© 1991 Karger AG. Basel 0042-1138/91/0477-0086 S 2.75/0
Urol Int 1991:47(suppl l):86-89
Restriction Fragment Length Polymorphism of the L-myc Gene and Susceptibility to Metastasis in Genitourinary Cancers Y. Kakehi3, Y. Takib, O. Yoshidac a 'D epartm ent of Urology. Kyoto University Faculty of Medicine, and ''Kyoto Municipal Hospital. Japan
Key Words. L-myc oncogene • Restriction fragment length polymorphism • Metastasis, susceptibility to genitourinary cancers
Abstract. We examined Southern blot analysis of genomic DNAs from 70 patients with sporadic renal cell carci noma, using the human L-myc oncogene fragment as a hybridization probe. Our purpose was to study the relationship between the restriction fragment length polymorphism (RFLP) of the L-myc and the frequencies of metastasis. The patients were classified into 3 genotypes according to the polymorphic patterns defined by two alleles (L-L: 17, L-S: 31, S-S: 22). The relative ratios of the 3 genotypes in the renal cancer patients were similar to those seen in healthy Japa nese. However, of 20 patients who exhibited distant metastases at diagnosis, only 2 belonged to the L-L type. The inci dence of distant metastasis in L-L type patients was significantly lower than that in L-S and S-S patients (p =0.068, by Fisher’s exact probability test). These results basically correspond to the previous findings in the lung cancer patients [Kawashima et al.: Proc. natn. Acad. Sci. USA 85: 2353-2356, 1988]. On the other hand, L-myc RFLP analysis in 50 prostatic cancer patients revealed that the incidence of metastasis at diagnosis did not correlate with L-myc genotypes. L-myc RFLP seems to be less promising in prostatic cancer than in lung or kidney cancer.
Not only in some hereditary cancers such as familial polyposis, retinoblastoma and Wilms tumor but also in sporadic cancers, a certain genetic background may relate to susceptibility or progression of the disease. To test this hypothesis, restriction fragment length polymorphism (RFLP) of some cellular oncogenes has been extensively studied. Krontiris et al. [1] reported in 1985 that the unique allelic restriction fragment of the Ha-ras-1 locus was associated with a susceptibility to several types of malignancies. However, other investigators could not find such a correlation. Lee et al. [2] in 1988 studied RFLP of epidermal growth factor receptor gene in both normal and tumor DNAs but its structural alteration was infrequent. In 1988, Kawashima et al. [3] at the National Cancer Center Research Institute, Tokyo, reported that there
was a close correlation of L-myc RFLP with the incidence of metastasis in 51 lung cancer patients. They further con firmed this association in 113 patients with lung cancer of various histological types. The L-myc gene is a third myc-related proto-oncogene which was isolated from a human small cell lung cancer cell line by Nau et al. [4] in 1985. Overexpression and gene amplification of L-myc were found in some human small cell lung cancer tissues. However, little has been clarified about its tissue distribution and biological function, yet.
Materials and Methods High-molecular-weight DNA was extracted from white blood cells or normal kidney tissues. In addition, DNA was also extracted from tumor tissues in 46 renal cancer patients. Southern blot analysis of Lmyc in genomic DNAs digested with EcoRI was done in the same con-
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
Introduction
87
L-myc RFLP in Genitourinary Cancers
1 2 3 4 5 N N T N T N N T
6 7 8 9 10 N T N T N T N T N T
11 12 13 18 N T N T N T N T
IS 16 17 18 19 20 N T N T N T N T N T N T
kb 23.7 — 9 36 6—
^ M
w
m
M l » - *
M
% 0 ......
u
w
W
M
-
V' w
w
-
W
W
v
w W
U
w
M
W
y
m
w
w
— — L
"
—
S
4.4 — 2.3— 2 0— -
21 22 23 2» 25 2« 27 28 29 30 N T N T N T N T N T N N T N T N T N T
*«BB**w— “B w -B -
81 N
T
82 M
N
T
31 32 33 N T N T N T
wC:
—
____
l V
«
I
m
|
a V
___________________________
* -3 5 ~ s
83 M
N
44 45 46 47 48 49 50 mouse N T N T N T N T N T N T N T D N A
T M S’
-
38 3S 38 37 36 39 80 N T N T N T N T N T N T N T
i f
----------------------^
w
«
—
s
-
>
-
W W
u
W
w
W
W W
W W
W
W
V
•j
'
-
-
ditions as described previously [5]. The 1.8-kb length human L-myc Smal-EcoRI fragment was prepared from the recombinant plasmid 6929 which was a kind gift of Dr. J .D. Minna through the Japanese Can cer Research Resources Bank, Tokyo.
Results In order to clarify whether the patients with cancers other than of lung origin are also genetically predisposed to metastasis, we firstly examined L-myc RFLP in renal cell carcinoma (RCC) patients. Figure 1 shows a re presentative Southern blot analysis of L-myc in normal and tumor tissue DNAs isolated from 50 RCC patients. There was no alteration in the RFLP pattern in tumor DNAs. All of the 70 RCC patients analyzed in the present study underwent nephrectomy at the Kyoto University
Hospital or its community hospitals during the period between 1986 and 1989. Twenty (29%) of them, however, had already had distant organ metastases at the time of surgery. The distribution of L-L, L-S and S-S was 17, 31 and 22, which was similar to that in 161 healthy Japanese [Yamada, pers. commun.]. Table 1 shows the relationship between L-myc RFLP and the presence or absence of distant metastasis in 70 RCC patients. The incidence of metastasis identified by the time of nephrectomy in the L-L type patients was sig nificantly lower than that in the L-S and S-S type patients. Table 2 shows the nuclear grade and T stage of primary tumor and the grade of renal vein tumor thrombus in rela tion to L-myc RFLP. In both the 2 L-L type patients with metastasis, the primary tumors were high grade and high stage and both were accompanied by venous tumor thrombi. On the other hand, many of the L-S and S-S type
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
Fig. 1. Representative Southern blot analyses of L-myc gene in normal (N) and tumor (T) tissue DNAs isolated from 50 RCC patients. M = Metastasis.
Kakehi/Taki/Yoshida
88
Table 1. L-myc RFLP and distant métastasés at nephrectomy in 70 RCC patients
Table 4. L-myc RFLP and initial stages in 63 prostatic cancer pa tients Stage
L-myc RFLP L-L
L-S
S-S
Total M-
17 15
31 19
22 16
M+
2
12
6
L-L vs. L-S + S-S: p = 0.0680: L-L vs. L-S: p = 0.0476 (by Fisher's exact probability test). M - = Absence of métastasés: M + = presence of métastasés.
L-myc RFLP L-L
L-S
S-S
A 1(6) A 2 (l) B1 (2) B2 (2) C ( 12) D1 (5) D2 (35)
1 0 1 0 3 0 6
3 0 0 0 6 3 15
2 i i 2 3 2 14
Total (63)
11
27
25
Ref.: L-L:L-S:S-S = 0.209:0.496:0.295 in 161 healthy Japanese. Table 2. Pathological parameters of primary tumors L-S
S-S
14 3(2) 12 5(2) 13 4(2)
21(7) 9(5) 21(5) 9(7) 28(11) 3(1)
18(5) 4(1) 13(2) 9(4) 21(4) 2(2)
Numbers of metastasis cases are given in parentheses.
Table 3. Sites of distant metastasis Patient number
L-myc RFLP
Sites
35 69
L-L L-L
lung, bone lung.bone
8 12 15 20 22 40 42 43 46 50 57 70
L-S L-S L-S L-S L-S L-S L-S L-S L-S L-S L-S L-S
lung lung lung lung, brain bone bone bone, skin lung pleura adrenal glands lung, bone lung
5 18 24 26 33 65
S-S S-S S-S S-S S-S S-S
lung liver lung lung lung lung
patients had distant metastasis in spite of the low risk fac tors of the primary tumors. As for the site of distant métastasés, the lung was most frequently involved (14 cases), bone was second in frequency (table 3). Next, we analyzed L-myc RFLP in leukocyte DNAs isolated from 63 prostatic cancer (PC) patients during the period between May and November 1989. Fifty-seven patients had stage A2-D2 disease at initial admission, and were still under treatment during that period. Six stage A 1 patients examined are alive now without disease progres sion. Table 4 shows the L-myc RFLP in 63 PC patients with respect to the initial clinical stages. All stage D2 patients had bone métastasés. There was no significant association of L-myc RFLP with the incidence of meta static disease as was found in lung or kidney cancer patients. However, the relative ratio of L-L:S-S in PC patients of all stages or DI plus D2 stages was much smaller than that expected from the distribution of L-myc RFLP in 161 healthy Japanese.
Discussion Correlation of L-myc RFLP with susceptibility to metastasis was identified in 70 RCC patients, which was similar, though not as strong, to that observed in the lung cancer study. On the other hand, correlation of L-myc with metastasis was weak in PC patients. L-myc RFLP can be a useful DNA marker to predict the prognosis of RCC patients, although its biological basis remains to be eluci dated.
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
Grade 1-2 Grade 3 -4 pT stage 1-2 pT stage 3 -4 Tumor thrombus Tumor thrombus +
L-L
L-myc RFLP in Genitourinary Cancers
1
2
3
Krontiris, T .G .; DiMartino, N .A.; Colb, M.; Parkinson. D.R.: U nique allelic restriction fragments of the Ha-ras locus in leukocyte and tumor DNAs of cancer patients. Nature 313: 369-374 (1985). Lee, J.S.; Ro, J.S.; Eisbruch, A.; Shtalrid. M ; Ferrell. R .E.; Gutterman, J.U .; Blick, M.: Multiple restriction fragment length poly morphisms of the human epidermal growth factor receptor gene. Cancer Res. 48: 4045-4048 (1988). Kawashima, K.; Shikama. H.; Imoto. K.; lzawa. M.; Naruke, T.; Okabayashi. K.; Nishimura. S.: Close correlation between restric tion fragment length polymorphism of L-myc gene and metastasis of human lung cancer to the lymph node and other organs. Proc. natn. Acad. Sci. USA 85: 2353-2356 (1988).
4
5
Nau, M.M.; Brooks. B.J.; Batley, J.; Sausville, E .; Gazdar. A .F.; Kirsch, I.R .; McBride, O.W .; Bertness, V.; Hollis, G .F.; Minna, J.D .: L-myc, a new myc-rclatcd gene amplified and expressed in human small cell lung cancer. Nature 318: 69-73 (1985). Kakchi, Y.; Yoshida, O.: Restriction fragment length polymor phism of the L-myc gene and susceptibility to metastasis in renal cancer patients. Int. J. Cancer 43: 391-394 (1989).
Yoshiyuki Kakehi Department of Urology Kyoto University Faculty of Medicine Kyoto (Japan)
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
References
89
Urol Int 1991:47(suppl l):86-89
Restriction Fragment Length Polymorphism of the L-myc Gene and Susceptibility to Metastasis in Genitourinary Cancers Y. Kakehi3, Y. Takib, O. Yoshidac a 'D epartm ent of Urology. Kyoto University Faculty of Medicine, and ''Kyoto Municipal Hospital. Japan
Key Words. L-myc oncogene • Restriction fragment length polymorphism • Metastasis, susceptibility to genitourinary cancers
Abstract. We examined Southern blot analysis of genomic DNAs from 70 patients with sporadic renal cell carci noma, using the human L-myc oncogene fragment as a hybridization probe. Our purpose was to study the relationship between the restriction fragment length polymorphism (RFLP) of the L-myc and the frequencies of metastasis. The patients were classified into 3 genotypes according to the polymorphic patterns defined by two alleles (L-L: 17, L-S: 31, S-S: 22). The relative ratios of the 3 genotypes in the renal cancer patients were similar to those seen in healthy Japa nese. However, of 20 patients who exhibited distant metastases at diagnosis, only 2 belonged to the L-L type. The inci dence of distant metastasis in L-L type patients was significantly lower than that in L-S and S-S patients (p =0.068, by Fisher’s exact probability test). These results basically correspond to the previous findings in the lung cancer patients [Kawashima et al.: Proc. natn. Acad. Sci. USA 85: 2353-2356, 1988]. On the other hand, L-myc RFLP analysis in 50 prostatic cancer patients revealed that the incidence of metastasis at diagnosis did not correlate with L-myc genotypes. L-myc RFLP seems to be less promising in prostatic cancer than in lung or kidney cancer.
Not only in some hereditary cancers such as familial polyposis, retinoblastoma and Wilms tumor but also in sporadic cancers, a certain genetic background may relate to susceptibility or progression of the disease. To test this hypothesis, restriction fragment length polymorphism (RFLP) of some cellular oncogenes has been extensively studied. Krontiris et al. [1] reported in 1985 that the unique allelic restriction fragment of the Ha-ras-1 locus was associated with a susceptibility to several types of malignancies. However, other investigators could not find such a correlation. Lee et al. [2] in 1988 studied RFLP of epidermal growth factor receptor gene in both normal and tumor DNAs but its structural alteration was infrequent. In 1988, Kawashima et al. [3] at the National Cancer Center Research Institute, Tokyo, reported that there
was a close correlation of L-myc RFLP with the incidence of metastasis in 51 lung cancer patients. They further con firmed this association in 113 patients with lung cancer of various histological types. The L-myc gene is a third myc-related proto-oncogene which was isolated from a human small cell lung cancer cell line by Nau et al. [4] in 1985. Overexpression and gene amplification of L-myc were found in some human small cell lung cancer tissues. However, little has been clarified about its tissue distribution and biological function, yet.
Materials and Methods High-molecular-weight DNA was extracted from white blood cells or normal kidney tissues. In addition, DNA was also extracted from tumor tissues in 46 renal cancer patients. Southern blot analysis of Lmyc in genomic DNAs digested with EcoRI was done in the same con-
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
Introduction
87
L-myc RFLP in Genitourinary Cancers
1 2 3 4 5 N N T N T N N T
6 7 8 9 10 N T N T N T N T N T
11 12 13 18 N T N T N T N T
IS 16 17 18 19 20 N T N T N T N T N T N T
kb 23.7 — 9 36 6—
^ M
w
m
M l » - *
M
% 0 ......
u
w
W
M
-
V' w
w
-
W
W
v
w W
U
w
M
W
y
m
w
w
— — L
"
—
S
4.4 — 2.3— 2 0— -
21 22 23 2» 25 2« 27 28 29 30 N T N T N T N T N T N N T N T N T N T
*«BB**w— “B w -B -
81 N
T
82 M
N
T
31 32 33 N T N T N T
wC:
—
____
l V
«
I
m
|
a V
___________________________
* -3 5 ~ s
83 M
N
44 45 46 47 48 49 50 mouse N T N T N T N T N T N T N T D N A
T M S’
-
38 3S 38 37 36 39 80 N T N T N T N T N T N T N T
i f
----------------------^
w
«
—
s
-
>
-
W W
u
W
w
W
W W
W W
W
W
V
•j
'
-
-
ditions as described previously [5]. The 1.8-kb length human L-myc Smal-EcoRI fragment was prepared from the recombinant plasmid 6929 which was a kind gift of Dr. J .D. Minna through the Japanese Can cer Research Resources Bank, Tokyo.
Results In order to clarify whether the patients with cancers other than of lung origin are also genetically predisposed to metastasis, we firstly examined L-myc RFLP in renal cell carcinoma (RCC) patients. Figure 1 shows a re presentative Southern blot analysis of L-myc in normal and tumor tissue DNAs isolated from 50 RCC patients. There was no alteration in the RFLP pattern in tumor DNAs. All of the 70 RCC patients analyzed in the present study underwent nephrectomy at the Kyoto University
Hospital or its community hospitals during the period between 1986 and 1989. Twenty (29%) of them, however, had already had distant organ metastases at the time of surgery. The distribution of L-L, L-S and S-S was 17, 31 and 22, which was similar to that in 161 healthy Japanese [Yamada, pers. commun.]. Table 1 shows the relationship between L-myc RFLP and the presence or absence of distant metastasis in 70 RCC patients. The incidence of metastasis identified by the time of nephrectomy in the L-L type patients was sig nificantly lower than that in the L-S and S-S type patients. Table 2 shows the nuclear grade and T stage of primary tumor and the grade of renal vein tumor thrombus in rela tion to L-myc RFLP. In both the 2 L-L type patients with metastasis, the primary tumors were high grade and high stage and both were accompanied by venous tumor thrombi. On the other hand, many of the L-S and S-S type
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
Fig. 1. Representative Southern blot analyses of L-myc gene in normal (N) and tumor (T) tissue DNAs isolated from 50 RCC patients. M = Metastasis.
Kakehi/Taki/Yoshida
88
Table 1. L-myc RFLP and distant métastasés at nephrectomy in 70 RCC patients
Table 4. L-myc RFLP and initial stages in 63 prostatic cancer pa tients Stage
L-myc RFLP L-L
L-S
S-S
Total M-
17 15
31 19
22 16
M+
2
12
6
L-L vs. L-S + S-S: p = 0.0680: L-L vs. L-S: p = 0.0476 (by Fisher's exact probability test). M - = Absence of métastasés: M + = presence of métastasés.
L-myc RFLP L-L
L-S
S-S
A 1(6) A 2 (l) B1 (2) B2 (2) C ( 12) D1 (5) D2 (35)
1 0 1 0 3 0 6
3 0 0 0 6 3 15
2 i i 2 3 2 14
Total (63)
11
27
25
Ref.: L-L:L-S:S-S = 0.209:0.496:0.295 in 161 healthy Japanese. Table 2. Pathological parameters of primary tumors L-S
S-S
14 3(2) 12 5(2) 13 4(2)
21(7) 9(5) 21(5) 9(7) 28(11) 3(1)
18(5) 4(1) 13(2) 9(4) 21(4) 2(2)
Numbers of metastasis cases are given in parentheses.
Table 3. Sites of distant metastasis Patient number
L-myc RFLP
Sites
35 69
L-L L-L
lung, bone lung.bone
8 12 15 20 22 40 42 43 46 50 57 70
L-S L-S L-S L-S L-S L-S L-S L-S L-S L-S L-S L-S
lung lung lung lung, brain bone bone bone, skin lung pleura adrenal glands lung, bone lung
5 18 24 26 33 65
S-S S-S S-S S-S S-S S-S
lung liver lung lung lung lung
patients had distant metastasis in spite of the low risk fac tors of the primary tumors. As for the site of distant métastasés, the lung was most frequently involved (14 cases), bone was second in frequency (table 3). Next, we analyzed L-myc RFLP in leukocyte DNAs isolated from 63 prostatic cancer (PC) patients during the period between May and November 1989. Fifty-seven patients had stage A2-D2 disease at initial admission, and were still under treatment during that period. Six stage A 1 patients examined are alive now without disease progres sion. Table 4 shows the L-myc RFLP in 63 PC patients with respect to the initial clinical stages. All stage D2 patients had bone métastasés. There was no significant association of L-myc RFLP with the incidence of meta static disease as was found in lung or kidney cancer patients. However, the relative ratio of L-L:S-S in PC patients of all stages or DI plus D2 stages was much smaller than that expected from the distribution of L-myc RFLP in 161 healthy Japanese.
Discussion Correlation of L-myc RFLP with susceptibility to metastasis was identified in 70 RCC patients, which was similar, though not as strong, to that observed in the lung cancer study. On the other hand, correlation of L-myc with metastasis was weak in PC patients. L-myc RFLP can be a useful DNA marker to predict the prognosis of RCC patients, although its biological basis remains to be eluci dated.
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
Grade 1-2 Grade 3 -4 pT stage 1-2 pT stage 3 -4 Tumor thrombus Tumor thrombus +
L-L
L-myc RFLP in Genitourinary Cancers
1
2
3
Krontiris, T .G .; DiMartino, N .A.; Colb, M.; Parkinson. D.R.: U nique allelic restriction fragments of the Ha-ras locus in leukocyte and tumor DNAs of cancer patients. Nature 313: 369-374 (1985). Lee, J.S.; Ro, J.S.; Eisbruch, A.; Shtalrid. M ; Ferrell. R .E.; Gutterman, J.U .; Blick, M.: Multiple restriction fragment length poly morphisms of the human epidermal growth factor receptor gene. Cancer Res. 48: 4045-4048 (1988). Kawashima, K.; Shikama. H.; Imoto. K.; lzawa. M.; Naruke, T.; Okabayashi. K.; Nishimura. S.: Close correlation between restric tion fragment length polymorphism of L-myc gene and metastasis of human lung cancer to the lymph node and other organs. Proc. natn. Acad. Sci. USA 85: 2353-2356 (1988).
4
5
Nau, M.M.; Brooks. B.J.; Batley, J.; Sausville, E .; Gazdar. A .F.; Kirsch, I.R .; McBride, O.W .; Bertness, V.; Hollis, G .F.; Minna, J.D .: L-myc, a new myc-rclatcd gene amplified and expressed in human small cell lung cancer. Nature 318: 69-73 (1985). Kakchi, Y.; Yoshida, O.: Restriction fragment length polymor phism of the L-myc gene and susceptibility to metastasis in renal cancer patients. Int. J. Cancer 43: 391-394 (1989).
Yoshiyuki Kakehi Department of Urology Kyoto University Faculty of Medicine Kyoto (Japan)
Downloaded by: University of Queensland 130.102.42.98 - 7/7/2017 11:50:30 AM
References
89
