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.

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,7-9p18/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 μCi32P-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 1shows examples of allele loss.

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.

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.

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.

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%).14Thus, 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,22,23malignant melanoma,24-27 renal cell carcinoma,28 hepatocellular carcinoma,29salivary 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).1Several commonly deleted regions along 6q have been reported in lymphoma and lymphoblastic leukemia including 6q12-21,366q14-21,37 6q21,38 6q21-22,39,406q21-23,41 6q23,38 6q23-24,426q23.1-27,37 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.

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.

REFERENCES

1
Johnsson
B
Mertens
F
Mitelman
F
Cytogenetic deletion maps of hematologic neoplasms: Circumstantial evidence of tumor suppressor loci.
Genes Chrom Cancer
8
1993
205
2
Weinberg
RA
Tumor suppressor genes.
Science
254
1991
1138
3
Knudson
AG
Antioncogene and human cancer.
Proc Natl Acad Sci USA
90
1993
10914
4
Vogelstein
B
Fearon
ER
Kern
SE
Hamilton
SR
Preisinger
AC
Nakamura
Y
White
R
Allelotype of colorectal carcinomas.
Science
244
1989
207
5
Fearson
ER
Vogelstein
B
A genetic model for colorectal tumorigenesis.
Cell
61
1990
759
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
1992
477
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
1995
2699
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
1996
665
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
1997
1778
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
1997
665
11
Hatta
Y
Yamada
Y
Tomonaga
M
Koeffler
HP
Extensive analysis of the retinoblastoma gene in adult T-cell leukemia/lymphoma (ATL).
Leukemia
11
1997
984
12
Okamoto
T
Multi-step carcinogenesis model for adult T-cell leukemia.
Jpn J Cancer Res
80
1989
191
13
Sadamori
N
Cytogenetic implication in adult T-cell leukemia: A hypothesis of leukemogenesis.
Cancer Genet Cytogenet
51
1991
131
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
1992
1481
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
1991
428
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
1997
1256
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
1994
246
18
Hatta
Y
Yamada
Y
Tomonaga
M
Said
JW
Miyoshi
I
Koeffler
HP
Allelotype analysis of adult T-cell leukemia.
Blood
92
1998
2113
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
1990
2724
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
1992
5815
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
1993
551
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
1991
1705
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
1996
677
24
Trent
JM
Thompson
FH
Meyskens Jr
FL
Identification of a recurring translocation site involving chromosome 6 in human malignant melanoma.
Cancer Res
49
1989
420
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
1991
5449
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
1996
2527
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
1996
589
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
1991
820
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
1995
1725
30
Stenman
G
Sandros
J
Mark
J
Edstrom
S
Partial 6q deletion in a human salivary gland adenocarcinoma.
Cancer Genet Cytogenet
39
1989
153
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
1994
2322
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
1995
342
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
1996
599
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
1990
1626
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
1995
5377
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
1996
1418
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
1994
286
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
1993
2157
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
1997
282
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
1998
2618
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
1992
1781
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
1997
310
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
1991
1508