Clinical and Genetic Studies of Renal Cell Carcinomas in a Family with a Constitutional Chromosome 3; 8 Translocation: Genetics of Familial Renal Carcinoma

Methods

Patients

Our initial evaluation and treatment of affected family members in 1977 resulted in complete remission of renal cancer in five patients [3]. Follow-up of these patients included yearly or more frequent physical examinations, renal function studies, abdominal ultrasound studies, radiographs, and urinalyses. New lesions were further evaluated with computed tomographic scan, needle biopsy, and, if indicated, surgical excision. These follow-up studies identified recurrences of renal carcinoma in all five previously affected family members, and tissue specimens were obtained from three of them (Figure 1). Surgery yielded sterile non-tumor tissue and two renal tumor nodules from Patient III-1 and one pair of specimens (tumor and non-tumor) from III-14. Paired tissues were obtained at autopsy of Patient III-13, but tumor specimens were not available from Patients II-4 and III-2.

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Figure 1. Four generations (I to IV) with the translocation (T) are shown. Squares indicate men, circles indicate women, and diamonds indicate persons of either gender. Clear-cell carcinoma of the kidney had developed in 10 relatives, whose symbol is crosshatched if cancer had been the cause of death before 1979 and blackened if cancer recurred between 1979 and 1990. Persons who have died have a diagonal line across the symbol. During the follow-up period, all five patients (Patients II-4, III-1, 2, 13, and 14) who had been in remission of renal carcinoma have developed new foci of the neoplasm. Three of them have died of the cancer. In addition, two of these patients (Patients III-13 and 14) have developed thyroid carcinoma (C). Abridged pedigree of the family with a constitutional 3; 8 translocation.

Laboratory Studies

Karyotyping was done on the surgical specimens, and DNA from the tumor and non-tumor specimens was prepared for molecular genetic analyses. Previous studies have shown that translocation carriers in our family have one normal chromosome 3, a normal 8, a derivative 3, and a derivative 8 [3, 4]. The derivative chromosome 8 comprises the entire 8p, the portion of 8q (long arm) centromeric to 8q24 and the translocated chromosome 3 segment from 3p14 to the telomere. The derivative 3 contains the remainder of the rearranged chromosomes 3 and 8 (3q, proximal 3p, and distal 8q). To identify additional chromosome changes in the renal cancers of t(3; 8) carriers, metaphases were prepared from the larger tumor nodule of Patient III-1 and the sole tumor nodule of Patient III-14, using previously described methods [5, 15]. Each well-banded tumor metaphase and 10 metaphase cells of the normal kidney were analyzed.

Southern blot analyses were done on the three pairs of tumor and non-tumor specimens from Patients III-1 (two tumor nodules) and III-14 (one nodule), using standard techniques [5, 6, 15, 16]. High-molecular-weight DNA was extracted directly from these tissues. In addition, part of the larger tumor from patient III-1 was cultured before DNA was extracted in an attempt to reduce admixture of tumor cells with normal cells [16]. The pairs of tumor and non-tumor samples were hybridized to eight DNA probes that can identify restriction fragment length polymorphisms (RFLPs) on chromosome 3, and to one probe for an RFLP on chromosome 8 [5, 6, 17]. Restriction fragment length polymorphisms are sites of variation in DNA sequence among individuals that can be exploited to identify allele loss in tumor DNA when compared with corresponding non-tumor DNA. In family members who carry the 3; 8 translocation, the derivative chromosome 8 bears five translocated 3p RFLPs (D3S2, D3F15S2 [formerly DNF15S2], THRB, RAF1 and D3S18), as well as the D8S17 locus on chromosome 8 [17-19]. The other RFLPs (D3S4, D3S30, and D3S3) are on the derivative chromosome 3 [17-19].

The partially degraded DNA of patient III-13 was unsuitable for Southern blotting. Instead, closely spaced oligonucleotide primers were generated that encompass polymorphic sites at three loci (D3S12, MYL3, and D3S14) on chromosome 3. In Patient III-13, who carries the 3; 8 translocation, D3S12 and MYL3 are on the derivative chromosome 8 and D3S14 is on the derivative 3 (unpublished data). These loci were amplified using the polymerase chain reaction, and alleles were resolved by their DNA melting behavior in denaturing gradient gel electrophoresis [20, 21]. To quantitate allelic dosage, the denaturing gradient gels were scanned with a Betascope Model 603 Blot Analyzer (Betagen Corporation, Waltham, Massachusetts) [22]. The digital output was quantified using the Betagen image analysis program, “ANALYZE” [23].

Parental origin of deleted alleles in the tumors of Patients III-1 and III-14 was determined by genetic analyses of blood specimens obtained from their surviving parents, spouses, and offspring. Genotype, karyotype, and inheritance data were combined to determine whether the rearranged or non-rearranged chromosome 3 was altered in the renal cancer cells.

Results

Clinical Observations

Patient III-1. At age 41 in 1977, this woman was found by screening to have asymptomatic renal masses that were found to be renal cell carcinoma at bilateral partial nephrectomies. She remained free of tumor for 12 years but developed a mass in the left kidney remnant in 1989. A left total nephrectomy revealed two new foci of renal carcinoma. More than 2 years after surgery, she is in a complete clinical remission with a serum creatinine of 133 µmol/L (1.5 mg/dL).

Patient III-14. This woman had bilateral partial nephrectomies for multifocal renal cell carcinoma detected by screening at age 39 in 1977. Three years later the patient had a total thyroidectomy for a localized papillary thyroid carcinoma. In 1989 she was found to have two solid masses in the right kidney, and a right nephrectomy showed renal carcinomas. Additionally, five minute renal tumors were found on examination of 3- to 4-mm sections of the entire nephrectomy specimen. Presently, the patient has hypertension that is well controlled with medications, and her serum creatinine is 168 µmol/L (1.9 mg/dL).

Patient III-13. At age 44 in 1977, this woman had a left total nephrectomy and right partial nephrectomy after screening studies showed probable bilateral renal carcinoma. She had a second partial right nephrectomy in 1978 to excise three additional foci of renal cancer. Five years later, she had a total thyroidectomy and postoperative radiotherapy to the neck for multifocal papillary and undifferentiated thyroid carcinoma. In 1984, the right kidney remnant was removed because of multifocal renal carcinoma and adenomas. Ambulatory peritoneal dialysis was started to manage her anephric state. In 1986, metastatic renal carcinoma was diagnosed in a biopsy of a right supraclavicular mass. She developed paraplegia due to spinal cord compression by metastatic disease and died in 1989. An autopsy showed widespread renal cell carcinoma.

Patient II-4. This woman had a right total nephrectomy for multifocal renal carcinoma at age 46 in 1966. She remained clinically free of tumor for 16 years. In 1982, she was found to have multiple tumors in the left kidney and a metastasis in the lung. She died several months later, and an autopsy confirmed metastases from recurrent renal carcinoma.

Patient III-2. The proband in this family developed bilateral renal cell carcinoma in 1976 at age 37. He had a left total nephrectomy and right partial nephrectomy with transplantation of the kidney remnant into the iliac fossa. He remained free of tumor until 1988 when mediastinal adenopathy was observed. Mediastinoscopy and biopsy revealed unresectable metastatic renal carcinoma. Computed tomography did not detect any tumor in the transplanted remnant of the right kidney. He received bronchoscopic laser therapy for several episodes of hemoptysis, and subsequently, irradiation for brain metastases. He died in 1990, and an autopsy was refused.

Among previously unaffected 3; 8 translocation carriers, follow-up studies of a 44-year-old man revealed a solitary renal mass that on needle aspiration, proved to be a benign renal cyst. He and three other carriers, ages 41 to 46 years, have remained free of cancer. Children and younger adults in the family who carry the t(3; 8) have not reached ages associated with a substantial risk for renal carcinoma.

Laboratory Studies

Karyotypes of normal kidney cells of Patient III-1 show, as reported previously, the inherited 3; 8 translocation [3]. A total of 176 cells from her larger (4 cm³) tumor were analyzed. Six of these cells were karyotyped, 39 were completely analyzed, 49 were partially analyzed, and 82 cells were only examined for chromosome number. None of the sufficiently analyzed cells had a normal karyotype, and the mode was 43 chromosomes. Only one tumor clone was found (Figure 2). The tumor cells displayed clonal loss of the derivative chromosome 8, which carries the segment of chromosome 3 telomeric to the 3p14 translocation breakpoint. The non-rearranged chromosomes 3 and 8 and the derivative chromosome 3 appeared to be unchanged. The cancer cells showed other clonal changes, including translocations or deletions that involved chromosomes 13, 14, and 16. Karyotype of a 6-mm tumor nodule from Patient III-14 showed only the clonal loss of the entire derivative 8 chromosome.

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Figure 2. The tumor cells show clonal loss of the entire derivative chromosome 8. The karyotype is as follows: 43,XX,-3,+der (3)t(3;8)t(p14.2;q24), −8,−13,−14,−16,+der(16)t(13;?;16) (pterq>24::?::>13q1-g13qter). Representative karyotype of renal carcinoma cells of patient III-1.

The four pairs of tumor and non-tumor DNA (two from Patient III-1, and one each from Patients III-13 and III-14) confirm and extend the karyotype evidence for loss of the derivative chromosome 8. Southern blot analyses of the tumor DNA of Patients III-1 and III-14 reveal loss of one allele of each informative locus on the derivative chromosome 8 (Table 1 and Figure 3). Allelic loss in both renal cancers of Patient III-1 was detected at four loci (D3S2, THRB, RAF1, and D8S17) on her derivative chromosome 8. The tumor of Patient III-14 had lost an allele at three loci (D3F15S2, D3S18, and D8S17) on her derivative chromosome 8. By polymerase chain reaction analysis, Patients III-13, III-1, and III-14 showed allelic loss at two informative loci on their derivative 8 (D3S12 and MYL3). The findings suggest loss of the entire derivative chromosome 8 in the renal cancer specimens of all three patients. No tumor showed allelic loss at loci on the derivative chromosome 3 (D3S4, D3S30, D3S3, and D3S14), which is consistent with the cytogenetic finding of an intact derivative chromosome 3.

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Figure 3. Her normal (N) cells are heterozygous . The direct extract of her tumor (TUa) shows a diminished signal for allele 1, presumably due to the presence of admixed nontumor cells. Allele 1 is undetectable after the tumor specimen (TUb) was cultured for several days to remove nontumor components. Renal cancer cells of Patient III-1 show allelic loss at THRB restriction site on the derivative chromosome 8.[1, 2]

To further confirm loss of the derivative 8, the deleted alleles in the tumors of Patients III-1 and III-14 were shown to be on this rearranged chromosome. Patient III-1 is heterozygous [1, 2] at the D3S2 locus on the derivative chromosome 8, and both her tumors have lost allele 2 (see Table 1). Her unaffected mother is homozygous for allele 1 [1, 1] at D3S2, indicating that the deleted allele 2 was inherited from her father (see II-1 on Figure 1), who transmitted the derivative chromosome 8. Likewise, Patient III-14 is heterozygous [1, 2] for D3S15S2 on the derivative chromosome 8, and her tumor has lost allele 1. All four of her children (see IV 1 to 4 on Figure 1) carry the 3; 8 translocation. Three of them are also homozygous [1, 1] for D3S15S2; her husband and fourth child are heterozygous [1, 2]. Therefore, the deleted allele 1 in the renal cancer of III-14 had been co-inherited with the derivative chromosome 8.

Discussion

Tumor suppressor genes are one of two major classes of genes (the other class being oncogenes) involved in transforming normal cells into cancer cells [14, 24]. The hallmark of a tumor suppressor gene is loss of its function in a tumor cell, usually as a result of a point mutation, gene rearrangement, or chromosomal deletion [13, 14]. Thousands of genes are deleted in a typical human tumor, and isolation of tumor suppressor genes has been difficult. In nearly all renal clear-cell carcinoma specimens, cytogenetic and molecular genetic studies have revealed chromosome 3p deletions [5-11, 18]. Loss of 3p alleles occurs in small renal tumors, such as the 6-mm tumor nodule in Patient III-14, suggesting that the deletion can be a relatively early event in renal tumorigenesis [18]. Gene mapping studies of renal cancers have found several non-overlapping regions of chromosome 3p deletions, each of which might contain a renal cancer suppressor gene [11, 18, 25, 26]. In chromosome transfer experiments, the malignant phenotype can be suppressed by inserting a normal chromosome 3 into human renal cancer cell lines that are missing a chromosome 3 [27].

Knowledge of tumor suppressor genes (Rb, WT1, NF1, APC, and p53) has been gained through studies of rare families and individuals with an inherited mutation, particularly a cytogenetically visible deletion or translocation [14]. In our family, all evaluable patients with renal cancer have a constitutional 3; 8 translocation that might have disrupted a renal cancer suppressor gene at the 3p14 breakpoint [5-12, 18]. If so, the translocation provides a landmark for cloning this gene. Two other patients have been reported to develop renal carcinoma in association with constitutional translocations at other chromosome 3 breakpoints [28, 29]. These breakpoints might also alter genes directly involved in renal tumorigenesis. Alternatively, the rearrangements might produce unstable derivative chromosomes that are prone to loss.

The renal cancers in three of our patients (Patients III-1, III-13, and III-14) and in the two previously reported patients with constitutional translocations, have in common a second chromosomal mutation—loss of the derivative chromosome containing the distal portion of 3p [28, 29]. This finding was not predicted by the classical Knudson model for tumor suppressor genes, in which the tumor cells of our patients were expected to lose the corresponding second (normal) allele on the non-rearranged chromosome 3 [13, 14]. A simple explanation is that undetected point mutations or minute deletions have occurred at loci in the normal chromosome 3, but more complex alternatives cannot be excluded.

The gene for von Hippel-Lindau disease is on chromosome 3p, and linked to the RAF locus on 3p25 [15, 30, 31]. The renal cancers of some of these patients show deletion of the normal second von Hippel-Lindau allele on chromosome 3p [29]. Members of our family with renal carcinoma show no other features of this autosomal dominant disease, and their cancers have lost the derivative chromosome 8 [17]. However, the multi-step process of renal carcinogenesis in our family and in other patients might involve somatic (acquired) mutations in the von Hippel-Lindau gene and other genes on chromosomes 5q, 6q, 13q, and 17p [10, 12, 18].

This follow-up study shows that the renal cancers in our family tend to recur, even after remissions lasting up to 16 years. One patient had recurrence with metastatic lesions only, whereas the other four had lesions in the kidney remnant that appear to be new foci of renal carcinoma. Many forms of hereditary cancers feature the development of simultaneous and metachronous tumors, including bilateral cancers in paired organs such as kidney, breast, and eye [13, 32]. Bilateral renal carcinomas developed in 7 of 10 cases in our family, exceeding the low frequency of bilateral renal cancers among sporadic cases [11]. During follow-up, two patients (III-13 and III-14) also developed carcinomas of the thyroid without antecedent radiotherapy to the neck, although the association might be due to chance or to increased medical surveillance [33].

The three recent deaths from renal cancer in our family raise the question of bilateral total nephrectomies at initial diagnosis of the cancer. However, removal of both kidneys consigns the patient to lifelong dialysis, which is associated with multiple complications and median survival times of 5 years or less in several published series [34, 35]. Our three patients who died recently had clinical remission of renal cancer for 6 years (Patient III-13), 12 years (Patient III-2), and 16 years (Patient II-4). In Patient III-2, the evidence suggests that metastatic disease stemmed from his original renal cancer and would not have been prevented by total nephrectomies. Another patient (III-13) had all remaining kidney tissue removed 7 years after her initial surgery for renal cancer, but she died 5 years later from metastatic renal carcinoma. Two other family members (Patients III-1 and III-14) are presently in a second complete remission after additional surgery. On balance, we continue to favor complete excision of detectable tumors with preservation of the kidney remnant, which is monitored by computed tomographic scans or magnetic resonance imaging. We also continue to periodically screen younger, unaffected t(3; 8) carriers to seek renal cancers at early stages.

Abbreviation

RFPL: Restriction fragment length polymorphism