Kathleen M. Murphy

FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML

We examined 6 different FMS-like tyrosine kinase-3 (FLT3) inhibitors (lestaurtinib, midostaurin, AC220, KW-2449, sorafenib, and sunitinib) for potency against mutant and wild-type FLT3, as well as for cytotoxic effect against a series of primary blast samples obtained from patients with acute myeloid leukemia (AML) harboring internal tandem duplication (FLT3/ITD) mutations. We found that inhibition of FLT3 autophosphorylation in a FLT3/ITD specimen does not always induce cell death, suggesting that some FLT3/ITD AML may not be addicted to FLT3 signaling. Relapsed samples and samples with a high mutant allelic burden were more likely to be responsive to cytotoxicity from FLT3 inhibition compared with the samples obtained at diagnosis or those with a low mutant allelic burden. These FLT3 inhibitors varied to a considerable degree in their selectivity for FLT3, and this selectivity influenced the cytotoxic effect. These results have important implications for the potential therapeutic use of FLT3 inhibitors in that patients with newly diagnosed FLT3-mutant AML might be less likely to respond clinically to highly selective FLT3 inhibition.

K562/GM-CSF Immunotherapy Reduces Tumor Burden in Chronic Myeloid Leukemia Patients with Residual Disease on Imatinib Mesylate

Purpose: Chronic myeloid leukemia (CML) can be responsive to T-cell–mediated immunity. K562/granulocyte macrophage-colony stimulating factor (GM-CSF) is a GM-CSF producing vaccine derived from a CML cell line that expresses several CML-associated antigens. A pilot study was developed to determine if K562/GM-CSF immunotherapy could improve clinical responses to imatinib mesylate (IM) in patients with chronic myeloid leukemia. Experimental Design: Patients with chronic phase CML who achieved at least a major cytogeneic response but remained with persistent, measurable disease despite one or more years on imatinib mesylate were eligible. Each was given a series of four vaccines administered in three-week intervals, with or without topical imiquimod, while remaining on a stable dose of imatinib mesylate. CML disease burden was measured serially before and after vaccination. Results: Nineteen patients were vaccinated, with a median duration of previous imatinib mesylate therapy of 37 (13–53) months. Mean PCR measurements of BCR-ABL for the group declined significantly following the vaccines (P = 0.03). Thirteen patients had a progressive decline in disease burden, 8 of whom had increasing disease burden before vaccination. Twelve patients achieved their lowest tumor burden measurements to date following vaccine, including seven subjects who became PCR-undetectable. Conclusions: K562/GM-CSF vaccine appears to improve molecular responses in patients on imatinib mesylate, including achieving complete molecular remissions, despite long durations of previous imatinib mesylate therapy. Clin Cancer Res; 16(1); 338–47

Use of T-Cell Antibodies for Donor Dosaging in a Canine Model of in utero Hematopoietic Stem Cell Transplantation

Aim: Microchimerism following canine in utero hematopoietic stem cell transplantation (IUHSCT) development of T-cell dosing regimens. Objective: To investigate the use of anti-T-cell antibodies for cell dosing of the donor graft in a canine model of IUHSCT. Study Design: Canine IUHSCT was performed by ultrasound-guided intraperitoneal injection in days 35–38 of fetal canines with CD34+ cells at doses of 4.5 × 108 to 1.3 × 109 cells/kg and T cells (CD3+ CD5+) at doses of 8 × 106 to 8.8 × 108 cells/kg. Postnatal studies included tissue histology and polymerase chain reaction-based chimerism analysis. Results: Term survival was 86–100%. Microchimerism (0–2%) was detected in five of eight recipients in multiple tissues. Histopathology revealed no evidence of graft-versus-host disease (GVHD). Conclusion: Canine IUHSCT is a useful model to investigate the role of donor T cells in engraftment and GVHD. IUHSCT at early gestational ages with high doses of donor T cells in the graft yields microchimerism in multiple tissues without GVHD.

In utero Hematopoietic Stem Cell Transplantation in Canines: Exploring the Gestational Age Window of Opportunity to Maximize Engraftment

Objective: In utero hematopoietic stem cell transplantation (IUHSCT) is a promising therapy for a variety of congenital disorders. Our objective was to determine the optimal time in gestation for IUHSCT in a canine model. Methods: IUHSCT was performed in day 31-50 (term 63) fetal canines with CD34+ cells isolated from paternal bone marrow at doses of 0.09-3.4 × 109 CD34+ cells/kg and T cells (CD3+/CD5+) from paternal blood at 0.11-1.1 × 109 cells/kg. Engraftment was assayed using PCR-based chimerism analysis (SRY gene detection for female recipients, and unique microsatellite loci for both sexes). Results: Microchimerism and chimerism were present in multiple recipients across most gestational ages at transplant. Maximal engraftment was obtained in hematopoietic tissues in transplants performed at 42 days. At extremes of recipient gestational age, minimal to no engraftment was seen. Conclusion: Fetal age at the time of IUHSCT plays an important role in achieving engraftment in our canine model.

The regulation of urokinase plasminogen activator gene expression in macrophages

Urokinase plasminogen activator (uPA) is a specific serine protease which converts the zymogen plasminogen into plasmin, a protease of broad specificity. uPA has been implicated in extracellular proteolysis in numerous cellular systems involving cellular migration, tissue remodelling, or invasive growth and metastasis of tumours (1, 2). In macrophages uPA is thought to be involved in cellular migration during the inflammatory response (3, 4) and is bound to a specific receptor on the leading edge of monocytes migrating in vitro (5).

Abstract 1781: Use of SNP arrays to assess loss of heterozygosity in gliomas

Introduction: Loss of heterozygosity (LOH) of chromosome arms 1p and 19q in gliomas has diagnostic, prognostic, and therapeutic implications. The deletion of 1p and the codeletion of chromosome arms 1p and 19q correlate with the diagnosis of oligodendroglioma, increased chemosensitivity and improved prognosis. Clinically, LOH is most frequently determined by Fluorescent In Situ Hybridization (FISH) and short tandem repeat (STR) analysis. Each approach suffers from the limitation that the probes/primers interrogate only a small portion of the chromosomes. Small deletions can be misinterpreted as LOH of the entire chromosome arm. We have investigated the use of a single nucleotide polymorphism (SNP) array for identifying LOH in gliomas. Methods: Genomic DNA was extracted from 5 formalin-fixed paraffin-embedded tissue samples following microdissection, and from peripheral blood. Samples were run on an ABI 3100 following multiplex PCR amplification of 5 STRs on chromosome 1p (D1S199, D1S186, D1S162, D1S312, D1S226) and 3 STRs on chromosome 19q (D19S918, D19S112, D19S206). Array analysis was performed using the Affymetrix genome-wide human SNP array 6.0 platform (906,600 SNPs) according to protocol. Data were analyzed with Partek Genomics Suite. Results: Cases 1 and 4 showed no LOH on chromosomes 1p and 19q by STR analysis and SNP array. Cases 2 and 5 showed LOH on chromosomes 1p and 19q by STR analysis and SNP array. Case 3 showed partial centromeric LOH on chromosome 1p by STR analysis which was confirmed by SNP array. Case 1 was a 48 year old male with anaplastic mixed oligoastrocytoma. Additional SNP array findings included a deletion of chromosome arm 13q. Case 4 was a 31 year old female with a low grade glioneural tumor, and no additional findings were made by SNP array. Case 2 was a 47 year old female with anaplastic oligodendroglioma. Additional SNP array findings included losses on chromosomes 5, 6, 9,15, and whole gain of chromosome 11. Case 5 was a 12 year old male with anaplastic oligodendroglioma. SNP array found additional losses on chromosomes 2, 3, 6, 8, 9, 10, and 20. Case 3 was a 47 year old female with an astrocytoma. Analysis of the 3 most centromeric STR loci on 1p demonstrated allelic imbalance that was consistent with LOH. The 2 most telomeric STR loci demonstrated allele ratios consistent with heterozygosity (negative for LOH). SNP array confirmed partial centromeric LOH on 1p and identified losses on chromosomes 2, 6, 10, 13, 16, and 22. Conclusion: Analysis of LOH on chromosome arms 1p and 19q by STR analysis and SNP array were concordant. SNP array identified additional gains or losses in each sample that were not detected by the STR assay. Interestingly, SNP array analysis also identified LOH on chromosome 13q, which contains the BRCA2 gene locus, in cases 1 and 3. If a second-hit can be identified, this may present a novel approach for identifying glioma patients who may potentially benefit from Poly (ADP-Ribose) Polymerase (PARP) inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1781.