Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 613-616
Detailed Deletion Mapping of the Long Arm of Chromosome 6 in
Adult T-Cell Leukemia
By
Yoshihiro Hatta,
Yasuaki Yamada,
Masao Tomonaga,
Isao Miyoshi,
Jonathan W. Said, and
H. Phillip Koeffler
From the Division of Hematology/Oncology, Cedars-Sinai Research
Institute, UCLA School of Medicine, Los Angeles, CA; the Deparment of
Laboratoy Medicine and the Department of Hematology, Nagasaki
University School of Medicine, Nagasaki, Japan; the Department of
Medicine, Kochi Medical School, Kochi, Japan; and the Department of
Pathology, Center for the Health Science, UCLA School of Medicine, Los
Angeles, CA.
 |
ABSTRACT |
Previously, we have found that the loss of heterozygosity (LOH) was
frequently observed on chromosome 6q in acute/lymphoma-type adult
T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for
ATL may be present on chromosome 6q. To further define a region
containing this gene, we performed fine-scale deletional mapping of
chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly
informative microsatellite markers. LOH was found in 9 samples (40.9%)
at 1 or more of the loci examined. Of the 9 samples, 8 shared the same
smallest commonly deleted region flanked by D6S1652 and D6S1644
(6q15-21). The genetic distance between these two loci is approximately
4 cM. These results suggest that a putative tumor-suppressor gene on
chromosome 6q15-21 probably plays a very important role in the
evolution of acute/lymphomatous ATL. Our map provides key information
toward cloning the gene.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
RECENT STUDIES SUGGEST that functional
inactivation of tumor-suppressor genes may play an important role in
the pathogenesis of leukemia1 as well as certain
cancers.2,3 With tumor-suppressor genes located on
autosomes, deletion of the normal copy of the gene that can be
frequently detected by analysis for loss of heterozygosity (LOH) of
closely linked markers allows expression of any recessive mutation in
the other copy. Indeed, tumor-suppressor genes have been identified and
characterized from regions showing frequent LOH in
tumors.2,4,5
Adult T-cell leukemia (ATL) is one of the peripheral T-cell malignant
neoplasms of which human T-cell leukemia virus type I (HTLV-I) is the
etiologic agent. Although the evolution from chronic-type to
acute/lymphomatous ATL is a common clinical feature, the mechanism of
transformation is poorly understood. Recently, substantial evidence has
been acquired for a pathogenetic role of tumor-suppressor genes in the
progression of ATL; altered expression and structural abnormalitites in
the p53,6 p16/INK4A,7-9
p18/INK4C,10 and Rb genes,11 in the evolution
of acute/lymphoma-type ATL have been observed. However, additional
genetic alterations responsible for the transition from chronic to
acute/lymphoma type are probably present because abnormalities of
candidate tumor-suppressor genes were not identified in a sizable
fraction of ATL cases. Furthermore, statistical analysis suggests that
ATL arises after approximately five independent genetic
events.12
Recent evidence obtained by cytogenetic studies indicates that
chromosome 6q is often affected in acute/lymphoma-type
ATL,13,14 suggesting the existence of a putative
tumor-suppressor gene(s) for ATL on this chromosomal arm. However, the
critical region in chromosome 6q has not been mapped. The frequency and
region of the deletions are difficult to estimate by conventional
cytogenetic analysis because small interstitial deletions are beyond
the sensitivity of the technique. We have previously performed
allelotype analysis in acute/lymphomatous ATL and have detected
frequent LOH on the long arm of chromosome 6. To define a small region
on chromosome 6q containing a putative tumor-suppressor gene for ATL,
we performed an intensive deletional map using 24 microsatellite
markers spanning chromosome 6q in 22 paired samples from
acute/lymphoma-type ATL patients.
| |
MATERIALS AND METHODS |
Samples.
Twenty-two paired genomic DNA samples were obtained from the patients
with acute/lymphoma-type ATL after their informed consent. All the ATL
samples were ascertained by determining the monoclonal integration of
the HTLV-I proviral genome. The clinical subtypes of ATL were based on
the diagnostic criteria proposed by the Lymphoma Study Group of
Japan.15 The percentage of contaminating normal cells in
the acute/lymphoma-phase samples was at most 30% and usually less than
10%. The corresponding control DNAs were obtained from either their
peripheral blood after complete remission (n = 17) or during
their chronic phase (n = 5). DNA was extracted by a standard technique
with proteinase K digestion and phenol/chloroform extraction.
Allelic loss analysis.
The LOH analysis was performed by PCR-amplification of microsatellite
sequences as described before.16 The genetic map of chromosome 6 and chromosome 6-specific microsatellite markers, including their primer sequences and sizes used in this study, were
compiled from the Geneton human genetic linkage map.17 Some
markers have been assigned to the same location in a 0 cM cluster.
Primers for polymerase chain reaction (PCR) amplification of
microsatellite markers were obtained from Research Genetics (Huntsville, AL). PCR was performed in a final volume of 20 µL containing 25 ng DNA, 1.5 mmol/L MgCl2, 10 pmol/L of each
of the primers, 2 nmol/L of each of the four deoxyribonucleotide
triphosphates (dNTP; Pharmacia, Stockholm, Sweden), 1 unit of Taq DNA
polymerase (GIBCO-BRL, Gaithersburg, MD), and 2 µCi
32P-labeled deoxycytidine triphosphates (dCTP) (3000 µCi/mmol; New England Nuclear/Dupont, Boston, MA) with specified
buffer provided by the supplier. PCR consisted of 40 seconds at
94°C, 30 seconds at 55° to 57.5°C, and 1 minute at 72°C
for 27 to 33 cycles in a Programmable Thermal Controller (MJ Research
Inc, Water Town, MA) to examine the products in the linear range of
signals. For some of the markers, PCR reaction was performed in a
multiplex fashion to ascertain either LOH or duplication of the region; two primer sets were mixed under the conditions described above. PCR
products were mixed with a formamide gel-loading solution, heat
denatured at 94°C, separated on a denaturing 5% to 8%
polyacrylamide gel containing 8.3 mol/L urea, and visualized by
autoradiography. Allelic losses were defined by visual comparison of
the relative allelic ratios of the normal and tumor samples on the
autoradiographs. In some cases of weak radiographic intensity,
differences in the alleles in the tumor versus control DNA were
analyzed with respect to the number of normal cells compared with
malignant cells in representative slides from the tumors. In such
cases, the ratio of allele intensities was classified as LOH if it
roughly agreed with the percentage of the tumor cells in the sample.
When visible reduction of radiographic signal was equivocal, a
radioanalytic imaging detector (Ambis; Ambis Inc, San Diego, CA) was
used to confirm our interpretation. All positive results were repeated for confirmation.
| |
RESULTS |
We screened 22 paired ATL samples for LOH with a panel of 24 highly
informative microsatellite markers spanning chromosome 6q. All patietns
were informative at multiple loci on chromosome 6q. Allelic loss was
observed in 9 of 22 cases (40.9%): 4 (samples D, H, L, and T) of the
15 acute leukemias and 5 (samples E, F, G, P, and S) of the 7 lymphoma
type. The most frequent LOH (5 of 11 informative cases; 45.5%) was
observed at the D6S1601 locus. Figure 1
shows examples of allele loss.
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| Fig 1.
Representative autoradiographs showing LOH in patients E,
H, and S. Loss of one parental band was observed in the acute/lymphoma
ATL samples (arrows). L, DNA samples isolated from the lymphoma cells;
C, DNA samples isolated from the corresponding normal peripheral
leukocytes after complete remission; A, DNA samples isolated from the
leukemic cells in acute type.
|
|
Figure 2 shows the deletional map on
chromsome 6q as composed from the nine cases that had LOH on the arm.
Of the 9 samples, 8 shared the same smallest consensus region, which
was approximately 4 cM between markers D6S1652 and D6S1644 located at
the 6q15-21 chromosomal band. Allelic loss of the smallest commonly
deleted region on 6q was observed in both acute (3 of 15, 30.0%) and
lymphoma type (5 of 7, 71.4%) of ATL.
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| Fig 2.
Patterns of LOH on chromosome 6q in ATL. The nine samples
that showed LOH at one or more loci are presented. **Represent the
smallest region of shared LOH. A partial ideogram of chromosome 6q and
the relative positions of the markers used in this study are shown to
the left of the diagram.  Represents informative with retention of
both alleles;  , informative with LOH;  , not informative.
|
|
| |
DISCUSSION |
In ATL, chromosomal regions of nonrandom deletions have been identified
by cytogenetics including 6q,13 especially at band 6q21.14 Similarly, we have previously identified by
allelotyping using microsatellite markers that chromosomal arm 6q is
one of the most frequent sites of LOH in acute/lymphoma-type
ATL.18
The aim of the present study was to delineate precisely the critical
region that is deleted on the long arm of chromosome 6 to localize
further the tumor-suppressor gene involved in ATL. To narrow this
region, the LOH on the arm 6q in ATL was mapped using 24 polymorphic
markers. We have found that the frequency of LOH on 6q (40.9%) was
higher than that reported by cytogenetic analysis (23%).14
Thus, cytogenetic studies have probably missed some cases of small
interstitial deletions on 6q. Our study showed that eight of the nine
tumors with interstitial losses or partial losses of chromosome 6q had
a commonly deleted region between D6S1652 and D6S1644 at 6q15-21. The
distance between these two loci corresponds to 4 cM of physical
distance.
From several LOH studies, chromosome 6q appears to be involved in the
pathogenesis of a number of solid tumors including ovarian carcinoma,19-21 breast carcinoma,22,23
malignant melanoma,24-27 renal cell
carcinoma,28 hepatocellular carcinoma,29
salivary gland adenocarcinoma,30 small-cell lung
carcinoma,31 prostate carcinoma,32 and
parathyroid adenoma.33 However, the precise nature of these
molecular deletions has so far not been analyzed in detail. In
hematological malignancies, deletions involving the long arm of
chromosome 6 are observed primarily in lymphoid malignancies, ie, acute
lymphoblastic leukemias (ALL),34, 35 lymphoproliferative
disorders (LPD), and non-Hodgkin's lymphomas (NHL).1
Several commonly deleted regions along 6q have been reported in
lymphoma and lymphoblastic leukemia including 6q12-21,36 6q14-21,37 6q21,38 6q21-22,39,40
6q21-23,41 6q23,38 6q23-24,42
6q23.1-27,37 and 6q25-27.38,41 However, to
date, no altered tumor-suppressor gene responsible for these tumors has
been determined. Cloning of the candidate gene(s) will define whether a
single or multiple tumor-suppressor gene(s) is clustered on 6q and is
commonly involved in these types of tumors.
Deletions of chromosome 6q are correlated with a poor prognosis in
NHL.43 Although all of the patients in our series were not
treated uniformly, we did not find any significant association between
LOH of 6q and the observed proportion of treatment failures probably
because the survival time of all the individuals with acute/lymphoma-type ATL was very short.
Taken together, we have identified a commonly deleted region of LOH on
chromosome 6q15-21 that may play a pivotal role in development of ATL.
Studies are in progress to investigate further this region of interest.
| |
ACKNOWLEDGMENT |
The authors thank Kim Burgin and Marge Goldberg for their excellent
secretarial help.
| |
FOOTNOTES |
Submitted July 13, 1998;
accepted September 14, 1998.
Supported by Concern Foundation and the Parker Hughes Fund. H.P.K. is a
member of the Jonsson Comprehensive Cancer Center and holds the endowed
Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical
Center/UCLA School of Medicine.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Yoshihiro Hatta, MD, First Department of
Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchi,
Itabshi-ku, Tokyo, 173-8610, Japan.
| |
REFERENCES |
1.
Johnsson B, Mertens F, Mitelman F:
Cytogenetic deletion maps of hematologic neoplasms: Circumstantial evidence of tumor suppressor loci.
Genes Chrom Cancer
8:205, 1993[Medline]
[Order article via Infotrieve]
2.
Weinberg RA:
Tumor suppressor genes.
Science
254:1138, 1991[Abstract/Free Full Text]
3.
Knudson AG:
Antioncogene and human cancer.
Proc Natl Acad Sci USA
90:10914, 1993[Abstract/Free Full Text]
4.
Vogelstein B, Fearon ER, Kern SE, Hamilton SR, Preisinger AC, Nakamura Y, White R:
Allelotype of colorectal carcinomas.
Science
244:207, 1989[Abstract/Free Full Text]
5.
Fearson ER, Vogelstein B:
A genetic model for colorectal tumorigenesis.
Cell
61:759, 1990[Medline]
[Order article via Infotrieve]
6.
Sakashita A, Hattori T, Miller CW, Suzushima H, Asou N, Takatsuki K, Koeffler HP:
Mutation of p53 gene in adult T-cell leukemia.
Blood
79:477, 1992[Abstract/Free Full Text]
7.
Hatta Y, Hirama T, Miller CW, Yamada Y, Tomonaga M, Koeffler HP:
Homozygous deletions of the p15 (MTS2) and p16 (CDKN2/MTS1) genes in adult T-cell leukemia (ATL).
Blood
85:2699, 1995[Abstract/Free Full Text]
8.
Uchida T, Kinoshita T, Watanabe T, Nagai H, Murate T, Saito H, Hotta T:
The CDKN2 gene alterations in various types of adult T-cell leukaemia.
Br J Haematol
94:665, 1996[Medline]
[Order article via Infotrieve]
9.
Yamada Y, Hatta Y, Murata K, Kamihira S, Ikeda S, Tsukasaki K, Sugawara K, Hirakata Y, Ogawa S, Hirai H, Koeffler HP, Mine M, Tomonaga M:
Deletions of p15 and/or p16 genes as a poor prognosis factor in adult T-cell leukemia.
J Clin Oncol
15:1778, 1997[Abstract/Free Full Text]
10.
Hatta Y, Spirin K, Tasaka T, Morosetti R, Said JW, Yamada Y, Tomonaga M, Koeffler HP:
Analysis of p18 INK4C in adult T-cell leukaemia (ATL) and non-Hodgkin's lymphoma.
Br J Haematol
99:665, 1997[Medline]
[Order article via Infotrieve]
11.
Hatta Y, Yamada Y, Tomonaga M, Koeffler HP:
Extensive analysis of the retinoblastoma gene in adult T-cell leukemia/lymphoma (ATL).
Leukemia
11:984, 1997[Medline]
[Order article via Infotrieve]
12.
Okamoto T:
Multi-step carcinogenesis model for adult T-cell leukemia.
Jpn J Cancer Res
80:191, 1989[Medline]
[Order article via Infotrieve]
13.
Sadamori N:
Cytogenetic implication in adult T-cell leukemia: A hypothesis of leukemogenesis.
Cancer Genet Cytogenet
51:131, 1991[Medline]
[Order article via Infotrieve]
14.
Kamada N, Sakurai M, Miyamoto K, Sanada I, Sadamori N, Fukuhara S, Abe S, Shiraishi Y, Abe T, Kaneko Y, Shimoyama M:
Chromosome abnormalities in adult T-cell leukemia/lymphoma: A karyotype review committee report.
Cancer Res
52:1481, 1992[Abstract/Free Full Text]
15.
Shimoyama M, Members of the Lymphoma Study Group (1984-87):
Diagnostic criteria and classification of clinical subtypes of adult T-cell leukemia-lymphoma.
Br J Haematol
79:428, 1991[Medline]
[Order article via Infotrieve]
16.
Hatta Y, Takeuchi S, Yokota J, Koeffler HP:
Ovarian cancer has frequent loss of heterozygosity at chromosome 12p12.3-13.1 (region of TEL and Kip1 loci) and chromosome 12q23-ter: Evidence for two new tumor-suppressor genes.
Br J Cancer
75:1256, 1997[Medline]
[Order article via Infotrieve]
17.
Gyapay G, Morissette J, Vignal A, Dib C, Fizames C, Millasseau P, Marc S, Bernardi G, Lathrop M, Weissenbach J:
The 1993-1994 Geneton human genetic linkage map.
Nat Genet
7:246, 1994[Medline]
[Order article via Infotrieve]
18.
Hatta Y, Yamada Y, Tomonaga M, Said JW, Miyoshi I, Koeffler HP:
Allelotype analysis of adult T-cell leukemia.
Blood
92:2113, 1998[Abstract/Free Full Text]
19.
Lee JH, Kavangah JJ, Wildrick DM, Wharton JT, Block M:
Frequent loss of heterozygosity on chromosome 6q, 11, and 17 in human ovarian carcinoma.
Cancer Res
50:2724, 1990[Abstract/Free Full Text]
20.
Saito S, Saito H, Koi S, Sagae S, Kudo R, Saito J, Noda K, Nakamura Y:
Fine-scale deletion mapping of the distal long arm of chromosome 6 in 70 human ovarian cancers.
Cancer Res
52:5815, 1992[Abstract/Free Full Text]
21.
Foulkes WD, Ragoussis J, Stamp GWH, Allan GJ, Trowsdale J:
Frequent loss of heterozygosity on chromosome 6 in ovarian carcinoma.
Br J Cancer
67:551, 1993[Medline]
[Order article via Infotrieve]
22.
Devillee P, van Vliet M, van Sloun P, Kuipers Dijkshoon N, Hermans J, Pearson PL, Cornelisse CJ:
Allelotype of human breast carcinoma: A second major site for loss of heterozygosity is on chromosome 6q.
Oncogene
6:1705, 1991[Medline]
[Order article via Infotrieve]
23.
Theile M, Seitz S, Arnold W, Jandrig B, Frege R, Schlag PM, Haensch W, Winzer K-J, Barett JC, Scherneck S:
A defined chromosome 6q fragment (at D6S310) harbors a putative tumor supressor gene for breast cancner.
Oncogene
13:677, 1996[Medline]
[Order article via Infotrieve]
24.
Trent JM, Thompson FH, Meyskens Jr FL:
Identification of a recurring translocation site involving chromosome 6 in human malignant melanoma.
Cancer Res
49:420, 1989[Abstract/Free Full Text]
25.
Millikin D, Meese E, Vogelstein B, Witkowski C, Trent J:
Loss of heterozygosity for loci on the arm of chromosome 6 in human malignnt melanoma.
Cancer Res
51:5449, 1991[Abstract/Free Full Text]
26.
Ray ME, Su YA, Meltzer PS, Trent J:
Isolation and characterization of genes associated with chromosome-6 mediated tumor suppression in human malignant melanoma.
Oncogene
12:2527, 1996[Medline]
[Order article via Infotrieve]
27.
Healy E, Belgaid CE, Takata M, Vahlquist A, Rehman I, Rigby H, Rees JL:
Allelotypes of primary cutaneous melanoma and benign melanocytic nevi.
Cancer Res
56:589, 1996[Abstract/Free Full Text]
28.
Morita R, Ishikawa J, Tsutsumi M, Hikiji K, Tsukada Y, Kamidono S, Maeda S, Nakamura Y:
Allelotype of renal cell carcinoma.
Cancer Res
51:820, 1991[Abstract/Free Full Text]
29.
De Souza AT, Hankins GR, Washington MK, Fine RL, Orton TC, Jirtle RL:
Frequent loss of heterozygosity on 6q at the mannose 6-phosphate/insulin-like growth factor II receptor locus in human hepatocellular tumors.
Oncogene
10:1725, 1995[Medline]
[Order article via Infotrieve]
30.
Stenman G, Sandros J, Mark J, Edstrom S:
Partial 6q deletion in a human salivary gland adenocarcinoma.
Cancer Genet Cytogenet
39:153, 1989[Medline]
[Order article via Infotrieve]
31.
Merlo A, Gabrielson E, Mabry M, Vollmer R, Baylin SB, Sidransky D:
Homozygous deletion on chromosome 9p and loss of heterozygosity on 9q, 6p, and 6q in primary human small cell lung cancer.
Cancer Res
54:2322, 1994[Abstract/Free Full Text]
32.
Visakorpi T, Kallioniemi AH, Syvänen A-C, Hyytinen ER, Karhu R, Tammela T, Isola JJ, Kallioniemi O-P:
Genetic changes in primary reccurent prostate cancer by comparative genomic hybridization.
Cancer Res
55:342, 1995[Abstract/Free Full Text]
33.
Tahara H, Smith AP, Gaz RD, Cryns VL, Arnold A:
Genomic localization of novel candidate tumor suppressor gene loci in human parathyroid adenomas.
Cancer Res
56:599, 1996[Abstract/Free Full Text]
34.
Hayashi Y, Raimondi SC, Look AT, Behm FG, Kitchingman GR, Pui CH, River GK, Williams DL:
(1990) Abnormalities of the long arm of chromosome 6 in childhood acute lymphoblastic leukemia.
Blood
76:1626, 1990[Abstract/Free Full Text]
35.
Takeuchi S, Bartram CR, Wada M, Reiter A, Hatta Y, Seriu T, Lee E, Miller CW, Miyoshi I, Koeffler HP:
Allelotype analysis of childhood acute lymphoblastic leukemia.
Cancer Res
15:5377, 1995
36.
Guan XY, Horsman D, Zhang HE, Parsa NZ, Meltzer PS, Trent JM:
Localization by chromosome microdissection of a reccurent breakpoint region on chromosome 6 in human B-cell lymphoma.
Blood
88:1418, 1996[Abstract/Free Full Text]
37.
Menasce LP, Orphanos V, Santibanez-Koref M, Boyle JM, Harrison CJ:
Common region of deletion on the long arm of chromosome 6 in non-Hodgkin's lymphoma and acute lymphoblastic leukemia.
Genes Chromosomes Cancer
10:286, 1994[Medline]
[Order article via Infotrieve]
38.
Offit K, Parsa NZ, Gaidano G, Filippa DA, Louie D, Pan D, Jhanwar SC, Dalla-Favera R, Chaganti RSK:
6q deletions define distinct clinico-pathologic subsets of non-Hodgkin's lymphoma.
Blood
82:2157, 1993[Abstract/Free Full Text]
39.
Gerard B, Cave H, Guidal C, Dastugue N, Vilmer E, Grandchamp B:
Deletion of a 6 cM commonly deleted region in childhood acute lymphoblastic leukemia on the 6q chromosomal arm.
Leukemia
11:282, 1997
40.
Takeuchi S, Koike M, Seriu T, Bartram CR, Schrappe M, Reiter A, Park S, Taub HE, Miyoshi I, Koeffler HP:
Frequent loss of heterozygosity on the long arm of chromsomome 6: Identification of two distinct regions of deletion in childhood acute lymphoblastic leukemia.
Cancer Res
58:2618, 1998[Abstract/Free Full Text]
41.
Gaidano G, Hauptschein RS, Parsa NZ, Offit K, Rao PH, Lenoir G, Knowles DM, Chaganti RSK, Dalla-Favera R:
Deletions involving two distinct regions of 6q in B-cell non-Hodgkin lymphoma.
Blood
80:1781, 1992[Abstract/Free Full Text]
42.
Zhang Y, Weber-Matthiesen K, Siebert R, Matthiesen P, Schlegelberger B:
Frequent deletions of 6q23-24 in B-cell non-Hodgkin's lymphomas detected by fluorescence in situ hybridization.
Genes Chromosomes Cancer
18:310, 1997[Medline]
[Order article via Infotrieve]
43.
Offit K, Wong G, Filippa DA, Tao Y, Chaganti RSK:
Cytogenetic analysis of 434 consecutively ascertained specimens of non-Hodgkin's lymphoma: Clinical correlations.
Blood
77:1508, 1991[Abstract/Free Full Text]
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Abstract
Introduction
