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Translocations and deletions of the short arm of chromosome 12 [t(12p) and del(12p)] are common recurring abnormalities in a broad spectrum of hematologic malignant diseases. We studied 20 patients and one cell line whose cells contained 12p13 translocations and/or 12p deletions using fluorescence in situ hybridization (FISH) with phage, plasmid, and cosmid probes that we previously mapped and ordered on 12p12-13. FISH analysis showed that the 12p13 translocation breakpoints were clustered between two cosmids, D12S133 and D12S142, in 11 of 12 patients and in one cell line. FISH analysis of 11 patients with deletions demonstrated that the deletions were interstitial rather than terminal and that the distal part of 12p12, including the GDI-D4gene and D12S54 marker, was deleted in all 11 patients. Moreover, FISH analysis showed that cells from 3 of these patients contained both a del(12p) and a 12p13 translocation and that the affected regions of these rearrangements appeared to overlap. We identified three yeast artificial chromosome (YAC) clones that span all the 12p13 translocation breakpoints mapped between D12S133 and D12S142. They have inserts of human DNA between 1.39 and 1.67 Mb. Because the region between D12S133 and D12S142 also represents the telomeric border of the smallest commonly deleted region of 12p, we also studied patients with a del(12p) using these YACs. The smallest YAC, 964c10, was deleted in 8 of 9 patients studied. In the other patient, the YAC labeled the del(12p) chromosome more weakly than the normal chromosome 12, suggesting that a part of the YAC was deleted. Thus, most 12p13 translocation breakpoints were clustered within the sequences contained in the 1.39 Mb YAC and this YAC appears to include the telomeric border of the smallest commonly deleted region. Whether the same gene is involved in both the translocations and deletions is presently unknown.

Although approximately 70% of patients with acute promyelocytic leukemia (APL) are cured after treatment with all-trans retinoic acid (ATRA) and anthracycline based chemotherapy, those who relapse cannot be cured even with chemotherapy or arsenic trioxide salvage therapy. In order to study the molecular mechanisms of resistance to ATRA, liposomal ATRA, and arsenic trioxide, we have utilized a murine model of APL generated by “knocking in” the PML/RARα gene into the murine cathepsin G locus (Westervelt et al. Blood. 102(5):1857). Of the 76 single knock-in (SKI) mice followed for a median time of 368 days, 30 (39%) suffered from rapid-onset leukocytosis, anemia, thrombocytopenia, and hepato-splenomegaly. Spleens from these leukemic animals were removed and the tumors cells (> 90% CD34+/Gr-1+APL cells) were frozen in aliquots and banked for future passive transfer into genetically compatible recipients. The SKI mice exhibited the expected median time to death of 222 days. Attempts to expand these tumors in vitrofailed, thus precluding the use of in vitromethods to generate ATRA and arsenic resistant SKI APL tumor cells. An alternate in vitromodel of identifying de novoresistant SKI APL tumors to the drugs was established by incubating individual SKI APL tumors with ATRA, liposomal ATRA, and arsenic in vitrofor only 3 days and using ELISA to measure upregulation of MMP-9 (gelatinase B), a surrogate marker for neutrophil differentiation. ATRA, liposomal ATRA, and arsenic induced 6.2 ± 4.4, 5.7 ± 3.5, 1.7 ± 1.0 fold increase in accumulated levels of MMP-9, respectively. These data suggest that ATRA and liposomal ATRA are more potent inducers of terminal differentiation than arsenic in these APL tumors. More importantly, the SKI APL tumors were also assessed for in vivoresistance to these agents by injecting 1 x 106banked APL tumor cells into the peritoneal cavity of wild-type genetically compatible mice. The mice were then treated i.p. with either arsenic trioxide (0.4 mg q.d.), liposomal ATRA (0.4 mg q.d.), or diluent control. Doses were administered daily starting on day +5 until death. Tumors in the recipient mice were tracked by PCR for the transgene and FACS. The tumors proliferated in the peritoneal cavity (days 1–14) followed by migration to the bone marrow and spleen (days 14–28). By the eighth week post-injection, all mice suffered from leukocytosis and eventually died. Of the 22 banked tumors studied, only two tumors showed de novoresistance to ATRA both in vitroand in vivo. We will present data on preliminary RNA profiling and DNA sequencing of the PML-RARα transgenes in these APL tumors and attempt to correlate these results and the results of in vitroinduction of MMP-9. The SKI APL tumors will provide us with unique reagents to study the biology of resistance to ATRA and arsenic trioxide.