Myeloid Leukemia Tumors Research Articles

Abstract 5441: A rapid homogeneous immunoassay for the therapeutic drug management of imatinib in plasma using a novel selective monoclonal antibody

Abstract Objective: Imatinib is a potent and selective inhibitor of the Bcr-Abl and KIT protein tyrosine kinases. It is widely used for treatment of Philadelphia chromosome positive chronic myeloid leukemia (Ph+ CML) and KIT-positive gastrointestinal stromal tumor (GIST). Studies have shown a significant inter-patient variability in imatinib trough concentrations up to 16 fold and a correlation between systemic exposure of the drug and clinical outcome. The current physical methods for measuring imatinib require specially trained personnel, larger sample sizes, are expensive, time consuming and not amenable for widespread use. The objective of this study is to develop an automated immunoassay that provides a rapid, simple and inexpensive method to routinely measure exposure of imatinib in patients undergoing chemotherapy. Methods: A library containing five novel and highly selective antibodies to imatinib were covalently linked to carboxyl-modified nanoparticles to develop a prototype automated homogenous assay for use on the Beckman Coulter AU®400 clinical analyzer which uses a two reagent system and 2 uL of plasma to quantitate imatinib. The degree of aggregation of nanoparticles, inversely proportional to the concentration of imatinib in a sample, was monitored at 600 nm. Total imprecision and linearity of the assay were evaluated by testing normal human plasma (NHP) pools spiked with different levels of imatinib. The background of the assay was also determined by measuring >100 NHP blank samples. Immunoassay selectivity was characterized by testing metabolites including N-desmethylimatinib and other structure-related compounds of imatinib for cross-reactivity at relevant physiological concentrations. Results: With a quantitative range from 100 to 4,000 ng/mL and instrument auto-dilution, plasma samples with imatinib concentrations up to 10,000 ng/mL could be determined using this assay. Time-to-first result was 11 minutes and 400 samples per hour could be measured using a stored calibration curve. The total imprecision of NHP pools spiked with imatinib at four concentrations across the assay range was <10% CV and the assay was linear from 100 to 4,000 ng/mL. The background of the assay was <50 ng/mL. The assay cross-reactivities to N-desmethylimatinib and other cross-reactants were determined to have insignificant impact on the quantitation of imatinib. Conclusion: This immunoassay is suitable for determining imatinib concentrations in plasma with the advantages of speed, small sample size, no sample pretreatment and application on automated instrumentation, providing a tool for optimization of imatinib plasma trough levels with pharmacokinetic-guided dosing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5441. doi:10.1158/1538-7445.AM2011-5441

Increased prevalence of chronic fatigue among survivors of childhood cancers: A population‐based study

Fatigue is prevalent in adult cancer survivors but less studied in childhood cancer survivors. Aims were to assess fatigue levels, prevalence of chronic fatigue (CF) and the association of CF with health-related quality of life (HRQoL) in survivors of acute myeloid leukemia (AML), infratentorial astrocytoma (IA), and Wilms tumor (WT) in childhood. Seventy percent (398/567) of Nordic patients treated for AML, IA, and WT between 1985 and 2001 at age >1 year responded to a postal survey, encompassing the Fatigue Questionnaire and the Short Form 36 (SF-36). Participants were divided into two groups at time of study; younger (YG, 13-18 years) and older (OG, 19-34 years). Respondents (19-34 years, n = 763) from a Norwegian general population (GP) survey served as controls for the OG. The OG [mean age was 24 years (SD 3.3)] had higher fatigue levels compared to the YG and the GP, especially the females (P < 0.05). There was also a higher prevalence of CF in the OG than in the GP (14 vs. 6%, P < 0.001). Regardless of diagnosis, the OG with CF had poorer physical health (P < 0.05) on the SF-36 but better mental health (P < 0.05 and P = 0.001) relative to controls with CF. The prevalence of CF is higher among Nordic survivors of AML, IA, and WT than GP controls of similar age. CF is associated with impaired HRQoL in survivors. However, they reported better mental health than CF GP controls. This might indicate different underlying mechanisms of CF in the two populations.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Posterior reversible encephalopathy syndrome in childhood cancer

BackgroundPosterior reversible encephalopathy syndrome (PRES) is characterized by seizures, headaches, altered mental status, cortical blindness and typical transient lesions on magnetic resonance imaging. Patients and methodsWe describe seven childhood cancer patients with clinical and radiological symptoms of PRES, and reviewed all well-documented PRES cases reported during childhood cancer treatment. ResultsFifty-six children with PRES, including our 7 cases, were identified in the literature. Mean age at onset was 9 (range: 2–17) years. Primary diagnoses were acute lymphoblastic leukemia (n = 31), acute myeloid leukemia (n = 5), non-Hodgkin lymphoma (n = 7) and solid tumors (n = 13). PRES patients presented with seizures (n = 50), altered mental status (n = 20), visual disturbances (n = 24) and/or headaches (n = 17). PRES was associated with hypertension in 49 patients. About 86% of the patients had both clinical and radiological reversible symptoms. Four patients developed epilepsy, in one patient ataxia remained and one patient had a persistent mydriasis. ConclusionAlthough PRES has predominantly been described in leukemia patients, it occurs in children with solid tumors as well. Hypertension seems to be the most important trigger for the occurrence of PRES during childhood cancer treatment. Seizures are the most common accompanying sign. Symptoms and radiological findings normalize in ∼90% of the cases, but in 10% neurological symptoms remain.

Drug Transporters and Imatinib Treatment: Implications for Clinical Practice

Imatinib mesylate is approved for the treatment of chronic myeloid leukemia (CML) and advanced gastrointestinal stromal tumors (GIST). Unfortunately, in the course of treatment, disease progression occurs in the majority of patients with GIST. Lowered plasma trough levels of imatinib over time potentially cause disease progression, a phenomenon known as "acquired pharmacokinetic drug resistance." This outcome may be the result of an altered expression pattern or activity of drug transporters. To date, the role of both efflux transporters (ATP-binding cassette transporters, such as ABCB1 and ABCG2) and uptake transporters [solute carriers such as organic cation transporter 1 (OCT1) and organic anion transporting polypeptide 1A2 (OATP1A2)] in imatinib pharmacokinetics and pharmacodynamics has been studied. In vitro experiments show a significant role of ABCB1 and ABCG2 in cellular uptake and retention of imatinib, although pharmacokinetic and pharmacogenetic data are still scarce and contradictory. ABCB1 and ABCC1 expression was shown in GIST, whereas ABCB1, ABCG2, and OCT1 were found in mononuclear cells in CML patients. Several studies have reported a clinical relevance of tumor expression or activity of OCT1 in CML patients. Further (clinical) studies are required to quantify drug transporter expression over time in organs involved in imatinib metabolism, as well as in tumor tissue. In addition, more pharmacogenetic studies will be needed to validate associations.

V559A and N822I double KIT mutant melanoma with predictable response to imatinib?

Dear Editor, Since the initial report of KIT mutations in subtypes of melanoma, several reports of KIT-targeted therapies have been published (Carvajal et al., 2009; Hodi et al., 2008; Lutzky et al., 2008). Activating KIT mutations have been documented in a variety of neoplastic diseases including, in addition to melanoma, mastocystosis, acute myeloid leukemia, seminoma and gastrointestinal stromal tumors (Went et al., 2004). Herein, we report a case of a KIT double mutation in anorectal melanoma not previously described in melanoma, but with a response to imatinib mesylate that might have been expected given the knowledge from other cancers. The patient is an 87-year-old woman who developed constipation and a palpable rectal mass in the summer of 2009. Biopsy of the mass demonstrated an ulcerated melanoma invading the submucosa and muscle to a depth of approximately 11 mm with angiolymphatic invasion. Computerized tomography (CT) scans of the chest, abdomen and pelvis revealed metastatic disease in the pelvis, inguinal region and lungs. Substantial growth of the disease was documented on serial CT scans 3 months apart with the largest lung nodule increasing 44% (14 to 25 mm). The patient declined treatment with systemic chemotherapy and was not a candidate for the available clinical trials but she did express an interest in some form of therapy. Her melanoma was assessed for KIT mutational status. A double mutation was identified, comprising the exon 11 mutation V559A and the exon 17 mutation N822I. The patient subsequently initiated treatment with imatinib 400 mg daily in November 2009. Surveillance CT scans in February 2010 revealed that most metastatic lesions were stable to minimally decreased, including a right inguinal lymph node (16 to 14 mm), a right upper lobe lung nodule (20 to 18 mm), a right lower lobe lung nodule (25 to 23 mm) and a right ischioanal lesion (19 to 15 mm). There was complete disappearance of multiple subcentimetre lung nodules; however, there was also some growth of other lung nodules, the largest of which increased from 25 to 28 mm. The findings were consistent with a mixed response, although in accordance with RECIST criteria, the patient’s response would best be classified as stable disease ( 7% decrease in sum of diameters of target lesions). No repeat biopsy was attempted. The dose of imatinib was titrated from 400 to 800 mg with the aim of overcoming any possible resistance to therapy and improving the patient’s response. Unfortunately, the patient developed significant fatigue, dyspnea on exertion and lower extremity edema. Treatment was held, and after recovery from these acute symptoms, attempts were made to reinitiate imatinib at the lower dose of 400 mg. The patient was not able to tolerate resumption of therapy, and she experienced further clinical decline and progression of disease; ultimately, she succumbed to her metastatic melanoma in August, 2010. Efforts to define the genetic landscape of melanomas have led to the recognition that alterations in the KIT gene constitute the predominant mutation in melanomas arising from chronic sun-damaged skin and acral and mucosal anatomical locations (Curtin et al., 2005, 2006). KIT mutations in melanoma are now only beginning to be catalogued. The experience with KIT mutations in GISTs, however, is much more extensive, and this experience provides a conceptual framework for understanding the pathobiology of and therapeutic approaches to treating neoplastic diseases driven by KIT mutations (Corless and Heinrich, 2008). KIT mutations underlie approximately 85% of GISTs; and the vast majority ( 70%) occurs within exon 11 corresponding to the autoinhibitory juxtamembrane domain. KIT exon 9 lies within the extracellular ligand-binding region, and mutations within this exon account for 10 to 15% of KIT mutations in GIST. KIT mutations within the kinase domain (either exon 13 or exon 17) occur with much less frequency (<5%). To date, molecular analyses have identified approximately two dozen unique KIT mutations in melanoma (Woodman and Davies, 2010). Similar to GIST, the majority of KIT mutations in melanoma occur within the juxtamembrane autoinhibitory region encoded by exon 11. Unique to melanoma, though, the

Fanconi anemia manifesting as a squamous cell carcinoma of the mandible: a case report

Progressive bone marrow failure and development of malignancies, particularly acute myeloid leukemia and solid tumors the most important features of Fan- coni’s Anemia (FA). This paper reports the case of a 16-year-old patient with FA who developped squa- mous cell carcinoma of the mandible, ten years after the bone marrow transplantation (BMT).

Imatinib-Mesylate Induced Interstitial Pneumonitis in Two CML Patients

Imatinib mesylate, a selective inhibitor of BCR-ABL kinase activity, has demonstrated significant clinical efficacy in the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GISTs). It has become the standard of treatment for these diseases. Although the toxicity profile of imatinib is superior to that of interferon or other cytotoxic agents, some adverse events including edema, gastrointestinal toxicities and hematologic toxicities are commonly observed in the patients treated by imatinib. We present two cases of imatinib induced interstitial pneumonitis during the treatment of a chronic phase of CML.

The role of natural killer cells in chronic myeloid leukemia

Chronic myeloid leukemia is a neoplasia resulting from a translocation between chromosomes 9 and 22 producing the BCR-ABL hybrid known as the Philadelphia chromosome (Ph). In chronic myeloid leukemia a proliferation of malignant myeloid cells occurs in the bone marrow due to excessive tyrosine kinase activity. In order to maintain homeostasis, natural killer cells, by means of receptors, identify the major histocompatibility complex on the surface of tumor cells and subsequently induce apoptosis. The NKG2D receptor in the natural killer cells recognizes the transmembrane proteins related to major histocompatibility complex class I chain-related genes A and B (MICA and MICB), and it is by the interaction between NKG2D and MICA that natural killer cells exert cytotoxic activity against chronic myeloid leukemia tumor cells. However, in the case of chronic exposure of the NKG2D receptor, the MICA ligand releases soluble proteins called sMICA from the tumor cell surface, which negatively modulate NKG2D and enable the tumor cells to avoid lysis mediated by the natural killer cells. Blocking the formation of sMICA may be an important antitumor strategy. Treatment using tyrosine kinase inhibitors induces modulation of NKG2DL expression, which could favor the activity of the natural killer cells. However this mechanism has not been fully described in chronic myeloid leukemia. In the present study, we analyze the role of natural killer cells to reduce proliferation and in the cellular death of tumor cells in chronic myeloid leukemia.

Selective inhibition of PDGFR by imatinib elicits the sustained activation of ERK and downstream receptor signaling in malignant glioma cells

Clinical studies using the tyrosine kinase inhibitor, imatinib mesylate (Gleevec®), in glioblastoma, have shown no major inhibition of tumor growth or extension of survival for patients, unlike those in chronic myeloid leukemia (CML) and gastrointestinal stromal tumors. The molecular mechanisms of action of imatinib in glioblastoma cells are still not well understood. In this study, we investigated the effects of imatinib on the platelet derived growth factor receptor (PDGFR) downstream signaling pathways as well as on other cellular functions in human glioblastoma cells. NIH3T3 fibroblast and K562 CML cells were used for comparison. Western blot analysis demonstrated that imatinib was more effective in inhibiting the activated rather than the quiescent forms of the target proteins. Furthermore, the imatinib treatment induced the sustained activation of extracellular signal-regulated kinase (ERK 1/2) signaling as well as components of other downstream signaling pathways, such as PI3K/Akt, STAT3 and p38MAPK. Prior stimulation of the malignant cells with exogenous PDGF-BB partially abrogated this activation. Further analysis indicated that the activation of ERK induced by the imatinib treatment was related to the S-phase re-entry of the cell cycle in one of the three glioma cells. Imatinib significantly inhibited cell migration but not cell growth. The combination treatment of imatinib with a MEK or PI3K inhibitor resulted in significant growth inhibition but did not inhibit cell migration beyond the inhibition achieved with the imatinib treatment alone. The treatment of glioma cells with small interfering RNA inhibiting PDGFRB, however, evoked enhanced Akt signaling. These results indicate that the imatinib treatment of malignant glioma does not result in significant inhibitory effects and should be used with caution.

Imatinib mesylate enhances the malignant behavior of human breast carcinoma cells

Imatinib mesylate (Imatinib), clinically employed for chronic myeloid leukemia and gastrointestinal stromal tumors, is a selective inhibitor of the tyrosine kinases, c-abl, c-kit and PDGFRs. Due to the frequent expression of these genes in breast cancer cells, the clinical efficacy of Imatinib has recently been investigated in patients with advanced and metastatic breast cancer. Here, we have studied the effects of Imatinib on human MA-11 breast carcinoma cells, expressing both c-abl and PDGFRbeta, in vitro and in mouse xenografts. The effects of Imatinib mesylate on the human MA-11 breast carcinoma cell line were studied in vitro and in xenografts. Daily intraperitoneal treatment with 60 mg/kg Imatinib for 9 days of athymic nude mice pre-implanted subcutaneously with MA-11 cells did not result in an anti-tumor effect, but rather increased the take rate of 3 × 10(4) cells from 30.8 to 84.6% and caused the appearance of large abdominal masses in 30% of mice. To investigate the mechanism(s) of the observed effects of Imatinib on MA-11 tumors, we exposed the cells in vitro to Imatinib for 9 days. The surviving population, expanded in culture, showed increased motility and over-expressed a set of genes associated with aggressive behavior. Also, several genes belonging to the Wnt and the MAPK pathway were differentially expressed. In promoter activation assays, Imatinib increased the promoter activity driven by both Wnt and MAPK/ERK-1/2. Our data suggest caution in the clinical use of Imatinib in breast cancer patients; the comparison of Imatinib-surviving breast cancer cells with parental cells may help define the regulatory pathways involved in the increased malignancy of residual tumor cells that survive therapy, ultimately providing important therapeutic targets.

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.

Synchronous development of KIT positive acute myeloid leukemia in a patient with gastrointestinal stromal tumor

We report a case of synchronous occurrence of KIT-positive acute myeloid leukemia (AML) and gastrointestinal stromal tumor (GIST). A 63-year-old woman was hospitalized for dizziness, and abdominal computed tomography revealed an exophytic gastric mass and hepatic metastasis. The patient was diagnosed with GIST and was administered imatinib (400 mg/day) for the metastatic unresectable tumor. After 2 weeks of imatinib treatment, the patient developed pancytopenia, which persisted even after the drug was discontinued, thereby necessitating bone marrow biopsy. Biopsy examination indicated AML, and karyotyping revealed a complex karyotype. We did not observe point mutations at residues D816 and N822 of KIT. Therefore, the patient received standard induction chemotherapy, but on the 18th day after completion of chemotherapy, she died of septic shock and multi-organ failure. Since KIT plays an important role in both GIST and AML, we consider that both these malignancies may have been associated with each other.

Pulse-modulated second harmonic imaging microscope quantitatively demonstrates marked increase of collagen in tumor after chemotherapy

Pulse-modulated second harmonic imaging microscopes (PM-SHIMs) exhibit improved signal-to-noise ratio (SNR) over conventional SHIMs on sensitive imaging and quantification of weak collagen signals inside tissues. We quantify the spatial distribution of sparse collagen inside a xenograft model of human acute myeloid leukemia (AML) tumor specimens treated with a new drug against receptor tyrosine kinase (ABT-869), and observe a significant increase in collagen area percentage, collagen fiber length, fiber width, and fiber number after chemotherapy. This finding reveals new insights into tumor responses to chemotherapy and suggests caution in developing new drugs and therapeutic regimens against cancers.

Clonally Related B-NHL Populations and Cell Lines Harbor Subsets with Significant Phenotypic and Functional Heterogeneity.

Abstract 1699Poster Board I-725Many malignancies such as myeloid leukemia, breast, colon, and CNS tumors appear to harbor phenotypically and functionally distinct populations of cells with self-renewal, differentiative, and tumorigenic capacity. These unique populations, influencing tumor growth and persistence, have been termed “cancer stem cells,” or “tumor-initiating cells.” No such tumor-initiating capacity has been prospectively identified in non-Hodgkin lymphoma (NHL). Identification of these novel sub-populations in B-NHL is particularly challenging given the unique proliferative capacity, differentiation potential, and population diversity of the peripheral B cell subsets from which lymphomas arise. Our studies have focused on the identification of sub-populations that meet criteria for “lymphoma stem cells” (LySC) in B-NHL samples. Two experimental sources of NHL cells were used: i) primary human DLBCL (TXN190, TXN131, C10, C12) derived at our institution from malignant effusions, and, ii) continuously cultured, widely used DLBCL lines (OCI-Ly-7, -10, -19).Using multiparameter FACS, we initially performed immunophenotypic studies to investigate a broad range of cell surface markers. Notably, we observed significant heterogeneity in the surface antigen density for CD22, CD27, CD45R/B220, and CXCR5. These data suggest that subpopulations residing within NHL populations may possess functional heterogeneity. To test this concept, we first performed colony-forming unit (CFU) assays in methycellulose medium. Analysis of 7 specimens shows a CFU frequency ranging from .0003% to 10% of the overall population, demonstrating functional heterogeneity with regard to in vitro colony-forming ability. Surprisingly, we found that generation and successful re-plating of B-NHL colonies did not require the addition of exogenous factors such as PHA-conditioned medium, cytokine, or interleukin supplementation. Next, we performed flow cytometric separation of candidate subpopulations as a means of segregating functional potential. These studies showed that CD45R/B220 expression levels appear to correlate with colony-forming ability. B-NHL cell fractions with lower levels of expression of the murine pan-B cell marker CD45R/B220 appear to be enriched for CFU activity in methylcellulose culture compared to B220-high cells. In addition, CD45R/B220-low B-NHL has higher tumorigenic potential (assessed by kinetics of growth rate) in assays of subcutaneous tumor nodule generation. These studies indicate differential potential of NHL subpopulations, and may begin to establish a phenotype of LySC. Finally, we measured in vitro clonogenic potential using two assays – i) limiting dilution suspension culture, and - ii) CFU assays. Interestingly, the results from these two independent assays do not correlate well in B-NHL; clonogenic potential appears to be substantially higher in limiting-dilution suspension cultures when compared to methylcellulose assays. These findings indicate that the relationship between CFU frequency and growth potential in B-NHL populations may differ from those found in normal hematopoietic cell types, and suggest that some caution should be exercised in the interpretation of B-NHL CFU assays.The adaptation of assays useful for identification of tumor-initiating sub-populations has also been useful in defining subsets of clonally related B-NHL cells that appear immunophenotypically and functionally distinct in vitro and in vivo. Our studies support the notion that B-NHL tumor-initiating cells may comprise a unique population amenable to prospective immunophenotypic and/or functional identification. Implicit in the concept of these putative LySC is that the control, and ultimately the eradication, of such unique NHL populations may result in improved clinical outcomes.[SAW and RMR contributed equally to this work. Funded by the Lymphoma SPORE, P50 CA130805501A1, PI: Richard Fisher, MD.] DisclosuresNo relevant conflicts of interest to declare.

Diagnosis of Fanconi Anemia in An Asymptomatic Adult with Mosaicism and a Molecular Explanation.

Abstract 4213 INTRODUCTIONFanconi Anemia (FA) is a primarily autosomal recessive disorder with a defective DNA repair pathway associated with mutations in any of 13 genes. The majority of patients reported in the literature have one or multiple congenital anomalies, including low birth weight, short stature, café au lait spots, abnormal radii and/or thumbs, structural renal abnormalities, microcephaly, and deafness, among others. About 25% of reported patients had few or none of these findings. The hazard of severe aplastic anemia peaks at 10 years of age, and patients have very high risks of acute myeloid leukemia and specific solid tumors, such as head and neck and gynecologic squamous cell carcinomas. Only six patients have been reported who were diagnosed between 40 and 50 years of age. However, patients may present as adults with neoplasms, or remain asymptomatic and undiagnosed. METHODSWe report the diagnosis of FA in the oldest known patient, an asymptomatic 55 year old woman (Case 2), identified only because she was a potential stem cell donor for her 42 year old sister (Case 1) with severe aplastic anemia. RESULTSCase 1 had a history of thrombocytopenia at 26, and anemia and thrombocytopenia during pregnancy; physical exam was normal except for a slightly deformed thumb; blood lymphocyte chromosome aberrations were increased with both diepoxybutane (DEB) and mitomycin C (MMC). The patient died following an HLA-matched bone marrow transplant from a non-FA brother. Case 2, the other of two siblings of Case 1 who were HLA matches, had higher than normal chromosome breakage in blood (3 cells with multiple radials with MMC) but not in the FA range; skin fibroblasts were diagnostic of FA, confirming hematopoietic somatic mosaicism. She had a normal appearance, a history of hypothyroidism and mitral valve prolapse, five pregnancies with five children (one miscarriage, one set of twins), a near normal blood count (Hb 13.3 g/dl, MCV 99 fl, WBC 3500/ul, and platelets 139,000/ul), and was a regular blood donor. Bone marrow cellularity and morphology were normal, but cytogenetics showed a small clone (46,XX,add(11)(q23)[6]/46,XX[14]). Complementation analysis of Case 1 indicated group A (FA-A), and molecular analysis identified two mutations in the FANCA gene. One mutation, p.S1208S (c.3624C>T) was a splice site mutation occurring in exon 36 and has been previously described. The second mutation was a novel nonsense mutation in exon 23, p.S674X (c.2021C>A). Five siblings had normal breakage results; four were heterozygous for the nonsense mutation and one was negative for both mutations. Case 2, with mosaicism for FA, had the familial splice mutation in both blood and fibroblasts, and the familial nonsense mutation in fibroblasts, but was skewed heavily toward wild-type in blood. cDNA studies confirmed that the predicted splice mutation created an alternate splice site resulting in multiple transcripts, including exon skipping, which varied in different tissues. The molecular mechanism for the loss of the nonsense mutation in the blood is most likely due to back mutation at a hot spot, which occurred in a hematopoietic stem cell which then had a selective growth advantage. CONCLUSIONSReversion of one FANCA mutation probably occurred in a hematopoietic stem cell which was selected for and repopulated the peripheral blood. A plausible explanation for the lack of FA clinical features is the leaky splice mutation which may provide sufficient levels of protein for normal function in DNA repair. Family members should be tested for FA by chromosome breakage analysis in blood (and/or fibroblasts to identify those who may be mosaics). Those who have FA are at risk of syndrome-specific solid tumors, as well as aplastic anemia, myelodysplastic syndrome, and leukemia, if a non-gene corrected hematopoietic stem cell were to emerge. Even if asymptomatic, they should not be used as stem cell transplant donors for siblings with FA, because they may fail to repopulate the recipient marrow. FA is undoubtedly underdiagnosed in adults at this time. Disclosures:No relevant conflicts of interest to declare.

Imatinib Mesylate Induced Cardiotoxicity: a Cardiac Progenitor Cell Disease?.

Abstract 2200Poster Board II-177Cardiotoxicity is a limiting factor of successful anthracycline-based anticancer regimens. Recently, cardiac side effects have been also reported for Tyrosine Kinase Inhibitors, among which Imatinib Mesylate (IM) has been able to change the natural history of Chronic Myeloid Leukemia (CML) and Gastrointestinal Stromal Tumors. The mechanism of IM-related cardiac dysfunction has not been fully elucidated. Since we have demonstrated the presence of resident cardiac stem cells controlling homeostasis of the human and murine heart, we advanced the hypothesis that IM-induced cardiotoxicity involves myocardial cell turnover resulting in an impairment of the adaptive response of the heart to the toxic insult. To this purpose, we studied the in vitro genotoxic effect of 5mM, 25mM and 50 mM concentrations of IM on human and rat Cardiac Progenitor Cells (CPCs). Freshly isolated human BM CML cells carrying Bcr/Abl translocation served as control. In addition, to ascertain whether IM affects cardiac structure and function, young rats were treated by three weekly i.p. injections of 50, 100 and 200 mg/kg IM for 4 weeks when hemodynamic parameters and LV remodelling were analyzed. A dose and time dependent DNA damage was observed in both CPCs and CML after in vitro exposure to IM. As early as 6 hours after 50 mM IM, DNA double strand breaks formation (dSBs), as shown by immunostaining of Histone A2X, involved 50% and 70% of CPCs and CML, respectively. At later time points more than 80% of both cells died. However, low concentrations (5mM), corresponding to therapeutic doses, exerted a permissive reparative response on CPCs that persisted with time. As a result of this divergent effect, CPCs proliferation and survival increased from 12 to 24 h in spite of the presence of IM. In vivo studies showed that, following 200 mg/kg IM, all animals died within the first 2 weeks. In the presence of moderate anemia, diastolic dysfunction was present even at 50 mg/kg IM and a dose dependent impairment in Left Ventricular End Diastolic Pressure (LVEDP) and +/- dP/dT was observed. Anatomical parameters indicated that only higher doses IM resulted in a smaller LV with a concentric type of remodelling. In the absence of diffuse myocardial fibrosis and/or high levels of apoptosis, small foci of myocardial damage were observed only with 200 mg/kg IM. Preliminary data on myocardial cell turnover indicate that high doses of IM blunted the proliferative response.of cardiomyocytes to the cytotoxic injury. Conversely, in agreement with in vitro data, low IM doses were associated with a reparative response. In conclusion, IM, by inhibiting the multiple pathways modulated by tyrosine kinases, affects in a dose dependent manner CPCs and cardiac function suggesting possible alternative mechanisms of its effects on the cardiovascular system. Disclosures:No relevant conflicts of interest to declare.

High Dose Chemotherapy and Autologous Stem Cell Transplantation in the Primary Therapy of Advanced Follicular Lymphoma in Adults: A Systematic Review and Meta-Analysis of Randomized Clinical Trials.

Abstract 2314Poster Board II-291 Background:The impact of high dose therapy and autologous stem cell transplantation (ASCT) in the upfront management of adults with advanced follicular lymphoma (FL) on overall survival remains uncertain. A number of randomized clinical trials (RCTs) compared ASCT to conventional dose chemotherapy regimens. We performed a systematic review to address this question. Objective:To compare the overall survival (OS), event free survival (EFS), treatment related mortality (TRM), incidence of secondary myelodysplasia/acute myeloid leukemia (t-MDS/AML) and secondary solid tumors within the ASCT strategy and chemotherapy. Methods:We performed a search of MEDLINE, EMBASE, CENTRAL, American Society of Hematology (ASH), American Society of Clinical Oncology (ASCO), BIOSIS, PAPERSFIRST, PROCEEDINGS, clinical trials registries (National Cancer Institute, clinicaltrials.gov) and bibliographies of relevant studies for randomized controlled trials comparing myelo-ablative chemotherapy with ASCT to any chemotherapy in adults with untreated advanced (Ann Arbor stage III-IV) FL. Two reviewers recorded study characteristics, methodology and outcomes. Disagreement was resolved by discussion. Results:Seven trials were identified and included in the systematic review but only four with available data reporting 941 patients were included in the meta-analysis. Three trials excluded: one was never reported; the second was stopped early due to the increased toxicities in the transplant arm; and the third showed no significant difference in EFS or OS at an interim analysis with insufficient data for the meta-analysis. None of these studies has been published. In one of the 7 trials patients in both arms received rituximab during the induction treatment. High dose therapy and ASCT did not result in improved OS compared to standard therapy, Hazard Ratio (HR) 1.15 (0.85, 1.55). The HR for EFS was 0.61 (0.34, 1.11) with substantial heterogeneity, I2 =91%, Chi2 p value <0.00001. Only the duration of the follow up could explain this heterogeneity with statistically significant subgroup interaction, P <0.00001. The incidence of t-MDS/AML ranged from 1% to 7% in the ASCT arms of the four studies, with a difference in favour of standard chemotherapy, Risk Difference (RD) 4% (95%CI: -1%, 8%), and significant heterogeneity (Chi2 p value of 0.008, I2=75%) due to difference in duration of follow up (p=0.005 for the subgroup differences).There was no increase in mortality directly attributable to treatment (RD 0%, 95%CI: -2%, 1%) nor in secondary solid tumors (RD 1%, 95%CI: -2%, 4%). Conclusions:High dose therapy and ASCT as part of initial does not improve overall survival despite a trend towards better disease control, likely because of the effectiveness of second and subsequent lines of treatment for FL. T-MDS/AML occurs more commonly following ASCT and represents a significant risk to patients. With one exception, these studies were conducted before the introduction of monoclonal B cell antibodies. Future trials of ASCT in the context of current chemo-immunotherapy approaches to FL would further inform this issue. Disclosures:No relevant conflicts of interest to declare.

Clinical Scale Generation of Functional Human Natural Killer Cells From Umbilical Cord Blood CD34-Positive Cells for Immunotherapy.

Abstract 2666Poster Board II-642Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been isolated and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. The use of GBGM culture media in a clinical applicable protocol allows the ex vivo expansion and differentiation of CD34+ cells to more than 4-logs into CD56+CD3- NK cells with very high purity. These ex vivo-generated CD56+ cell products contain NK cell subsets expressing CD94/NKG2A and KIR as well express high levels of activating NKG2D and NCRs. Functional analysis showed that CD56+ cell products containing alloreactive NK cells efficiently kill myeloid leukemia and melanoma tumor cells as well as primary acute myeloid leukemia (AML) cells. We have currently translated the protocol to clinical scale production using a closed cell culture bioprocess. CD34+ selection from frozen UCB samples (n=9) using the CliniMACS device was optimized and the selection resulted in a CD34+ enrichment with a mean number of 2,2 ± 1,6 × 106 cells and a purity of 71 ± 14 % CD34+ cells. Validation experiments using the CliniMACS selected cells showed, that the generation of sufficient numbers of NK cells without contaminating T-cells or B-cells under a closed system process is feasible. The cell cultures using bags show a mean expansion of 1273 ± 506 fold (range 759–1770 fold, which generated 8,6×108 − 1,9×109 NK cells from cord blood-derived CD34+ cells within maximal 6 weeks of culture. The mean purity of the NK cell product was 71 ± 9 % (range 63–80%) of the total cells in the bag cultures. These NK cell products will be used for immunotherapy in elderly AML patients. This study is a phase I dose escalation study in a series of 12 AML patients who have successfully achieved CR (<5% blasts in the bone marrow) after standard intensive chemotherapy. Disclosures:Spanholtz:Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment.

Alloreactive NK-Cells Cure Tumor Stem Cell Containing Breast Cancer.

Abstract 4082Poster Board III-1017 BackgroundPatients with metastasized solid tumors can not be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Acute myeloid leukemia tumor stem cells can be killed by alloreactive T- or NK-cells, as patients have a higher survival chance after allogeneic transplantation. In the past we published the curative effectiveness of allogeneic spleen and bone marrow transplantation in breast cancer bearing mice. Our current aim was to study if either alloreactive T- or NK-cells are able to cure these mice. In addition we wanted to know if the tumors contain slow cycling tumor stem cells. MethodsThe 4T1 breast cancer cell line was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank of CB6F1 (H-2b/d) mice. At day 12 mice were treated with 2× 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY), followed by transplantation of spleen and bone marrow cells. B6CBAF1 (H-2b/k) mice were used as allogeneic donors. These donors harbour alloreactive NK-cells towards CB6F1 recipients as determined by an in vivo NK-cell alloreactivity assay. NK-cell or T-cell depleted grafts were obtained from donors injected with anti-AsialoGM1 or a combination of anti-CD4 and anti-CD8 respectively some days before spleen and bone marrow harvest. Tumor stem cells were defined as slow cycling (i.e. label retaining) Hoechst extruding cells. 4T1 tumor cells were labelled with Vybrant CM-Dil (Dil) and then injected s.c. in CB6F1 mice. Fourteen days later tumors were excised. Part of the tumor was prepared as single cell suspension. Subsequently the presence of Dil retaining tumor stem cells was determined by fluorescence microscopy. The other part of the tumor was snap frozen and studied by histochemistry and fluorescence microscopy. For in vitro cytotoxicity testing 4T1 cells were again labelled with Dil and then seeded in 96 well plates at 30 cells/well. Chemotherapeutics were added in the different plates: 4-OH-CY (the active metabolite), cisplatine and doxorubicin, in concentrations far above the lowest maximal lethal dose as determined in MTT assays. Wells were checked weekly for growth and presence of tumor stem cells. Hoechst staining was used in these cultures to underscore that Dil retaining chemorefractory cells are indeed stem cells. [Display omitted] ResultsNinety percent of CB6F1 mice injected with 4T1 breast cancer cells developed a tumor growing progressively to a size that made it necessary to sacrifice them. Growth in mice treated with radio- and chemotherapy only or additionally with CB6F1 spleen and bone marrow cells was delayed for 10 days compared with tumor growth in untreated mice, but the incidence was equally high (90% and 80% respectively). In contrast, but in concord with our previous published results, only 10% of 4T1 breast cancer bearing mice transplanted with haploidentical spleen and bone marrow cells did show progressive tumor growth (follow-up 99 days at the time of abstract submission, p<0.004 log-rank test)). Similarly, mice transplanted with spleen and BM cells from T-cell depleted donors did not develop tumors at all (p<0.004 log-rank test), in contrast to recipients of in vivo NK-cell depleted grafts (90% tumor incidence).Label retaining tumor stem cells are unmistakably present in tumors at the day of transplantation, evidenced by the presence of a minute number of Dil retaining cells in single cell suspension of the tumor and in tumor tissue sections. More direct evidence for chemo-resistance of 4T1 tumor stem cells is derived by results of in vitro co-culture experiments. When Dil labelled 4T1 cells are plated in 30 cells/well concentration, approx. 25% of the wells contain one or two label retaining cells while the other wells comprise non-label retaining cells only. After incubation with chemotherapeuticals growth is observed only in wells containing a label retaining cell. These label retaining cells are also Hoechst negative. ConclusionsThis report provides the first evidence that tumor stem cells can be eliminated in vivo by alloreactive NK cells resulting in cure of chemorefractory breast cancer. It is also shown in vitro that only tumor stem cell containing tumor colonies resist the effect of various chemotherapeuticals commonly used in oncology as they do in vivo. These results strongly encourage the exploration of clinical alloreactive NK therapy in patients with metastasized solid tumors. Disclosures:No relevant conflicts of interest to declare.