Int. J. Cancer: 50, 6-9 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the InternationalUnion Against Cancer Publicationde I Union InternationaleContre le Cancer
ASSOCIATION BETWEEN RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF THE L-myc GENE AND LUNG METASTASIS IN HUMAN BREAST CANCER Marie-Helkne CHAMPEME,Ivan BIECHE,Alain LATIL,Kame1 HACENEand Rosette LIDEREAU' Centre RenC Huguenin, 5 rue Gaston Latouche, F-92211 St Cloud, France. EcoRl restriction fragment length polymorphism (RFLP) of the L-rnyc gene was examined in leukocyte DNAs isolated from 381 breast cancer patients. No differences in the patterns of L-rnyc RFLP were found between breast cancer patients and healthy individuals. However, among 97 patients who relapsed, a statistical correlationwas found between L-rnyc RFLP and lung metastases (p < 0.05). These results are in close agreement with previous findings in patients with cancer of the lung, bone or kidney, and suggest that L-myc RFLP may be a useful marker for predicting lung metastasis in some human cancers.
The normal human genome contains a limited number of specific genes (proto-oncogenes, anti-oncogenes, growth factor genes, etc.) that, after activation processes, are more o r less directly involved in the genesis and progression of human malignant tumors (Bishop, 1988). Genetic susceptibility to cancer has been evoked as one of the multiple factors participating in the malignant transformation process (Knudson, 1985), but the specific genes involved have been identified only for some cancers (Friend et al., 1986; Lee et aL, 1987; Nigro et al., 1989; Gessler et al., 1990). In this respect, RFLP analysis is a useful tool for studying the involvement of such genes. The best documented example concerns the Ha-ras locus where rare alleles, detected by cleavage of DNA with MspI/HpaII, are found more frequently in patients with various cancer types than in healthy controls (Krontiris et al., 1985; Lidereau et al., 1986; Hayward et al., 1988a). However, other workers have found no such association between particular Ha-ras alleles and cancer (Thein et al., 1986; Gerhard et al., 1987; Ishikawa et al., 1987; Wyllie et al., 1988). Recently, a TaqI RFLP at the same human proto-oncogene locus was reported to be present at a higher frequency in human malignant melanoma (Radice et al., 1987; Hayward et al., 1989). Other RFLPs specific for a number of different gene loci have been associated with increased risks of various human cancers: TaqI RFLP of the TGFa gene in cutaneous malignant melanoma (Hayward et al., 19886) and EcoRI RFLP of the c-mos locus in breast cancer (Lidereau et al., 1985), intracranial cancer (Diedrich et al., 1988), human myeloid leukemia (Revoltella et al., 1985) and esophageal cancer (Hollstein et al., 1986), in contrast with the results of Chenevix-Trench et al. (1989) in non-Hodgkin's lymphoma patients. EcoRI-restricted human DNAs show fragment length polymorphism of L-myc defined by 2 alleles: 10.0-kb and 6.6-kb fragments (Nau et al., 1985; Bieche et al., 1990). The presence of the S fragment (6.6-kb) is associated with susceptibility to local metastasis in lung cancer (Kdwashima eta/., 1988), distal metastasis in renal cancer (Kakehi and Yoshida, 1989) and osteosarcomas in male patients (Kato et al., 1990). We report here that L-myc RFLP may prove useful in identifying breast cancer patients at a high risk of developing lung metastases. MATERIAL AND METHODS
Leukocytes and DNA analysis Peripheral blood leukocytes were obtained from 381 primary breast cancer patients followed at the Centre RenC
Huguenin, France, between 1978 and 1985. Cells were stored at -70°C until extraction of high-molecular-weight DNA by conventional methods. For each sample, 10 p,g of D N A was digested with EcoRI, fractionated by electrophoresis on an 0.8% agarose gel and transferred to a nylon membrane by standard blotting procedures. The filters were hybridized with "P-labelled human L-myc probe prepared by nick translation. The membranes were then washed and exposed to autoradiography at -70°C for an appropriate period.
Human L-myc probe The L-myc probe, a 1.8-kb SmaI-EcoRI fragment cloned in pJB327, was a kind gift from Dr. J.D. Minna (Nau et al., 1985). Statistical analysis All results obtained in these studies were analyzed for statistical significance by the Chi-square test with Yates' correction for adjustment of the continuity of the Chi-square distribution where appropriate. Differences between the 2 populations were judged significant at a confidence level greater than 95% ( p < 0.05). RESULTS
The EcoRI RFLP, recognized by the 1.8-kb SmaI-EcoRI fragment of the pJB327 plasmid, consists of a 10.0-kb fragment (long fragment, L) and a 6.6-kb fragment (short fragment, S). In order to analyze the allele distribution in our breast cancer population, 381 lymphocyte DNAs from breast cancer patients seen in the Centre Rene Huguenin between 1978 and 1985 were analysed by Southern blot electrophoresis (Fig. 1). Ninety-nine (26.0%) patients were homozygous for the L fragment (L-L type), 86 (22.6%) were homozygous for the S fragment (S-S type) and 196 (51.4%) were heterozygous (L-S type). This allele distribution fits the prediction of HardyWeinberg's law. The frequency (48.3%) of the S allele is in agreement with the results obtained in a normal population (Nau et al., 1985). Therefore, L-myc RFLP is probably unrelated to the incidence of breast cancer. Moreover, L-myc RFLP is not associated with classical prognostic factors, including lymph-node involvement, steroid receptor status and tumor size (results not shown). In an effort to identify a possible correlation between L-myc RFLP and disease progression, we studied the subset of 97 patients among the 381 breast cancer patients who relapsed. The S allele was as frequent as the L allele in this smaller group (52.1% and 47.9%). Unlike lung and renal cancer, in which the S allele is associated with metastases, breast cancer showed no statistically significant relationship between L-myc RFLP and the incidence of relapse. It should, however, be pointed out that the present findings require confirmation in studies involving a larger number of subjects. 'To whom correspondence and reprint requests should be addressed. Received: April 24,1991 and in revised form May 30,1991
7
RFLP OF L - m y c AND BREAST CANCER METASTASES
FIGURE 1- L-myc RFLP. Leukocyte DNAs were digested with EcoRI enzyme, separated by electrophoresis and transferred to nylon membranes. The membranes were then hybridized with the ’*P-labelled human L-rnyc probe. Samples from 9 patients are shown. Sizes fin kilobases) of allelic fragments are indicated on the left. The genotype of patients 1, 2,4,9 and 14 is S-S, that of patients 3,5,7,8, 11, 12, 13 and 15L-S, and tharof patients 6 and 10 L-L.
-
TABLE I - RELATIONSHIP BETWEEN L-myc RFLP AND SITES OF RELAPSE’ IN 97 BREAST CANCER PATIENTS
Type of relapse
Genotypes (number of patients) L-L (24) L-S (45) S-S (28)
Bone
12’
16
9
Lung
1
10
9
Liver
4
8
4
2 10
3 17
3 7
5
15
7
Other sites’ Multiple metastases‘ Recurrences5
p value
NS (x’ = 1.98)‘ D < 0.05 (x’ = 6.31) NS (x’ = 0.15) NS (x’ = 1.85) NS (x’ = 1.38)
‘In 97 breast cancer patients, 144 sites of relapse were detected. Some patients developed more than one event.-’Number of patients.-’Brain, lymph node and akin.-‘At least 2 relapses within a 2-month period, or more than 3 relapses diagnosed in a single patient.-’Patients with recurrences were completely metasta~is-free.-~x’analysis was performed to compare genotype distribution in a specific-relapse-site group of patients with the distrihution in patients who had relapses elsewhere.
However, with regard to the sites of relapse (Table I), we observed a significant difference in the genotype distribution of the L-myc gene only for lung metastasis (x’ = 6.31, df = 2, p < 0.05). As shown in Table 11, one of 24 (4.2%) patients with the L-L genotype developed a lung metastasis, whereas 19 of 73 (26%) patients with S-S or L-S had a lung relapse. Compared to the overall group of patients with S-S and L-S genotypes, those with the L-L genotype showed a significantly lower incidence of lung metastasis (x:~,,, = 4.02, df = 1, p < 0.05). We did not observe such an imbalance in RFLPs of other loci on various chromosomes (eg. c-Ha-ras, c-mos, c-met and TGFa). DISCUSSION
In this study, no significant difference was found in the distribution of the L-rnyc genotypes between breast cancer patients and healthy individuals. Our results thus fail to support the hypothesis that the L-myc locus is involved in a
TABLE I1 - L-myc RFLP CORRELATION WITH LUNG METASTASIS AMONG Y7 BREAST CANCER PATIENTS WHO RELAPSED
Genotypes
L-L L-s s-S
Lung metastasis patient Yes No
1 (4.2%) 10 (22.2%) 9 (32.1%)
23 (95.8%) 35 (77.8%) 19 (67.9%)
~~~~l numbcr of patients
24 45
pvalue
p < 0.05 (XZ‘
Publication of the InternationalUnion Against Cancer Publicationde I Union InternationaleContre le Cancer
ASSOCIATION BETWEEN RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF THE L-myc GENE AND LUNG METASTASIS IN HUMAN BREAST CANCER Marie-Helkne CHAMPEME,Ivan BIECHE,Alain LATIL,Kame1 HACENEand Rosette LIDEREAU' Centre RenC Huguenin, 5 rue Gaston Latouche, F-92211 St Cloud, France. EcoRl restriction fragment length polymorphism (RFLP) of the L-rnyc gene was examined in leukocyte DNAs isolated from 381 breast cancer patients. No differences in the patterns of L-rnyc RFLP were found between breast cancer patients and healthy individuals. However, among 97 patients who relapsed, a statistical correlationwas found between L-rnyc RFLP and lung metastases (p < 0.05). These results are in close agreement with previous findings in patients with cancer of the lung, bone or kidney, and suggest that L-myc RFLP may be a useful marker for predicting lung metastasis in some human cancers.
The normal human genome contains a limited number of specific genes (proto-oncogenes, anti-oncogenes, growth factor genes, etc.) that, after activation processes, are more o r less directly involved in the genesis and progression of human malignant tumors (Bishop, 1988). Genetic susceptibility to cancer has been evoked as one of the multiple factors participating in the malignant transformation process (Knudson, 1985), but the specific genes involved have been identified only for some cancers (Friend et al., 1986; Lee et aL, 1987; Nigro et al., 1989; Gessler et al., 1990). In this respect, RFLP analysis is a useful tool for studying the involvement of such genes. The best documented example concerns the Ha-ras locus where rare alleles, detected by cleavage of DNA with MspI/HpaII, are found more frequently in patients with various cancer types than in healthy controls (Krontiris et al., 1985; Lidereau et al., 1986; Hayward et al., 1988a). However, other workers have found no such association between particular Ha-ras alleles and cancer (Thein et al., 1986; Gerhard et al., 1987; Ishikawa et al., 1987; Wyllie et al., 1988). Recently, a TaqI RFLP at the same human proto-oncogene locus was reported to be present at a higher frequency in human malignant melanoma (Radice et al., 1987; Hayward et al., 1989). Other RFLPs specific for a number of different gene loci have been associated with increased risks of various human cancers: TaqI RFLP of the TGFa gene in cutaneous malignant melanoma (Hayward et al., 19886) and EcoRI RFLP of the c-mos locus in breast cancer (Lidereau et al., 1985), intracranial cancer (Diedrich et al., 1988), human myeloid leukemia (Revoltella et al., 1985) and esophageal cancer (Hollstein et al., 1986), in contrast with the results of Chenevix-Trench et al. (1989) in non-Hodgkin's lymphoma patients. EcoRI-restricted human DNAs show fragment length polymorphism of L-myc defined by 2 alleles: 10.0-kb and 6.6-kb fragments (Nau et al., 1985; Bieche et al., 1990). The presence of the S fragment (6.6-kb) is associated with susceptibility to local metastasis in lung cancer (Kdwashima eta/., 1988), distal metastasis in renal cancer (Kakehi and Yoshida, 1989) and osteosarcomas in male patients (Kato et al., 1990). We report here that L-myc RFLP may prove useful in identifying breast cancer patients at a high risk of developing lung metastases. MATERIAL AND METHODS
Leukocytes and DNA analysis Peripheral blood leukocytes were obtained from 381 primary breast cancer patients followed at the Centre RenC
Huguenin, France, between 1978 and 1985. Cells were stored at -70°C until extraction of high-molecular-weight DNA by conventional methods. For each sample, 10 p,g of D N A was digested with EcoRI, fractionated by electrophoresis on an 0.8% agarose gel and transferred to a nylon membrane by standard blotting procedures. The filters were hybridized with "P-labelled human L-myc probe prepared by nick translation. The membranes were then washed and exposed to autoradiography at -70°C for an appropriate period.
Human L-myc probe The L-myc probe, a 1.8-kb SmaI-EcoRI fragment cloned in pJB327, was a kind gift from Dr. J.D. Minna (Nau et al., 1985). Statistical analysis All results obtained in these studies were analyzed for statistical significance by the Chi-square test with Yates' correction for adjustment of the continuity of the Chi-square distribution where appropriate. Differences between the 2 populations were judged significant at a confidence level greater than 95% ( p < 0.05). RESULTS
The EcoRI RFLP, recognized by the 1.8-kb SmaI-EcoRI fragment of the pJB327 plasmid, consists of a 10.0-kb fragment (long fragment, L) and a 6.6-kb fragment (short fragment, S). In order to analyze the allele distribution in our breast cancer population, 381 lymphocyte DNAs from breast cancer patients seen in the Centre Rene Huguenin between 1978 and 1985 were analysed by Southern blot electrophoresis (Fig. 1). Ninety-nine (26.0%) patients were homozygous for the L fragment (L-L type), 86 (22.6%) were homozygous for the S fragment (S-S type) and 196 (51.4%) were heterozygous (L-S type). This allele distribution fits the prediction of HardyWeinberg's law. The frequency (48.3%) of the S allele is in agreement with the results obtained in a normal population (Nau et al., 1985). Therefore, L-myc RFLP is probably unrelated to the incidence of breast cancer. Moreover, L-myc RFLP is not associated with classical prognostic factors, including lymph-node involvement, steroid receptor status and tumor size (results not shown). In an effort to identify a possible correlation between L-myc RFLP and disease progression, we studied the subset of 97 patients among the 381 breast cancer patients who relapsed. The S allele was as frequent as the L allele in this smaller group (52.1% and 47.9%). Unlike lung and renal cancer, in which the S allele is associated with metastases, breast cancer showed no statistically significant relationship between L-myc RFLP and the incidence of relapse. It should, however, be pointed out that the present findings require confirmation in studies involving a larger number of subjects. 'To whom correspondence and reprint requests should be addressed. Received: April 24,1991 and in revised form May 30,1991
7
RFLP OF L - m y c AND BREAST CANCER METASTASES
FIGURE 1- L-myc RFLP. Leukocyte DNAs were digested with EcoRI enzyme, separated by electrophoresis and transferred to nylon membranes. The membranes were then hybridized with the ’*P-labelled human L-rnyc probe. Samples from 9 patients are shown. Sizes fin kilobases) of allelic fragments are indicated on the left. The genotype of patients 1, 2,4,9 and 14 is S-S, that of patients 3,5,7,8, 11, 12, 13 and 15L-S, and tharof patients 6 and 10 L-L.
-
TABLE I - RELATIONSHIP BETWEEN L-myc RFLP AND SITES OF RELAPSE’ IN 97 BREAST CANCER PATIENTS
Type of relapse
Genotypes (number of patients) L-L (24) L-S (45) S-S (28)
Bone
12’
16
9
Lung
1
10
9
Liver
4
8
4
2 10
3 17
3 7
5
15
7
Other sites’ Multiple metastases‘ Recurrences5
p value
NS (x’ = 1.98)‘ D < 0.05 (x’ = 6.31) NS (x’ = 0.15) NS (x’ = 1.85) NS (x’ = 1.38)
‘In 97 breast cancer patients, 144 sites of relapse were detected. Some patients developed more than one event.-’Number of patients.-’Brain, lymph node and akin.-‘At least 2 relapses within a 2-month period, or more than 3 relapses diagnosed in a single patient.-’Patients with recurrences were completely metasta~is-free.-~x’analysis was performed to compare genotype distribution in a specific-relapse-site group of patients with the distrihution in patients who had relapses elsewhere.
However, with regard to the sites of relapse (Table I), we observed a significant difference in the genotype distribution of the L-myc gene only for lung metastasis (x’ = 6.31, df = 2, p < 0.05). As shown in Table 11, one of 24 (4.2%) patients with the L-L genotype developed a lung metastasis, whereas 19 of 73 (26%) patients with S-S or L-S had a lung relapse. Compared to the overall group of patients with S-S and L-S genotypes, those with the L-L genotype showed a significantly lower incidence of lung metastasis (x:~,,, = 4.02, df = 1, p < 0.05). We did not observe such an imbalance in RFLPs of other loci on various chromosomes (eg. c-Ha-ras, c-mos, c-met and TGFa). DISCUSSION
In this study, no significant difference was found in the distribution of the L-rnyc genotypes between breast cancer patients and healthy individuals. Our results thus fail to support the hypothesis that the L-myc locus is involved in a
TABLE I1 - L-myc RFLP CORRELATION WITH LUNG METASTASIS AMONG Y7 BREAST CANCER PATIENTS WHO RELAPSED
Genotypes
L-L L-s s-S
Lung metastasis patient Yes No
1 (4.2%) 10 (22.2%) 9 (32.1%)
23 (95.8%) 35 (77.8%) 19 (67.9%)
~~~~l numbcr of patients
24 45
pvalue
p < 0.05 (XZ‘
