Xin Mao

Molecular cytogenetic analysis of cutaneous T-cell lymphomas: identification of common genetic alterations in Sézary syndrome and mycosis fungoides

Background Data on genome‐wide surveys for chromosome aberrations in primary cutaneous T‐cell lymphoma (CTCL) are limited.

Comparative genomic hybridization analysis of primary cutaneous B-cell lymphomas: Identification of common genomic alterations in disease pathogenesis

To investigate genetic alterations in primary cutaneous B‐cell lymphomas (PCBCLs), we have analyzed 29 cases of PCBCL. Comparative genomic hybridization showed chromosome imbalances (CIs) in 12 cases (41%). The mean number of CIs per sample was 2.05 ± 2.97, with gains (1.48 ± 2.38) more frequent than losses (0.56 ± 1.40). The common regions of gains were 18/18q (50%), 7/7p (42%), 3/3q (33%), 20 (33%), 1p (25%), 12/12q (25%), and 13/13q (25%), whereas loss of 6q was frequent (42%). Among the different subsets of PCBCLs, CI was seen in 50% of diffuse large‐cell lymphomas (DLCLs), 33% of marginal zone lymphomas, and 8% of follicle center cell lymphomas and unclassified lymphomas. A similar pattern of CI was observed in these lymphomas, but loss of 6q and gains of 3/3q were present only in DLCLs. Microarray‐based genomic analysis of four DLCL cases identified oncogene gains of SAS/CDK4 (12q13.3) in three cases and MYCL1 (1p34.3), MYC (8q24), FGFR2 (10q26), BCL2 (18q21.3), CSE1L (20q13), and PDGFB (22q12–13) in two cases, whereas losses of AKT1 (14q32.3), IGFR1 (15q25–26), and JUNB (19p13.2) were identified in three cases, and losses of FGR (1p36), ESR (6q25.1), ABL1 (9q34.1), TOP2A (17q21–22), ERBB2 (17q21.2), CCNE1 (19q13.1), and BCR (22q11) were each identified in two cases. In addition, real‐time–polymerase chain reaction detected amplification of BCL2 in 5 of 29 cases. These findings suggest that there are complex but consistent genetic alterations associated with the pathogenesis of PCBCLs.

Molecular cytogenetic characterization of Sézary syndrome.

Sézary syndrome (SS) is a rare form of erythrodermic cutaneous T‐cell lymphoma with hematological involvement and a poor prognosis. At present little is known about the molecular pathogenesis of this malignancy. To address this issue, we analyzed 28 SS cases through the use of molecular cytogenetic techniques. Conventional cytogenetic analysis showed 12 of 28 cases with clonal chromosome abnormalities (43%). Seven cases had aberrations affecting chromosomes 1 and 17; five demonstrated rearrangement of chromosomes 10 and 14; four presented with an abnormality of 6q. Multiplex‐fluorescence in situ hybridization (M‐FISH) revealed complex karyotypes in 6 of 17 cases (35%), and recurrent der(1)t(1;10)(p2;q2) and der(14)t(14;15)(q;q?) translocations were each identified in two cases, and confirmed by dual‐color FISH. There was an overall difference in the incidence of clonal abnormalities detected by G‐banded karyotyping and M‐FISH. In addition, comparative genomic hybridization studies revealed chromosome imbalances (CIs) in 9 of 20 cases (45%), with a mean DNA copy number change per sample of 1.95 ± 2.74, and losses (mean: 1.25 ± 1.77) more frequent than gains (mean: 0.7 ± 1.26). The most common CIs noted were loss of 1p, followed by losses of 10/10q, 17p, and 19, and gains of 17q and 18. Furthermore, in conjunction with this study a systematic literature review was conducted, which showed a high frequency and consistent pattern of chromosome changes in SS. These findings suggest that chromosomal instability is common in SS, although there are specific chromosomal abnormalities that appear to be characteristic, and the identification of two different recurrent chromosome translocations provides the basis for further studies.

Genetic alterations in primary cutaneous CD30+ anaplastic large cell lymphoma.

Primary cutaneous CD30+ anaplastic large cell lymphoma (C‐ALCL) represents a distinct clinical subtype of CD30+ anaplastic large cell lymphomas. The etiology and underlying molecular pathogenesis of C‐ALCL remain unclear. This study aimed to investigate genetic changes in C‐ALCL. Comparative genomic hybridization (CGH) analysis of 23 DNA samples from 15 C‐ALCL cases identified chromosome imbalances (CI) in 10 samples from eight cases (43%). The mean number of CI per sample was 2.09 ± 3.86, with gains (2.00 ± 3.85) more common than losses (0.09 ± 0.29). The most frequent CI were gains of 1/1p and 5 (50%) and 6, 7, 8/8p, and 19 (38%). Microarray‐based CGH analysis of six DNA samples from five cases with CI revealed genomic imbalances (GI) in all of the cases studied. This included oncogene copy number gains of FGFR1 (8p11) in three cases, and NRAS (1p13.2), MYCN (2p24.1), RAF1 (3p25), CTSB (8p22), FES (15q26.1), and CBFA2 (21q22.3) in two cases. Real‐time PCR analysis of nine DNA samples from eight cases with CI and GI detected amplifications of CTSB and RAF1 in seven cases (88%), REL (2p13p12) and JUNB (19p13.2) in six cases (75%), and MYCN and YES1 (18p11.3) in four cases (50%). Immunohistochemical staining of paraffin sections from six cases demonstrated expression of JUNB protein in five cases and BCL2 in three cases. These results reveal a consistent pattern of genetic alterations in C‐ALCL and provide the molecular basis for further investigation of this disease.

BCL2 and JUNB abnormalities in primary cutaneous lymphomas.

Background  BCL2 is upregulated in nodal and extranodal B‐cell non‐Hodgkins lymphomas, with a consequent antiapoptotic effect. However, loss of BCL2 has also been noted in some malignancies, suggesting a different molecular pathogenesis.

Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma

Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture. Candidate drivers of cSCC development were derived by first defining a set of in vitro cancer genes and then comparing their expression in a range of clinical data sets containing normal skin, cSCC and the benign hyper-proliferative condition psoriasis. A small interfering RNA (siRNA) screen of the resulting 21 upregulated genes has yielded targets capable of reducing xenograft tumor volume in vivo. Small-molecule inhibitors for one target, Polo-like kinase-1 (PLK1), are already in clinical trials for other malignancies, and our data show efficacy in cSCC. Another target, C20orf20, is identified as being overexpressed in cSCC, and siRNA-mediated knockdown induces apoptosis in vitro and reduces tumor growth in vivo. Thus, our approach has shown established and uncharacterized drivers of tumorigenesis with potent efficacy as therapeutic targets for the treatment of cSCC.

A genomic and expression study of AP-1 in primary cutaneous T-cell lymphoma: evidence for dysregulated expression of JUNB and JUND in MF and SS.

Activator protein 1 (AP‐1) consists of a group of transcription factors including the JUN and FOS family proteins with diverse biological functions. This study assessed the genomic and expression status of the AP‐1 transcription factors in primary cutaneous T‐cell lymphoma (CTCL) by using immunohistochemistry (IHC), Affymetrix expression microarray, real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) and fluorescent in situ hybridization (FISH). IHC showed JUNB protein expression in tumor cells from 17 of 33 cases of Sezary syndrome (SS) and JUND protein expression in 16 of 23 mycosis fungoides cases. There was no correlation between JUNB and CD30 expression. However, 7 of 12 JUNB‐positive SS cases expressed both phosphorylated and total extracellular signal‐regulated kinase (ERK) 1/2 mitogen‐activated protein kinase (MAPK) proteins. Expression microarray showed over threefold increased expression of JUNB in three of six SS patients and similar findings were also noted after re‐analysis of previously published data. Real‐time RT‐PCR confirmed the overexpression of JUNB in four SS cases and of JUND in three of four cases. FISH showed increased JUNB copy number in four of seven SS cases. These findings suggest that deregulation of AP‐1 expression in CTCL is the result of aberrant expression of JUNB and possible JUND resulting from genomic amplification and constitutive activation of ERK1/2 MAPK in this type of lymphoma.

Molecular and cytogenetic analysis of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary tumor occurring in the central nervous system of adults. Although progress has been made in clinical management of this tumor, little is known about the molecular defects underlying the initiation and progression of GBM. To address these issues, we have characterized five cases of GBM using cytogenetics, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and direct sequencing. All of these tumors were observed to have clonal chromosome aberrations. Complicated chromosome translocations including der(18)t(2;4;12;18), der(X)t(X;10)(q27.1;p12.1) and der(10)t(10;15)(p11.23;q11.2), and der(1) (:1p31-->1q44::7q11. 3-->7qter) were seen in three tumors. Loss of the CDKN2 gene was noted in four tumors. A gain of copy number of the Cathepsin L gene was seen in two tumors. Amplification of the CDK4, MDM2, and GLI/CHOP genes was noted in two tumors, and amplification of the PDGFR gene was detected in one tumor. Mutation of exon 5 of the TP53 gene was found in three tumors. No mutation of the BCL10 gene was detected in five cases of GBM analyzed, although deletion of chromosome 1p was seen in two tumors. These results provide information for further investigation of GBM.

Allelotype of Uterine leiomyomas

Uterine leiomyomas are the most common benign tumor that arise from smooth muscle cells of the myometrium. Little is known about the etiology and pathogenesis of this tumor. To investigate the molecular pathogenesis of these tumors, we have conducted an allelotype of 102 leiomyomas from 12 patients, using 67 fluorescently-tagged oligonucleotide primers amplifying microsatellite loci covering all autosomes. No areas of the genome showed frequent loss of heterozygosity (LOH); however, the highest rate of LOH (9%) was observed on 7q, consistent with previous cytogenetic observations. Uterine leiomyomas are sometimes multiple. In general, multiplicity of other types of neoplasm is associated with genetic predisposition to the disease. Because multiple tumors were available from each of the 12 patients studied, we looked for evidence of allele-specific LOH, which might indicate the presence of an underlying predisposition gene. However, no evidence for allele-specific LOH was detected, indicating that if cases of multiple uterine leiomyoma are due to an underlying predisposition gene, it is unlikely to be a recessive oncogene.

A case of adult T‐cell leukaemia/lymphoma characterized by multiplex‐fluorescence in situ hybridization, comparative genomic hybridization, fluorescence in situ hybridization and cytogenetics

Adult T‐cell leukaemia/lymphoma (ATLL) is a neoplasm of mature helper (CD4) T lymphocytes. Little is known, however, about the chromosome aberrations associated with the pathogenesis of this malignancy. Using molecular cytogenetic techniques we, therefore, investigated a 44‐year‐old man who had a 7‐year history of ATLL with cutaneous involvement mimicking primary cutaneous T‐cell lymphoma. Conventional cytogenetics revealed gross chromosomal changes with chromosome numbers ranging from 71 to 82. There were structural abnormalities of chromosomes 7 and 9, partial deletions of chromosomes 1, 3, 5 and 6, and loss of chromosomes 2, 4, 9, 11–14, 21 and 22. Multiplex‐fluorescence in situ hybridization (M‐FISH) identified two derivative chromosomes, der(6)t(6;7)(q16;q21) and der(7)t(6;7)(q16;q21)ins(6;12)(q2?;?), and a deletion of chromosome 1p. Conventional FISH confirmed the M‐FISH findings. Comparative genomic hybridization of the blood revealed gains of DNA copy number at 1q12–25, 6p24–25, 9p23, 16p13–q13, 17q11–21, 19p13 and 20q13 and loss at 11p15 while lymph nodes showed gains at 3p22–24, 3q27–29, 7q36 and 15q26 and losses at 2p24–25, 2q37, 10p14–15, 11p15, 13q33–34 and 16p13.3. No DNA copy number changes were seen in a skin lesion. These results show the extent of genetic abnormalities within this malignancy.