopenia Grade 4) with infectious complications in 4 pts (including PICC line infection in 2 pts, and Herpes zoster infection and neck abscess formation in one pt each) and neutropenic fever in one patient without documented infection. 6 pts developed grade 3 or 4 leukopenia with prolonged cytopenias in 2 pts requiring withholding and/or discontinuation from treatment. No patient manifested reactivation of CMV infection. One patient developed a fungal otitis externa remote from study during the terminal phase of CTCL. In conclusion, alemtuzumab has shown impressive responses in patients with refractory E-CTCL and could be considered for first-line therapy. The persistence of MRD suggests that a longer duration of therapy or sequential use of other biologic agents may enhance responses or improve durability.

Description: AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values

Description: Cytogenetic analysis was performed on peripheral blood lymphocyte cultures from 19 patients with mycosis fungoides (MF )/Sézary syndrome (SS) stimulated with either phytohemagglutinin, a conventional mitogen, or a combination of interleukin-2 (IL-2) plus IL-7. The use of both PHA-stimulated and IL-2 plus IL-7–stimulated cultures enhanced the ability to identify clonal abnormalities. Clonal abnormalities were observed in 11 patients (53%) including one with monosomy for the sex chromosome as the sole abnormality. Five of the 11 patients with clonal abnormalities had normal peripheral white blood cell counts, indicating detectability of clones in the absence of frankly leukemic disease. The presence of clonal abnormalities correlated with advanced stage disease and a significantly reduced survival duration from the time of cytogenetic studies. Clonal abnormalities involving chromosomes 1 and 8 were observed in six cases. In five cases with aberrations of chromosome 1, loss of material involved the region between 1p22 and 1p36. In an additional case, a reciprocal translocation involving 1p33 was observed. Clonal abnormalities involving chromosomes 10 and 17 were observed in 5 cases, clonal abnormalities involving chromosome 2 in 4 cases, and clonal abnormalities involving chromosomes 4, 5, 6, 9, 13, 15, 19, and 20 in 3 cases. In 2 cases a der(8)t(8; 17)(p11; q11) was observed. Regions of the genome that encode T-cell receptors were not involved in abnormalities. The region between 1p22 and 1p36 is identified as a region of the genome that requires detailed analysis toward the identification of potential gene(s) involved in the process of malignant transformation and/or progression in MF/SS.

Description: Background- Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder of the brain caused by the reactivation of latent JC polyoma virus. Rituximab (Rituxan, Mabthera) is a B-cell depleting, monoclonal antibody which has been linked to reactivation of the hepatitis B virus. HIV infection, purine analog therapy, hematopoietic transplant and lymphoma have previously been associated with PML. We evaluated the characteristics of patients with B-cell lymphoproliferative disorders who developed PML after exposure to rituximab, and the quality of case reports available in the medical literature and at the FDA. Methods- Data sources included 1 observation at Northwestern Memorial Hospital, 14 reports obtained from the FDA MedWatch database, 9 case reports from the medical literature, and 21 reports from the manufacturer, Genentech, that were submitted to the FDA. Reports that did not confirm the diagnosis of PML, were associated with HIV infection, or rituximab use for a rheumatologic indication were excluded. Results- Of 47 unique case reports screened, 3 were excluded for rituximab use in rheumatologic disease, 2 for pre-existing HIV, and 13 for inadequate confirmation of the PML diagnosis. In the 29 remaining cases, survival of PML was reported in only one patient. Diagnosis was made by brain biopsy (n=6), autopsy (n=6), or MRI of brain AND positive JC virus by PCR (n=17). Five cases were seen in hematopoietic transplant recipients, 4 autologous and 1 allogeneic. Of the 24 patients who were not transplanted, 10 had purine analog exposure. Thirteen of the 14 patients that did not receive transplant or a purine analog received an alkylating agent, with 7 receiving standard R-CHOP. The median age of the 29 patients was 63.5 years (range 32–89), 15 were female and 14 male. Indications for rituximab were: large B-cell lymphoma (n=6), follicular lymphoma (n=6), CLL (n=5), Waldenstrom (n=2) and other non-Hodgkin lymphomas (n=10). Median time to diagnosis of PML from first and last rituximab doses was 10 and 5 months respectively. The median number of rituximab doses was 6. Quality comparison of source data is contained in table 1. Overall completeness ratio for literature and observed reports vs. FDA and manufacturer reports was 1.48. Conclusions: We have found 29 cases of rituximab-associated PML, of which 14 were not associated with transplantation or purine analog exposure, suggesting an association of rituximab therapy independent of these other treatment related risk factors. FDA and manufacturer reports are inferior to those available in published case reports or through active surveillance. Further examination of the relationship of rituximab to PML, using an active surveillance strategy, is warranted. Type of Information Literature and Observed Cases (n=10) %Reporting FDA and Manufacturer Cases (n=19) %Reporting Completeness Ratio Outcome 90 63 1.42 Date of Lymph 80 68 1.18 Diagnosis Date of Death 78 53 1.47 HIV Status 60 16 3.75 Specific Lymphoma Type 100 84 1.19 Dose of Rituximab 80 63 1.27 Date of 1st Dose 70 94 .74 Date of Last Dose 70 84 .83

Description: Without a HSCT, CLL remains an incurable disease. A dramatic response to intensive chemo-immunotherapy may be accompanied by the irreversible long term consequences to the bone marrow, limiting further therapeutic options. For this reason we have initiated a clinical trial combining Rituximab (RIT) and Alemtuzumab (ALEM), two monoclonal antibodies with established activity and side effects profiles, as an initial therapy for patients with CLL requiring intervention. Methods This trial will enroll 21 previously untreated CLL patients and be considered promising if ≥ 85% of them respond. Therapy duration is 18 weeks. Subcutaneous (SQ) ALEM dose escalation: 3 mg – 10 mg – 30 mg on days 1, 3, 5, is followed by the 30 mg 3 times/week for 17 weeks. RIT, 375 mg/m2/dose IV is administered every other week staring on the 3d week for 8 cycles. All patients received PCP, herpes virus, and fungal prophylaxis as well as CMV viral DNA monitoring. Responses were based on NCI-WG 1996 criteria, however, lymphadenopathy and organomegaly was also assessed by serial CT scans. MRD was measured in peripheral blood and bone marrow aspirate using flow cytometry for CD19+/CD5+/CD23 lymphocytes. Results Since September 2005, 13 patients have been enrolled and the interim analyses are available for the 11 patients who have completed the therapy. All patients met ECOG criteria for treatment requirement. Median age was 54 years (29 – 75) with 6 males and 7 females. The median time from the diagnosis to treatment was 13 months (1–56 months). Clinical stage (Rai) was I in 4, II in 4, and IV in 5 patients. Median β2 microglobulin was 2.54 (0.34–4.29). Median WBC was 42 ×109/L (16 – 119), Hgb 13.3 g/dL (10.7 – 15.6), and platelet count 144 × 109/L (67 – 302). Cytogenetic analysis, by FISH panel, was 13q in 6 patients, trisomy 12 in 5 patients, and 13q/11p in 2 patients. 3 patients were Zap70+ and 1 patient was Zap70+ and CD38+. All of the patients responded with 2 CR, 8 PR, and 1 SD. By NCI-WG 1996 criteria, without CT scans, 8 patients (72%) achieved CR and 3 patients (27%) achieved PR. At the completion of the study 9 patients (81%) had no evidence of MRD by flowcytometry, 1 had too few cells to access clonality, and 1 was MRD positive. Median duration of the response has not been reached with a median follow-up of 15 months (9–23+). 3 patients had increased lymphadenopathy by CT scan (by 5, 6, and 8 month after completion of therapy), none reached the definition of disease progression. 4 patients had CMV reactivation by PCR, 2 of whom had symptom of malaise, none suffered organ involvement, and all of them cleared the infection with gancyclovir administration. No other serious infectious complications were documented. All patients developed grade 1–2 skin rash at the site of ALEM injection after the 1st dose of 3mg only; none required intervention. All patients developed grade 3–4 lymphopenia; neutropenia: grade 2 in 2, grade 3 in 4, and grade 4 in 1 patients; anemia: grade 1 in 9, grade 2 in 2 patients. 5 patients have not achieved full T cell recovery by 22, 21, 18, 15, and 9 months. The other 6 evaluable patients achieved T cell recovery by 1 (n=2), 4 (n=2), 7, and 10 months. Patients who suffered CMV reactivation achieved faster T cell recovery. None of the patients developed an autoimmune phenomena or Richter’s transformation Summary Combination of ALEM and RIT is well tolerated and active regimen for patients with CLL and may represent a viable alternative to the combination chemotherapy.

Description: Standardized response criteria are essential for interpretation of clinical data, comparisons among clinical trials, development of new therapies, and approval of agents by regulatory agencies. In 1999, an International Working Group (IWG) developed recommendations for response assessment for non-Hodgkin’s lymphomas (NHL) that were adopted internationally by study groups and regulatory agencies and, subsequently, by clinical trials groups for Hodgkin lymphoma (HL) as well (Cheson et al, J Clin Oncol, 17:1244, 1999). Since their publication, several observations compelled a reassessment and, ultimately, revision of those guidelines, e.g., the availability of FDG-PET scans, new insights into lymphoma pathology and biology, the failure of the IWG guidelines to include HL and extranodal NHL, and features of the original guidelines that were found to be unclear as they were implemented into clinical trials. Most notable of these was the interpretation of the response category of Complete Remission unconfirmed (CRu). In the context of the IHP, a group of international lymphoma investigators with expertise in medical hematology/oncology, radiation oncology, nuclear medicine and imaging, pathology, biostatistics, and pediatrics were convened to revise the IWG guidelines. Committees focused on Response Criteria, Pathology/Biology, Endpoints, and Clinical Features, and discussions were initiated to undertake a major revision of the IWG guidelines. The important modifications that will be presented included, but were not limited to, integration of PET according to recent data (Juweid et al, J Clin Oncol, 23:4652, 2005), to facilitate the distinction between persistent tumor and scar/fibrosis, virtually eliminate the designation of CRu, and improve prediction of outcome. Guidelines were provided for the specific indications where this test can be currently recommended. Other proposals involved the role of flow cytometry and assessment of minimal residual disease. Response criteria for extranodal sites were also incorporated. Adoption of these revised guidelines by study groups will further improve the conduct and interpretation of clinical trials leading to more effective therapies for patients with lymphomas.

Description: 8-Aminoadenosine (8-NH2-Ado), a ribosyl nucleoside analog, in preclinical models of multiple myeloma inhibits phosphorylation of proteins in multiple growth and survival pathways, including Akt. Given that Akt controls the activity of mammalian target of rapamycin (mTOR), we hypothesized that 8-NH2-Ado would be active in mantle cell lymphoma (MCL), a hematological malignancy clinically responsive to mTOR inhibitors. In the current study, the preclinical efficacy of 8-NH2-Ado and its resulting effects on Akt/mTOR and extracellular-signal–regulated kinase signaling were evaluated using 4 MCL cell lines, primary MCL cells, and normal lymphocytes from healthy donors. For all MCL cell lines, 8-NH2-Ado inhibited growth and promoted cell death as shown by reduction of thymidine incorporation, loss of mitochondrial membrane potential, and poly (adenosine diphosphate-ribose) polymerase cleavage. The efficacy of 8-NH2-Ado was highly associated with intracellular accumulation of 8-NH2-adenosine triphosphate (ATP) and loss of endogenous ATP. Formation of 8-NH2-ATP was also associated with inhibition of transcription and translation accompanied by loss of phosphorylated (p-)Akt, p-mTOR, p-Erk1/2, p-phosphoprotein (p)38, p-S6, and p-4E-binding protein 1. While normal lymphocytes accumulated 8-NH2-ATP but maintained their viability with 8-NH2-Ado treatment, primary lymphoma cells accumulated higher concentrations of 8-NH2-ATP, had increased loss of ATP, and underwent apoptosis. We conclude that 8-NH2-Ado is efficacious in preclinical models of MCL and inhibits signaling of Akt/mTOR and Erk pathways.

Description: Glucocorticoid (GC) is an effective anti-myeloma agent by directly inducing apoptosis and growth inhibition in myeloma cells. The anti-myeloma action of GC is mediated by its intracellular receptor, the glucocorticoid receptor (GR). The response to GC is variable among myeloma patients and resistance to glucocorticoid treatment develops almost invariably. The mechanism regulating glucocorticoid-sensitivity in not well understood, although it has been hypothesized that the regulation might occur at both the receptor (GR) and post-receptor level. In MM.1 myeloma cell lines, we observed significant down-regulation of GR expression as the cells develop GC-resistance, suggesting that GR expression level is an important determinant of GC-response. Here we present evidence that GR expression level directly correlates with the clinical outcome in myeloma patients who were treated with GC-containing regimens. First, using a quantitative real-time PCR analysis, we performed a retrospective analysis of GR gene expression on bone marrow plasma cell samples from 35 patients with newly diagnosed myeloma who were then treated on the ECOG study E1A00. This is a phase 3 trial which randomized patients to dexamethasone (dex) alone vs. dex in combination with thalidomide. Pre-treatment expression levels of GR-alpha, GR-beta and GR-P splicing isoforms were measured for 35 patients, of whom, 10 had post-treatment samples available. Interestingly, a large variation of the GR expression levels was observed across the patient samples, with the GR-alpha being the predominant isoform. However, a clear correlation between GR expression and treatment response was not seen, likely due to the small sample size. To access a larger patient pool, we retrospectively analyzed the GR expression levels using the cDNA microarray data from the Arkansas group on 351 newly diagnosed myeloma patients treated with Total Therapy 2, of which dexamethasone was an essential component throughout the treatment course. Of the 351 patients, both event-free (EFS) and overall survivals (OS) were compared between 49 patients (14%) with low levels of GR expression and the rest (86%) with high levels of GR expression. Strikingly, the cases with low levels of GR expression were associated with statistically significantly worse EFS and OS compared to those with high levels of GR expression, with the 5-year EFS of 20% vs. 52% and OS of 34% vs. 68%, P 〈 0.01. In summary, we have shown for the first time that the baseline level of glucocorticoid receptor expression in newly diagnosed myeloma patients predicts their clinical response to GC-containing regimen and the overall clinical outcome.