ALBERT

Description: The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.

Description: Purpose Ovarian cancer is the most lethal gynecologic cancer. Chemotherapy, the standard of care, has hematologic toxicity, primarily neutropenia. G-CSF is currently used to support white blood cell (WBC) and absolute neutrophil counts (ANC). Prior clinical trials from China suggest that acupuncture could ameliorate chemotherapy-induced leukopenia; the proposed mechanism is an increase in G-CSF levels. In the current study, we investigated the effect of acupuncture, administered during myelosuppressive therapy, on WBC and ANC counts in ovarian cancer patients. Patients and methods Twenty-one newly diagnosed or recurrent ovarian cancer patients were randomized to receive active versus sham acupuncture while undergoing standard IV platinum and taxane-containing chemotherapy. A standardized protocol with 9 acupuncture points was employed with manual and electroacupuncture stimulation. The frequency of acupuncture treatment was 2–3 times per week for a total of 10 sessions, starting 1 week before the 2nd cycle of chemotherapy. WBC and ANC counts were checked weekly at five time points. Serum G-CSF was collected four times during the study. Results Of 587 patients screened, 21 patients were enrolled and received either acupuncture or sham treatment. Patients in both the active and control arms had similar patient characteristics and treatment. Both median WBC and ANC values at nadir in the acupuncture arm were higher than in the control arm, but the differences were not statistically significant, after adjusting for the baseline difference. However, the median WBC in the acupuncture arm at recovery was statistically significantly higher than the control arm, after adjustment (8,600 cell/μL, range: 4,800–12,000 vs. 4,400 cell/μL range: 2,300–10,000) (p=0.045). The recovering median ANC in the patients receiving acupuncture also was higher, but this difference was not statistically significant (p=0.094). The median serum G-CSF at baseline for patients in the active vs. control arm was similar (37.3 pg/mL, range 28.6–393.3 vs. 32.0, range 11.8–211.3, respectively) (p=0.291). At the second time point, the 1st day of the 2nd cycle, the acupuncture group had a higher G-CSF value than the control group (p=0.121). At nadir, the acupuncture group still had a slightly higher G-CSF value than in the control group (p=0.796). However, at the recovery day, the 1st day of 3rd cycle, the G-CSF value in the acupuncture group was lower than in the control arm (p=0.729). No statistical significance in G-CSF value was found at each time point between the two groups. Conclusion The acupuncture protocol used in this study was feasible and safe. We report trends of higher WBC and ANC values during one cycle of myelosuppressive chemotherapy in ovarian cancer patients, suggesting a potential myeloprotective effect of acupuncture. However, current data do not support an acupuncture effect on G-CSF production. These findings warrant a larger study to explore the observed clinical trends and other potential underlying mechanisms.

Description: Background: SEER data indicate that African Americans (AA) have a lower incidence of DLBCL but a higher mortality rate than Caucasians (C). To investigate this, we conducted a single-center analysis of AA and C patients with DLBCL at UAB. UAB is a primary care facility and the main tertiary referral center in Alabama. It is located in Jefferson County where the average African American population of 39% is nearly triple the national average. Methods: After IRB approval, patients diagnosed with DLBCL from 1995 to 2007 were identified from pathology, referral, and UAB tumor registry databases. Baseline demographic data including race, age at diagnosis, stage, treatment administered, response to treatment, and survival were extracted. Serum LDH levels and performance status were not consistently available in medical records and therefore not included in the final analysis. Patients with monomorphic post-transplant lymphoproliferative disease and primary CNS lymphoma were excluded. Associations between race and stage, treatment (first-line rituximab, anthracycline, or combination therapy), response, and outcome were analyzed using Chi-squared or Cochran Mantel-Haenszel statistical analysis. Results: A total of n=309 (n=32 AA and n=277 C) patients were identified. AA patients were diagnosed at a significantly younger age than C patients [median age 49 (range: 22–90) vs. 61 (range: 16–91), respectively; p=0.0131]. AA patients also presented with advanced stage disease (Ann Arbor stage III–IV) more frequently (69% vs. 52%, p=0.046). No difference in extranodal disease presentation or in complete response rate to first-line therapy was noted. As expected, those with early stage disease, age

Description: In this report the role played by human immunodeficiency virus type-1 (HIV-1) in the pathogenesis of HIV-1-related thrombocytopenia was investigated. CD34+ hematopoietic stem/progenitor cells were purified from the bone marrow (BM) of HIV-1(+) thrombocytopenic patients, HIV- 1(+) nonthrombocytopenic individuals, HIV-1(-) patients with immune thrombocytopenic purpura, and HIV-1(-) normal donors. CD34+ cells from HIV-1(+) thrombocytopenic individuals alone showed a reduced capacity to give rise to megakaryocytic colonies (CFU-Meg) and also a progressive and significant decline in cell number when placed in liquid culture containing recombinant human interleukin-3 (rIL-3). This decline involved not only megakaryocyte but also erythroid and granulocyte/macrophage progenitors. The defects in megakaryocyte colony formation and CD34+ cell growth did not result from a productive HIV-1 infection of CD34+ cells. Moreover, HIV-1 DNA was absent from CD34+ cells in 10 of 12 thrombocytopenic patients examined. On the other hand, the decreased survival/proliferation of CD34+ cells in liquid culture, within the HIV-1(+) thrombocytopenic patients, was correlated with the presence of HIV-1 p24 antigen in BM plasma. These results demonstrate an impairment of CD34+ cells in HIV-1(+) individuals presenting thrombocytopenia as the only hematologic manifestation. Furthermore, these findings suggest that increased viral replication in the BM microenvironment may cause this impairment and possibly contributes to HIV-induced thrombocytopenia.

Description: Background: Viral infections are the leading cause of non-relapse mortality after unrelated umbilical cord blood transplants (UCBT). Post-transplant lymphopenia, lack of established antiviral memory, and lack of cytotoxic function among infused UCB lymphocytes are jointly responsible for ineffective immunity. We have previously demonstrated ex vivo T cell expansion and maturation starting from a very small fraction (10%) has hindered T cell expansion. We hypothesized that the presence of cytokines essential for T cell homeostasis, in particular IL-7 may aid expansion and survival, beads with different densities of CD3/CD28 antibodies may impact UCB T cell expansion. Research Design: From frozen/thawed specimens we tested 2 different sources of CD3/CD28 artificial beads (historical control beads vs ClinExVivo®, (Invitrogen Corporation), ± IL-7. Methods: Thawed UCB samples were centrifuged over Ficoll gradient. T cells were positively selected with EasySep®, (StemCell Technologies) and were incubated in gas permeable bags with CD3/CD28 beads in 5% serum replete media + 100u/ml IL-2 ± 10ng/ml of IL7. Media & cytokines were replenished to maintain a concentration of ∼0.75 ×106 cells/ ml. Automated cell count, trypan blue viability assessed overall cell growth and viability at each feeding . ‘Lyse no wash’ FACS staining with Trucount® beads quantified viable CD3+ T cells. 4-color FACS was employed to characterize the evolution of surface and intracellular immunophenotype. Cytotoxicity profile of the day 0 and D12-14 progeny was tested by Europium release assay (Delfia® assay) against a Burkitt’s lymphoma cell line (IM9). Two-tailed paired Student t-tests analyzed the impact of the experimental variables. Results: At the end of 12–14 day expansion periods, analyzing all cytokine conditions, we could demonstrate an average of 43-fold viable CD3+ T cell expansion using control APC beads (n=8), while T cells on ClinExVivo® beads expanded on average 94-fold (p=0.11, n=11). Addition of IL7 to the culture media afforded significantly better expansion with the control bead condition (mean 43 vs 26 fold, p=0.02). IL7 also enhanced T cell expansion with ClinExVivo® beads (mean 147 fold vs 49 fold, though statistically NS). There were significantly more viable T cells generated with ClinExVivo® beads when all timepoints were analyzed, irrespective of the absence (p=0.017) or presence (p=0.05) of IL7. We also analyzed the mechanism how IL7 may potentiate overall T cell expansion. T cells entering cell cycle was enhanced (though not significantly) as demonstrated with intracellular Ki-67 staining (65% T cells in cycle with IL7 versus 52% without, p=0.2). IL7’s overall salutary effect on total expansion may be best explained by simultaneously reduced T cell apoptosis as quantified by activated ic Caspase-3 detection (9.3% versus 7.6% without vs with IL7, respectively, p=0.08) regardless of the source of beads. Granzyme A, B, perforin expression was enhanced comparably, while cytotoxicity was absent against IM9 cells regardless of ± IL-7. Conclusions: These results demonstrate significant expansion of UCB T cell that is further improved by utilizing ClinExVivo® clinical grade APC beads and IL7. These results pave the way to donor-derived UCB T cell expansion suitable for DLI to boost immunity.

Description: Camptothecin (CPT), a natural alkaloid isolated from Camptotheca acuminata, has potent broad spectrum antitumor activity by inhibiting type I DNA topoisomerase. It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with refractory/relapsed lymphoma, but with only modest activity. For intravenous administration, we have developed IT-101, a ca. 40 nm diameter nanoparticle that is an assembly of cyclodextrin-based polymer conjugates of 20-(S)-CPT. The purpose of this study is to compare the preclinical efficacy of IT-101 with CPT-11 both in vitro and in vivo. Fluorescence microscopy studies demonstrated that incubation of a human lymphoma cell line with IT-101 resulted in the uptake and intracellular accumulation of CPT. Based on the demonstration of in vitro cytotoxity of IT-101 against multiple human lymphoma cell lines, we initiated experiments in xenograft models of lymphoma. In subcutaneous human xenograft models, a single cycle of three weekly doses of intravenous IT-101 at 10 mg/kg (CPT equivalents) showed significantly potent antitumor activity against Daudi, Karpas 299, and L540 cell lines compared to three weekly doses of intraperitoneal CPT-11 at its maximum tolerated dose in mouse of 100 mg/kg (P 〈 0.0001, P = 0.0072, and P 〈 0.0001, respectively). In the same animal models, IT-101 led to pathologically complete remissions in 78%, 44%, and 78% of animals inoculated with Daudi, Karpas 299, and L540 cell lines, respectively. In disseminated human xenograft models, IT-101, administered using the same dosing schedule, significantly prolonged the survival of animals intravenously injected with either Daudi or Karpas 299 cell lines when compared to CPT-11 (P 〈 0.0001 and P = 0.0049, respectively). The promising present results in a variety of lymphomas provide the basis for a phase I/II clinical trial in patients with refractory/relapsed lymphoma.

Description: Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin lymphoma. MCL is characterized by the chromosomal translocation t(11;14) leading to overexpression of cyclin D1 (CCND1), a regulator of the cell cycle. NF-κB plays a central role in key cellular processes, including cell cycle regulation and apoptosis, and it has been shown to be constitutively activated in MCL cell lines and in patient biopsy samples and to play a key role in the growth and survival of MCL cells. In an exploratory study, we evaluated the hypothesis that germline variation in candidate genes from the cell cycle and NF-κB pathways predict overall survival in MCL. Methods: We genotyped 235 SNPs from 29 cell cycle genes and 447 SNPs from 55 NF-κB genes in 39 MCL patients aged 38–64 years who participated in a population-based study conducted from 1998–2000 through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles county. Tagging single nucleotide polymorphisms (SNPs) were selected from HapMap. Stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site. To assess the statistical significance of a gene, we first used principal components analysis to capture the genetic variation of the SNPs within a gene. Cox proportional hazards analysis was then used to assess the association between the top principal components from a gene with overall survival. Results: Through early 2005, there were 22 deaths in 39 patients (56%). The median follow-up of the 17 surviving patients was 56 months (range 36–70). Eleven of the 55 genes (TNFSF14 p=0.0007, NF-κBIA p=0.003, IL1R2 p=0.01, TNFRSF25 p=0.02, TNFSF13B p=0.02, FAS p=0.03, TLR2 p=0.04, TNFSF10D p=0.04, TNFSF10 p=0.06, MAP3K2 p=0.06) in the NF-κB pathway were associated with overall survival. In the cell cycle pathway, only 3 of the 29 genes (CASP5 p=0.06, CASP9 p=0.09, BCL2 p=0.09) were associated with overall survival, and a previously reported SNP in CCND1 (rs603965) showed no association (p=0.5). Permutation p-values for observing at least this number of associated tests in each pathway due to chance (from multiple testing) was p=0.8 for the cell cycle pathway and p=0.03 for NF-κB pathway. The small sample size precluded multi-gene modeling. Conclusion: In a population series of MCL patients, we found suggestive evidence that host genetic variation in candidate NF-κB pathway genes affects overall survival. These results complement tumor expression data implicating this pathway in the pathogenesis of MCL. In addition, such findings further support the value of developing therapeutic targets from this pathway.

Description: The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocation partner for AF10 in morphologically distinct subsets of acute leukemia with a recurring 10;11 translocation. As the name implies, CALM is involved in the regulation of clathrin-mediated endocytosis. We hypothesize that expression of the CALM-AF10 protein disrupts endocytosis of growth receptors, leading to constitutive activation and, thereby, contributes to the development of leukemia. The CALM-AF10 fusion transcripts are characterized by two breakpoints in CALM at positions 1926 and 2091. Using the 293T human renal epithelial cell line as a model, we demonstrated that CALM1926-AF10 localized diffusely to the cytoplasm, whereas CALM2091-AF10 had a more prominent punctate appearance. This difference in distribution was likely attributable to protein domains in CALM rather than AF10, which is a transcription factor that localizes to the nucleus. The difference in localization extended to their effects on endocytosis. Whereas ∼4–8% of cells expressing truncated CALM2091, CALM1926, or AF10 displayed impaired transferrin uptake, this value was ∼60% in cells expressing CALM2091-AF10 and only ∼13% in cells expressing CALM1926-AF10. Next, we used a retroviral transduction-transplantation murine model to test the impact of CALM-AF10 expression on the development of leukemia. Mice transplanted with either CALM1926-AF10 or CALM2091-AF10 transduced fetal liver cells developed disease within a short period of time (∼8 to 21 weeks post-transplantation). Interestingly, expression of CALM1926-AF10 led to the development of a myeloproliferative disease (MPD)-like leukemia, with a predominance of mature neutrophils, whereas expression of CALM2091-AF10 led to the development of an acute myeloid leukemia (AML) with 〉30% immature myeloid blasts, with one exception. One mouse transplanted with progenitor cells expressing CALM2091-AF10 developed a MPD accompanied by a granulocytic sarcoma; however, examination of RNA from the spleen of this diseased mouse revealed that it had undergone a splicing event consistent with the size expected for the 1926 breakpoint. Both diseases were transplantable to secondary recipients confirming the leukemic nature of the CALM-AF10 expressing cells. Previous studies show that patients with the t(10;11) (p13;q14-21) develop either AML or T cell-acute lymphoblastic leukemia (T-ALL). Most of the patients with T-ALL express the CALM1926-AF10 fusion. Although there are differences in presentation between humans and mice, the hematological disease may be a reflection of the conditions of the mouse model. To identify possible secondary cooperating mutations, spectral karyotyping analysis of the mouse AMLs and MPDs was performed. However, to date we have not identified any significant abnormalities. We hypothesize that defective endocytosis synergizes with transcriptional deregulation, leading to an aggressive form of leukemia.

Description: Background. Intracellular one-carbon transfer reactions are essential for nucleotide synthesis and methylation of biologic compounds including DNA. Previous studies have linked genetic variants in one-carbon metabolism genes with risk of developing NHL, but little is known regarding the impact of these variants on disease outcome. We evaluated the hypothesis that inherited genetic variation in genes involved in one-carbon metabolism is associated with overall survival in DLBCL. Methods. We genotyped 30 single nucleotide polymorphisms (SNPs) from 18 candidate one-carbon metabolism genes in 215 DLBCL patients who participated in a population-based case-control study conducted from 1998–2000 using the SEER (Surveillance, Epidemiology and End Results) cancer registries in the Detroit, Iowa, Los Angeles and Seattle. Stage, B-symptoms, first course of therapy, date of last follow-up and vital status through early 2005 were obtained from cancer registry files. Cox proportional hazards analysis was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals for the association between individual SNPs and overall survival, adjusting for age, demographic and clinical factors. We also used parallel modeling strategies to identify the best summary multi-SNP risk score to predict survival. Results. The median age at diagnosis was 57 years (range, 20–74), and 50 (23%) of the patients died during follow-up, with a median follow-up of 57 months (range, 31–78 months) for surviving patients. After adjusting for demographic and clinical variables, SNPs in SHMT1 (rs1979276; HRCT/TT=2.47, 1.31–4.67), BHMT (rs585800; HRAT/TT=2.02, 1.16–3.54), and TCN1 (rs526934; HRTT=1.86, 1.04–3.33) were the strongest and most robust predictors of survival. A summary score of the number of deleterious genotypes (0–3) from these three genes was strongly associated with survival (p=7.9 x 10−5) after accounting for demographic and clinical variables (HR=2.58 per deleterious genotype, 95% CI 1.75–3.80). A risk score combining the three SNPs with clinical and demographic variables (score of 0 to 5) was even more strongly associated with survival (p=1.4 x 10−13); the Kaplan Meier survival curves are shown in the Figure. In a time-dependent ROC analysis, the combined risk score had a concordance index of 0.75 at 5 years of follow-up (95% CI 0.69–0.81). Conclusion: Host genetic variation in the one-carbon metabolism genes SHMT1, BHMT, and TCN1, individually and particularly in combination, was associated with overall DLBCL survival after accounting for clinical and demographic factors, supporting a role for this pathway in disease progression. Future work should evaluate interactions of genes from this pathway with dietary nutrients and therapeutic agents in DLBCL prognosis. Figure Figure

Description: Background: Follicular lymphoma, derived from germinal center B cells, is one of the most common types of lymphoma. Follicular lymphoma like many other malignancies demonstrates genomic instability, a phenomenon where tumor cells accumulate various genetic changes including point mutations, genomic deletions and gene amplifications. Such genomic aberrations may contribute to cancer phenotype such as transformation. Previous studies of follicular lymphoma have shown a combination of cytogenetic - aneuploidy type events from gross chromosomal alterations to smaller deletions and amplifications. For example, deletions in chromosome 6q may be associated with follicular lymphoma aggressiveness. Efforts to identify these changes have used traditional karyotyping, comparative genomic hybridization (CGH) or traditional genetic markers (microsatellites) for loss of heterozygosity (LOH) analysis. Characterization of of genomic instability in follicular lymphoma has not been thoroughly investigated on a genomic scale. Objectives: We have employed a novel genomic technology relying on molecular inversion probes (MIPs) to conduct a large scale analysis of gene copy aberrations in follicular lymphoma. MIPs technology enables querying any gene sequence in the genome at a high resolution for allele-specific gene copy alterations and polymorphisms simultaneously, a significant improvement over other genomic methods such as array CGH. We are using this genomic technology to delineate concordant gene copy aberrations in follicular lymphoma that may influence a variety of clinical outcomes using a probe set with over 7,000 cancer-related genes among 25,000 loci. Methods: DNA was extracted from a cohort of follicular lymphoma clinical samples obtained at the time of diagnosis and prior to treatment. B-cell lymphoma cells were purified using a Rosette purification scheme, genomic DNA was isolated from the purified tumor cell populations from 37 individual lymphoma samples and the cohort was analyzed with a MIP cancer probe set described above. Each probe contains two unique recognition sequences to a genomic DNA target and a unique barcode sequence. The molecular inversion probe assay was conducted. The final products were hybridized to a generic barcode microarray overnight and interrogated by an Affymetrix scanner. Barcode array data was preprocessed and gene copy number was extrapolated for each molecular inversion probe. Concordant gene copy aberrations were determined across the sample set. Results: Follicular lymphoma samples from pretreated patients have common patterns of chromosomal losses, gains and multiple gene deletions with large overlap in some areas of deletion. Common large scale chromosomal deletions and amplifications included regions of chromosome 6 and 18. Multiple individual genes were found to be commonly amplified or deleted as well including BTK which is suspected to play a role in follicular lymphoma. Conclusions: MIP technology has been used to analyze follicular lymphoma for highly sensitive and specific gene copy analysis. We identified concordant gene copy alterations found across the majority of samples in the cohort, suggesting that these aberrations may play a role in follicular lymphoma development. We are pursuing additional studies to confirm these findings.