Genome-wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic recombination in acute myeloid leukemias.

Genome-wide analysis of single nucleotide polymorphisms in 64 acute myeloid leukemias has revealed that approximately 20% exhibited large regions of homozygosity that could not be accounted for by visible chromosomal abnormalities in the karyotype. Further analysis confirmed that these patterns were due to partial uniparental disomy (UPD). Remission bone marrow was available from five patients showing UPD in their leukemias, and in all cases the homozygosity was found to be restricted to the leukemic clone. Two examples of UPD11p were shown to be of different parental origin as indicated by the methylation pattern of the H19 gene. Furthermore, a previously identified homozygous mutation in the CEBPA gene coincided with a large-scale UPD on chromosome 19. These cryptic chromosomal abnormalities, which seem to be nonrandom, have the characteristics of somatic recombination events and may define an important new subclass of leukemia.

Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event.

Basal cell carcinoma is the most common human cancer with increasing incidence reported worldwide. Despite the aberrant signaling role of the Hedgehog pathway, little is known about the genetic mechanisms underlying basal cell carcinomas. Towards a better understanding of global genetic events, we have employed the Affymetrix Mapping 10K single nucleotide polymorphism (SNP) microarray technique for "fingerprinting" genomewide allelic imbalance in 14 basal cell carcinoma-blood pair samples. This rapid high-resolution SNP genotyping technique has revealed a somatic recombination event-uniparental disomy, leading to a loss of heterozygosity (LOH), as a key alternative genetic mechanism to allelic imbalances in basal cell carcinomas. A highly conserved LOH region at 9q21-q31 was found in 13 of 14 (93%) basal cell carcinomas. Further statistical and fluorescence in situ hybridization analyses confirmed that the 9q LOH was a result of uniparental disomy in 5 of 13 (38%) basal cell carcinomas. De novo mutations in the Patched 1 gene (PTCH) were found in 9 of 13 (69%) basal cell carcinomas with 9q LOH. A second important locus, containing LOH at 6q23-q27 was found in 5 of 14 (36%) basal cell carcinomas, suggesting that the presence of an additional putative tumor suppressor gene may be contributing to basal cell carcinoma development. This study shows that the rate of 9q LOH in basal cell carcinomas has been previously underestimated. Furthermore, we provide the first evidence that uniparental disomy due to somatic recombination constitutes one of the mechanisms of LOH in basal cell carcinoma tumorigenesis.

3'CBFbeta deletion associated with inv(16) in acute myeloid leukemia.

Recent reports have shown that concomitant submicroscopic deletions can occur in association with chromosomal translocations/inversions in several leukemia subtypes. Detectable by fluorescence in situ hybridization (FISH), these losses of sequence include deletion of the 5' region of the ABL gene and the 3' region of BCR in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL), as well as the 5' region of ETO in acute myeloid leukemia (AML) French-American-British type M2 associated with t(8;21), 3'MLL in AML and ALL, and 3' core-binding factor beta (CBFbeta) in AML associated with inv(16). While it has been widely reported that submicroscopic deletions of the derivative 9 in CML have an adverse prognostic impact, the clinical significance, if any, of deletions associated with t(8;21), inv(16)/t(16;16), or MLL rearrangement is yet to be determined. We analyzed a series of 39 patients diagnosed with AML who had cytogenetically detectable inv(16)/t(16;16) by using a FISH probe for the CBFbeta region to determine the incidence of the 3'CBFbeta deletion. Deletions were detected in three patients (8%), all associated with inv(16), bringing the number of cases reported so far to seven. The prognostic significance of this finding remains unclear.

Comparative genomic hybridization and multiplex-fluorescence in situ hybridization: an appraisal in elderly patients with acute myelogenous leukemia.

Comparative genomic hybridization (CGH) and multiplex-fluorescence in situ hybridization (M-FISH) were used to evaluate the presentation karyotype in 15 and 18 patients respectively, aged >/=60 years, with acute myeloid leukemia (AML). Conventional G-banded analysis was performed in all patients prior to evaluation. Comparative genomic hybridization confirmed the G-banded karyotype fully in 12 patients and partially in two patients. Normal CGH profiles were observed in patients with a normal karyotype and in one patient with a balanced chromosomal translocation as the sole cytogenetic aberration. Multiplex-fluorescence in situ hybridization provided additional information in two patients with a complex karyotype, but failed to detect a telomeric translocation in one patient. Eight patients with normal G-banded karyotypes appeared normal by M-FISH. These results demonstrate that both CGH and M-FISH analysis correlate well with the G-banded karyotype in AML. Furthermore, although additive cytogenetic data was not provided by either technique in cases with normal karyotype, DNA copy number change and cryptic translocations below the resolution of CGH and M-FISH may still be the initiating event for leukemogenesis for these patients.

Fluorescence in situ hybridisation analysis of bone marrow trephine biopsy specimens; an additional tool in the diagnostic armoury.

Fluorescence in situ hybridisation (FISH) analysis is now widely employed in the diagnosis and risk stratification of a wide range of malignant diseases. While this technique is used successfully with formalin-fixed paraffin-embedded (FFPE) sections from numerous tissue types, FISH analysis of FFPE tissue sections from trephine biopsy specimens has been less widely reported, possibly due to technical limitations relating to the decalcification protocols employed. During the last 4 years FISH analysis has been carried out successfully in 42 out of 55 (76%) consecutive trephine biopsy specimens received as part of the standard diagnostic service at our institution. Samples decalcified using EDTA-based protocols were analysed successfully in 31/31 cases (100%), whereas only 11/24 samples (46%) decalcified using formic acid-based protocols were successful. In our experience, FISH analysis of trephine biopsy specimens is a highly reproducible technique and a very useful adjunctive tool in the diagnostic armoury; however, its use in a standard diagnostic setting relies on the use of EDTA-based decalcification protocols.

Fluorescence in situ hybridisation analysis of formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of non-Burkitt high grade B-cell non-Hodgkin's lymphoma: a single centre's experience.

AIMS: In recent years the genetic aberrations associated with diffuse large B-cell lymphoma and the new subtype described in the 2008 revision of the WHO classification, 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma' have been increasingly well defined. Recurrent genetic abnormalities include rearrangements involving MYC (8q24), BCL2 (18q21) and BCL6 (3q27); as the prognostic and therapeutic implications associated with these abnormalities are clarified their accurate identification at diagnosis is becoming increasingly critical. We describe our experience of using a panel of fluorescence in situ hybridisation (FISH) probes on formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of 162 patients with non-Burkitt high grade B-cell non-Hodgkin's lymphomas (HG-BNHL). METHODS: BCL6, IGH-BCL2 and MYC status were determined prospectively in sequential patients presenting with HG-BNHL, with respect to the presence of rearrangements and copy number changes. Small numbers of samples were analysed retrospectively or were studied at relapse in previously untested patients. RESULTS: FISH analysis was successful in 160/162 (99%) cases, with abnormalities detected in 118/160 (74%). CONCLUSIONS: FISH analysis of formalin-fixed paraffin-embedded tissue sections is a highly reproducible technique with an excellent success rate for the detection of genetic abnormalities which will play an increasingly important role in improving risk stratification of patients with HG-BNHL.