Carol N. Phillips

Chromosome and growth factor abnormalities in melanoma

Cultures from metastatic melanomas of 15 patients had detailed melanoma growth stimulatory activity (MGSA) and cytogenetic analysis. The presence of melanoma cells was confirmed by microscopic identification of melanin, tyrosinase activity, and electron microscopy characterization of melanosomes. The MGSA is found in cytoplasmic granules after immunocytochemical stain. Three of the cultures did not produce MGSA and showed no distinctive cytogenetic differences. Breakpoints in derivative chromosomes were concentrated in region 1p1, and among all cultures chromosome 1 was the most frequently rearranged. It also has a low copy number of normal homologs. Chromosomes 18, X, and Y were never derivative, and chromosomes 2 and 4 were rarely so. Thus the cytogenetic data indicate that 4q13-21, the hybridization site for MGSA cDNA, is spared from gross change, although it could be under the influence of another site on chromosome 1 that is lost or rearranged. The ratio of abnormal to normal autosomes (mean per cell) in no culture exceeded 0.5, and for no autosome exceeded 0.8, suggesting a limit to the rearrangement tolerated for cell survival. If the Y is retained, the X:Y ratio varies around a normal figure of 1. The ratio of autosomes to sex chromosomes varies around a normal figure of 22. These data suggest stability of the X chromosome in cells undergoing multiple rearrangements of the autosomes.

Detection of BCR-abl in acute leukemia by molecular and cytogenetic methods

Background: The Philadelphia chromosome (Ph), t(9;22)(q34;q11), is detected by karyotyping in a minority of patients with acute leukemia. Ph results in fusion of the c-abl oncogene on chromosome 9 with the breakpoint cluster region BCR gene on chromosome 22. The purpose of this study was to compare reverse trascriptase-polymerase chain reaction (RT-PCR) for BCR/abl fusion to cytogenetic methods for Ph detection in patients with acute leukemia. Methods and Results: Peripheral blood and bone marrow samples from cases of adult acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) were examined for Ph by RT-PCR, karyotyping, and fluorescence in situ hybridization (FISH). Using total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electorphoresed, and probed for BCR/abl fusion. Patient cells and SUPB15 and K562 cell lines were used as breakpoint controls. Karyotyping was done by standard Giemsa banding. FISH was performed on bone marrow smears using digxigenin-labeled DNA probes for major and minor bcr breakpoints (corresponding to involvement of major bcr exons 2/3 and minor bcr exon 1, respectively) and biotin-labeled DNA probes for abl. Rhodamine-conjugated antidigoxigenin and fluorescein-conjugated avidin yielded red and green fluorescent signals, respectively. A total of 32 samples from patients with AML were studied, 20 from patients with de novo or relapsed AML and 12 from patients in remission. Five of 32 cases of AML (16%) were RT-PCR+/Ph+ all with major bcr breakpoints between exons 3 and 4. One of the 32 cases (3%) was RT-PCR+/Ph+; this case was the only positive remission sample. Of the four T-PCR+/Ph- cases, one showed t(2;17), one showed t(9;11), and two had a normal karyotype. FISH was done in three RT-PCR+ cases, yielding positive results with the major probe in two. A total of 22 samples from patients with ALL were studies, 15 from patients with de novo or relapsed ALL and seven from patient remission. Seven of 22 cases of ALL (32%) were RT-PCR+, four with major-bcr breakpoints between exons 3 and 4, and three with breakpoints in the minor-bcr. Two of the 22 (9%) cases were RT-PCR+/Ph+. Of the five RT-PCR+/Ph- cases, two showed a 22q- but lacked the typical Ph break on chromosome 9, 1 showed a 12p-, and two had a normal karyotype. FISH was performed in four RT-PCR+ cases, yielding positive results with the major probe in two cases and with the minor probe in two cases. Conclusions: RT-PCR is more sensitive than karyotyping, detecting masked Ph or translocations not found by cytogenetic analysis. FISH is a helpful adjunctive test when used to confirm BCR/abl fusion in RT-PCR+/Ph- cases.