E. Frascella

Improved Long-Distance Polymerase Chain Reaction for the Detection of t(8;14)(q24;q32) in Burkitt's Lymphomas

The t(8;14)(q24;q32), involving MYC gene (8q24) and the immunoglobulin heavy chain (IgH) locus (14q32), represents about 75% of all translocations in Burkitts lymphoma (BL). Due to the great variability of the breakpoint region, a standard polymerase chain reaction assay is not sufficient for the detection of this chromosomal translocation. The availability of new and more efficient DNA polymerases that allow the amplification of genomic fragments many kilobase-pairs long, makes it possible to identify the t(8;14) in BL cells by long-distance polymerase chain reaction (LD-PCR). We have established a simplified and efficient LD-PCR for the detection of t(8;14)(q24;q32) that relies on the use of one primer specific for MYC exon II combined, in different reactions, with four primers for the IgH locus: three for the constant regions Cmu, Cgamma, and Calpha, and one for the joining region (JH). We first studied seven BL cell lines and optimized LD-PCR reaction for analysis of tumor specimens. Five of seven cell lines were positive for the t(8;14), whereas two lines derived from endemic BL were negative, as expected. Of 15 biopsies obtained from pediatric BL and subsequently analyzed, 13 (87%) were positive for the translocation detected by LD-PCR and showed a product ranging in size from 2.0 to 9.5 kb. Cmu region was involved in 6 cases, Cgamma and Calpha in 2 cases each, and JH in 3 cases. Interestingly, 2 of the tumors positive for JH showed a second, larger PCR product with the Calpha- and Cgamma-specific primer, respectively. We established that our LD-PCR method could detect 10(-3) BL cells within a population of hematopoietic cells lacking the translocation. In conclusion, our LD-PCR method represents a fast, highly sensitive, and specific tool to study sporadic BL and to detect minimal disease and residual disease in patients affected by t(8;14)-positive lymphomas.

MAGE, BAGE and GAGE gene expression in human rhabdomyosarcomas

MAGE, BAGE and GAGE genes encode tumor‐associated antigens that are presented by HLA class I molecules and recognized by CD8+ cytolytic T lymphocytes. These antigens are currently regarded as promising targets for active, specific tumor immunotherapy because MAGE, BAGE and GAGE genes are expressed in many human cancers of different histotype and are silent in normal tissues, with the exception of spermatogonia and placental cells. MAGE, BAGE and GAGE gene expression has been extensively studied in different tumors of adults but is largely unknown in many forms of pediatric solid cancer. Using RT‐PCR, we analyzed MAGE‐1, MAGE‐2, MAGE‐3, MAGE‐4, MAGE‐6, BAGE, GAGE‐1,‐2 or ‐8 and GAGE‐3,‐4,‐5,‐6 or ‐7b gene expression in 31 samples of pediatric rhabdomyosarcoma, the most frequent form of malignant soft tissue tumor in children. MAGE genes were expressed in a substantial proportion of patients (MAGE‐1, 38%; MAGE‐2, 51%; MAGE‐3, 35%; MAGE‐4, 22%; MAGE‐6, 35%), while expression of BAGE (6%); GAGE‐1, GAGE‐2 and GAGE‐8 (9%); and GAGE‐3, GAGE‐4, GAGE‐5, GAGE‐6 and GAGE‐7B (16%) was less frequent. Overall, 58% of tumors expressed at least 1 gene, and 35% expressed 3 or more genes simultaneously. Our data suggest that a subset of rhabdomyosarcoma patients could be eligible for active, specific immunotherapy directed against MAGE, BAGE and GAGE antigens.

Normal and Rearranged PAX3 Expression in Human Rhabdomyosarcoma

PAX3, a member of the PAX-gene family, encodes a nuclear transcription factor that is transiently expressed in the neural tube and in muscle progenitor cells and regulates embryonal development in the mouse. Together with the FKHR gene it is involved in the t(2;13)(q35;q14), a specific translocation associated with alveolar rhabdomyosarcoma (ARMS). As a consequence of the rearrangement two chimeric transcripts originate: FKHR-PAX3 and PAX3-FKHR. We studied the expression of wild type PAX3 and the chimeric transcripts originating from the t(2;13) in a series of 23 rhabdomyosarcomas (RMS) of childhood, by reverse transcriptase polymerase chain reaction (RT-PCR). Wild type PAX3 was detected in 48% of the RMS, whereas another 39% were positive only after nested PCR. Normal adult-skeletal muscle showed a very weak expression of PAX3, but fetal muscle did not express PAX3. PAX3-FKHR was found in 11 of 15 alveolar RMS, 7 of which were positive also for the reciprocal transcript, whereas no RMS expressed FKHR-PAX3 alone. These results confirm that the PAX3-FKHR transcript is specifically associated with the alveolar RMS and that it is a more sensitive marker of the t(2;13) than the reciprocal product FKHR-PAX3. Furthermore, the finding of PAX3 expression with or without PAX3-FKHR transcript in the great majority of the cases raises the question of whether PAX3 expression could play a role in the pathogenesis of RMS.

MYCN expression in human rhabdomyosarcoma cell lines and tumour samples

The MYCN oncogene encodes a phosphoprotein that acts as a transcription factor and is involved in the regulation of cell proliferation and differentiation in normal as well as in cancer cells.MYCN amplification and expression have been reported in various tumours, including neuroblastoma and lung cancer, but little is known about its expression in human rhabdomyosarcoma. MYCN expression and amplification were studied in five alveolar and five embryonal rhabdomyosarcoma cell lines and in 19 tumour biopsies. All the cell lines studied expressed MYCN RNA, as demonstrated by northern blot analysis and RT‐PCR, but the oncogene was amplified in only one. Similarly, MYCN protein was detected in all cell lines by western blot analysis, with higher levels of expression in alveolar than in embryonal rhabdomyosarcoma cells. RT‐PCR analysis of tumour samples demonstrated 18/19 cases positive for MYCN RNA. Although MYCN expression was higher in alveolar than in embryonal rhabdomyosarcoma cell lines, no clear relationship between histology and level of MYCN expression could be established in this tumour series. These data suggest that MYCN expression is a common feature of rhabdomyosarcoma, independent of gene amplification and without a clear relationship with specific histological and clinical features. Copyright

Concomitant Amplif ication and Expression of PAX7-FKHR and MYCN in a Human Rhabdomyosarcoma Cell Line Carrying a Cryptic t(1;13)(p36;q14)

Alveolar rhabdomyosarcoma (ARMS) is associated with the specific chromosomal translocation (2;13)(q35;q14) or its rarer variant t(1;13)(p36;q14), which produces the fusion gene PAX7-FKHR. Here we describe the human cell line RC2, derived from an ARMS, which harbors a cryptic t(1;13)(p36;q14) and concomitantly shows amplification of the PAX7-FKHR fusion gene and of the MYCN oncogene. The t(1;13) and MYCN oncogene were studied by standard cytogenetic analysis and molecular techniques. The reverse transcriptase polymerase chain reaction demonstrated the expression of PAX7-FKHR mRNA in RC2 cells, although karyotype analysis failed to demonstrate a t(1;13)(p36;q14) chromosomal translocation or a derivative 13 chromosome. Double minute chromosomes were detected in all the metaphases studied. Fluorescence in situ hybridization analysis revealed multiple copies of the PAX7-FKHR fusion gene localized exclusively on a subset of double minutes, whereas multiple copies of MYCN were identified on other double minute chromosomes. Southern-blot analysis demonstrated that RC2 cells contain approximately 20 copies of the MYCN oncogene. So far no continuous RMS cell line carrying the t(1;13)(p36;q14) has been described, and PAX7-FKHR and MYCN amplifications have always been reported to occur separately in rhabdomyosarcoma (RMS). The availability of an ARMS cell line that harbors the t(1;13)(p36;q14) constitutes a useful tool for further understanding the role of the PAX7-FKHR fusion gene in RMS oncogenesis and may improve knowledge of the possible relation between PAX7-FKHR and MYCN amplification.

Clinical features of childhood acute myeloid leukaemia with specific gene rearrangements.

Specific gene rearrangements seem to distinguish distinct subsets of acute myeloid leukaemia (AML) with different features and prognosis, and some reports suggest that the epidemiological distribution of AML could vary among countries. To date, cytogenetic examination has been used to study the frequency of these genetic alterations in a large series of children with AML; nevertheless, in a proportion of cases, these gene rearrangements may be cryptic and undetectable by conventional cytogenetic techniques. To evaluate the frequency of specific gene rearrangements, the corresponding clinical morphological features at diagnosis and the potential prognostic impact on patients’ long-term survival, we screened by RT-PCR five different chimaeric transcripts (AML1-ETO, CBFb-MYH11, PML-RARa, MLL-AF9 and BCR-ABL) in a series of 270 Italian children with AML, treated with AIEOP-LAM 87– 92, BFM 83-93 and AIDA protocols between 1988 and 1998, and whose RNA were available and morphology had been centrally reviewed. Of the cases, 45% were positive for one fusion gene. The frequency of AML1-ETO, CBFb-MYH11, PML-RARa and MLLAF9 were 14, 6.3, 20 and 4%, respectively, while BCR-ABL was observed in only two cases. The frequency of AML1-ETO, MLLAF9 and CBFb-MYH11 found was comparable with those of t(8;21), t(9;11) and inv(16) reported previously, suggesting that these gene rearrangements do not have geographic heterogeneity. On the contrary, PML-RARa-positive AML represented 20% of our cohort. This is a higher frequency than that reported in other studies of children from Northern Europe or the United States, but the increased frequency of promyelocytic leukaemia in Italian AML children has already been described. Cytogenetic analysis was successfully carried out in 153 patients, while it failed or was not performed in 117 patients. Abnormalities were identified in 90/153 (59%), the frequency of t(8;21), inv(16), t(15;17) and t(9;11) was 9.8, 2, 17 and 2%, respectively; the 63 remaining cases (41%) had a normal karyotype. In the t(8;21)-positive AML cases, the most common associated alterations involved chromosome 9. Among patients with normal karyotype, RT-PCR identified 17/63 (27%) chimaeric transcripts, and six further cases with translocations undetected by cytogenetics were found among the 43 children reported to have an abnormal karyotype. Considering t(8;21), inv(16), t(15;17) and t(9;11) together, 130/153 (85%) patients, who had a successful analysis were correctly classified cytogenetically. Then, in our experience, cytogenetics showed comparable specificity (100%), but lower sensitivity (67%) than RT-PCR. A general agreement exists in considering RT-PCR more sensitive than conventional cytogenetics in identifying specific gene rearrangements and the fusion genes studied have been described in the absence of identifiable translocations. Nevertheless, the molecular technique is not capable of identifying associated abnormalities. Therefore, since the genetic studies in childhood AML allow for the identification of patient subgroups who probably can benefit from a more tailored therapy and the fusion gene identified can represent a useful target for the minimal residual disease study, ideally all cases should be studied by RT-PCR and cytogenetics: two complementary methods in the genetic characterisation of leukaemia. Clinical data collected included FAB subtypes, gender, age, WBC count at diagnosis and extramedullary involvement, whenever available (Table 1). The low number of M7-AML was due to the scarce number of cells collected in megakaryoblastic leukaemia. The median age at diagnosis for all patients was 7.8 (range 0– 19.5). Cases with AML1-ETO and PML-RARa fusion genes were older patients (median age 8.2 and 9.5 years, respectively), while the median age of patients with CBFb-MYH11 and MLLAF9 was 6.7 and 3.2 years, respectively. A strong, although not exclusive, association between M3, M4 and M5 FAB subgroups and the presence of PML-RARa, CBFb-MYH11 and MLL-AF9 gene rearrangements, respectively, was found. The AML1-ETO-positive cases represented 43% of M2 AML; however, this gene rearrangement was frequently found in M1 patients as well. As a whole, 92% (34/37) of AML1ETO-positive AML were classified as FAB-M1 or FAB-M2. The median WBC count at diagnosis was significantly higher in CBFb-MYH11-positive cases, while PML-RARaand AML1ETO-positive cases had the lowest values. Also, among M2 and M1 plus M2 FAB subgroups this value was significantly lower in AML1-ETO-positive AML than in negatives (16 000/ml vs 30 800/ ml, P1⁄4 0.027; 16 000/ml vs 34 200/ml, P1⁄4 0.0005). Extramedullary disease involved the liver (41%), spleen (37%), node (15%), CNS (8%) and other sites (9%). The FABM3 subtype and the presence of PML-RARa or AML1-ETO fusion genes were significantly associated with low frequency of extramedullary involvement (P1⁄4 0.0002, P1⁄4 0.002 and P1⁄4 0.0047, respectively), while a correlation with the presence of extramedullary disease was found with the presence of MLLAF9, M4 and M5 FAB subgroups and a WBC count at diagnosis higher than 20 000/ml (P1⁄4 0.024, P1⁄4 0.017, P1⁄4 0.018 and P1⁄4 0.046, respectively). CNS involvement was found in 19 patients and, in five of them, was the sole extramedullary site involved. A significant correlation was found only between CNS disease and a WBC count of X50 000/ml at diagnosis (P1⁄4 0.002). In our series, the genetic subgroups showed a characteristic clinical and morphological profile with regard to FAB subtype, age distribution, WBC count at diagnosis, extramedullary Received 6 February 2004; accepted 4 May 2004; Published online 17 June 2004 Correspondence: E Frascella, Pediatric Hematology-Oncology, Department of Pediatrics, University of Padova, via Giustiniani 3, Padova 35128, Italy; Fax: þ39 0498211462; E-mail: emanuela.frascella@unipd.it Leukemia (2004) 18, 1427–1450 & 2004 Nature Publishing Group All rights reserved 0887-6924/04

Expression of type I interferon receptor in solid tumors of childhood.

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