Comparison of bone marrow high mitotic index metaphase fluorescence in situ hybridization to peripheral blood and bone marrow real time quantitative polymerase chain reaction on the International Scale for detecting residual disease in chronic myeloid leukemia.

BACKGROUND: Recently, an International Scale was proposed for standardizing BCR-ABL transcript measurements and reporting in the assessment of minimal residual disease by real-time quantitative polymerase chain reaction (RQ-PCR). Here we present the setting up of the International Scale conversion factors for a national laboratory by performing both a cross-analysis of a set of standard samples from a reference laboratory and an analysis of bone marrow and peripheral blood samples at diagnosis (from 32 and 27 patients, respectively). DESIGN AND METHODS: A total of 222 bone marrow and 173 peripheral blood mononuclear cell samples from 96 patients with chronic myeloid leukemia were analyzed with RQ-PCR according to Europe Against Cancer protocols. Additionally, 291 bone marrow samples were analyzed with high mitotic index metaphase fluorescence in situ hybridization (metaphase FISH). RESULTS: Major molecular response according to the International Scale in BCR-ABL/GUS transcript levels corresponded to a ratio of 0.035% in peripheral blood and 0.034% in bone marrow, yielding the same conversion factor of 2.86 for both types of sample. Based on metaphase FISH, values of 10%/-1.0 log, 1%/-2.0 log and 0.1%/-3.0 log on the International Scale, corresponded to 13%, 2%, and 0.3% of Philadelphia chromosome positive cells in bone marrow, respectively. CONCLUSIONS: In conclusion, conversion factors can be determined either by cross-analyzing a number of samples with a laboratory that has already established the International Scale or utilizing sufficient numbers of reference samples from chronic myeloid leukemia patients at diagnosis, or using the upcoming international standards.

Characterization of 10 vulvar carcinoma cell lines by karyotyping, comparative genomic hybridization and flow cytometry.

OBJECTIVE AND METHODS: Ten vulvar squamous cell carcinoma cell lines established at the University of Michigan (UM-SCV-1A, -1B, -2, -3, -4, -6, -7) and at the University of Turku (UT-SCV-1, -2, -3) were characterized by G-banding karyotyping, comparative genomic hybridization (CGH), and deoxyribonucleic acid (DNA) flow cytometry. RESULTS: All cell lines had hyperdiploid DNA content as measured by flow cytometry. The DNA index (DI) remained relatively stable through different passages in 9 of 10 cases. DIs of UM-SCV-3 and UT-SCV-2 were near-diploid, as were the corresponding karyotypes. The 10 SCVs showed remarkable genetic similarities with respect to consistent chromosome rearrangements. Loss of 3p, noted in 8/10 SCVs, was narrowed to the smallest common region at 3p11-3p13. Loss of 8pter-p11 was observed in 10/10 cell lines. Loss of 11qter-q23 was present in UM-SCV-1 and -2, and in all four recently karyotyped SCVs. Other consistent losses include Xpter-p11 in 6/10, and 18qter-q11 in 7/10 cell lines. Common gains included gain of 8q in 8/10 and 3q in 6/10. Consistent copy number imbalances were confirmed by CGH; concerning loss of 3p, in 63%, to loss of 8p in 70%, to gain of 3q in 83%, and to gain of 8q in 75% of the cell lines. CONCLUSIONS: CGH and karyotyping showed concordance in defining copy number imbalances, thus supporting the accuracy of CGH to detect chromosome imbalances in tumors that cannot be karyotyped.

Single copy heterozygote integration of HPV 33 in chromosomal band 5p14 is found in an epithelial cell clone with selective growth advantage.

Infection with human papillomavirus (HPV) of specific high-risk type triggers a series of events in target cells, which will eventually lead to development of genital neoplasia. The integration of high-risk HPV DNA into the cell genome has been regarded as a crucial event in tumor progression. With respect to different HPV types, the knowledge of HPV integrated loci is still limited. We have now determined the genomic variation and chromosomal location of HPV 33 DNA in the cell line UT-DEC-1, established from a vaginal mild dysplasia lesion. The viral sequence of the cell line was determined, and a variant of the prototype HPV 33 strain was identified, showing nucleotide substitutions resulting in amino acid changes in the E2, L2 and E4 open reading frames. In late passage UT-DEC-1 cells, a deletion of more than half of the 3' part of E1 and major parts of the E2 and E4 genes provided evidence for integration. The flanking sequences of the integration site were completely homologous to published sequences from chromosomal band 5p14, and remained unchanged in all subclones established from late passage cells. There were no chromosomal deletions or gross rearrangements at the integration site, and only a single heterozygotic copy of HPV 33 was detected. The karyotype of late passage cells showed only minor changes compared with early passage cells. During passaging of the cell line, there were progressive changes towards a malignant phenotype, and in parallel to this, the cells carrying episomal HPV 33 of the early passages was completely superseded by cells containing the integrated virus. Thus, our results show that this single copy heterozygote integration of HPV 33 into chromosome band 5p14 appears to be associated with emergence of cells escaping senescence, and with growth advantage compared with cells carrying episomal virus.