Hans Wessels

Identification of RUNX1/AML1 as a classical tumor suppressor gene

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

Paragangliomas of the head and neck region show complete loss of heterozygosity at 11 q22-q23 in chief cells and the flow-sorted dna aneuploid fraction

Nonchromaffin paragangliomas of the head and neck region, also known as glomus tumors, are usually benign neoplasms consisting of clusters of chief cells surrounded by sustentacular cells arranged in so-called Zellballen. Most of the patients have a familial background. In a previous study, examining all chromosome arms, we found loss of heterozygosity (LOH) predominantly at the chromosome 11q22-q23 region, where the disease causing gene PGL1 has been located by linkage analysis. However, all tumors showed only partial loss of allele signal intensities, and it was not clear whether this represented allelic imbalance or cellular heterogeneity. In the current study, we have performed LOH analysis for the 11q22-q23 region on DNA-aneuploid tumor cells, enriched by flow sorting, and on purified chief cell fractions obtained by single-cell microdissection. Complete LOH was found for two markers (D11S560 and CD3D) in the flow-sorted aneuploid fractions, whereas no LOH was found in the diploid fractions of three tumors. The microdissected chief cells from two of these tumors also showed complete LOH for both markers, indicating that the chief cells are clonal proliferated tumor cells. These results indicate that the PGL1 gene is likely to be a tumor suppressor gene, which is inactivated according to the two-hit model of Knudson. Furthermore, it shows that chief cells are a major if not the sole neoplastic component of paragangliomas.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor β (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2u2009Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Genome wide molecular analysis of minimally differentiated acute myeloid leukemia

This study used single nucleotide polymorphism (SNP)-array technology to study copy number changes and to determine regions of loss of heterozygosity in minimally differentiated acute myeloid leukemia. Several chromosomal regions were found to be deleted or duplicated, and mutations in 163gene were the most frequent mutations detected. Background Minimally differentiated acute myeloid leukemia is heterogeneous in karyotype and is defined by immature morphological and molecular characteristics. This originally French-American-British classification is still used in the new World Health Organization classification when other criteria are not met. Apart from RUNX1 mutation, no characteristic molecular aberrations are recognized. Design and Methods We performed whole genome single nucleotide polymorphism analysis and extensive molecular analysis in a cohort of 52 patients with minimally differentiated acute myeloid leukemia. Results Many recurring and potentially relevant regions of loss of heterozygosity were revealed. These point towards a variety of candidate genes that could contribute to the pathogenesis of minimally differentiated acute myeloid leukemia, including the tumor suppressor genes TP53 and NF1, and reinforced the importance of RUNX1 in this leukemia. Furthermore, for the first time in this minimally differentiated form of leukemia we detected mutations in the transactivation domain of RUNX1. Mutations in other acute myeloid leukemia associated transcriptions factors were infrequent. In contrast, FLT3, RAS, PTPN11 and JAK2 were often mutated. Irrespective of the RUNX1 mutation status, our results show that RAS signaling is the most important pathway for proliferation in minimally differentiated acute myeloid leukemia. Importantly, we found that high terminal deoxynucleotidyl transferase expression is closely associated with RUNX1 mutation, which could allow an easier diagnosis of RUNX1 mutation in this hematologic malignancy. Conclusions Our results suggest that in patients without RUNX1 mutation, several other molecular aberrations, separately or in combination, contribute to a common minimally differentiated phenotype.

Translocations (X;10)(p22;q24) and (1;10)(q21;q11) in a Follicular Adenoma of the Thyroid without Apparent Involvement of the RET Protooncogene

We report here the cytogenetic analysis of a follicular adenoma of the thyroid which revealed an abnormal clone with a t(X;10)(p22;q24) and a t(1;10)(q21;q11) together with normal cells. Fluorescence in situ hybridization (FISH) with YACs 273E3 and 344H4, which are located on 10q11.2 and are specific for the RET protooncogene, showed no abnormalities. It would therefore appear that this gene is not involved in the particular tumor, as has been reported in a number of papillary thyroid carcinomas. Several chromosomal aberrations have been suggested as been specific for follicular thyroid adenoma. However, until now, only a few such cases have been reported which involve structural abnormalities of chromosomes 10q11.2 and 10q24. We believe this to be the first report of a follicular thyroid adenoma with a t(X;10)and a t(1;10).

Sublocalization of the breakpoints of a t(5;16) in myelodysplasia

As a first step in characterizing a t(5;16)(q31;p11.2) in a patient with the diagnosis refractory anemia with ring sideroblasts, a cell fusion was carried out between bone marrow cells from the patient and the Chinese hamster cell line A3. Using PCR and FISH analysis on hybrid lines containing the human derivative 16 chromosome, the breakpoints could be mapped between the markers TCF-7 and IL-9 on chromosome 5 and OL-7 and s30A4 on chromosome 16, both regions spanning approximately 1 Mb. Since the breakpoint on 5q has occurred in a region that is frequently deleted in myeloid malignancies, the gene disrupted by this translocation could also be implicated in this aberration.

Characterization of a New Human B Cell Line (Bonna-12) with Trisomy 9 and Trisomy 12 Chromosomal Abnormality

A new EBV positive human B-cell line, BONNA-12 was established from splenic cells of a patient with a hairy cell leukemia (HCL). BONNA-12 cells grew spontaneously and formed colonies in semisolid media. Although the BONNA-12 cell line was identical with the patients spleen cells by HLA analysis and Southern blot examination of minisatellite DNA patterns, the immunoglobulin heavy and light chain rearrangement patterns differed from the original HCL. Cytogenetic analysis of the BONNA-12 cell line demonstrated in the major cell clone a 47, X, -Y, +9, +12 karyotype. Trisomy 12 is a characteristic abnormality in chronic lymphocytic leukemia that also rarely occurs in HCL. The BONNA-12 cell line is of potential value in the study of trisomy 12 in chronic B cell malignancies.