Papillary renal cell carcinoma

Most renal cell carcinomas are characterized by constant loss of the 3p13-pter chromosome segmentand afrequentgain ofthe 5q22-qter segment. A comparative histologic and cytogenetic investigation oflarge series ofrenal cell carcinomas now shows thatpurely papillary tumors differfrom the more common nonpapillaryform not only in their morphologic characteristic, but also in karyotype changes observed. All ofthe 1 1 papillary tumors of this studyfailed to show any rearrangement ofthe critical 3p segment, and trisomy of the 5q22-qter segment has never beenfound. The gain ofchromosome 17 was detected only in papillary renal cell carcinomas. Other nonrandom karyotype changes occurred with the same incidence in both types of tumor. Thus, some karyotype alterations in renal cell carcinomas couldperhaps be regarded as a genetic mechanism responsible for the phenotype of conversed tubular cells. (Am J Pathol 1989, 134: 27-34)

Most renal cell carcinomas are characterized by constant loss of the 3p13-pter chromosome segment and afrequentgain ofthe 5q22-qter segment. A comparative histologic and cytogenetic investigation oflarge series ofrenal cell carcinomas now shows that purely papillary tumors differfrom the more common nonpapillaryform not only in their morphologic characteristic, but also in karyotype changes observed. All ofthe 1 1 papillary tumors of this studyfailed to show any rearrangement ofthe critical 3p segment, and trisomy of the 5q22-qter segment has never beenfound. The gain ofchromosome 17 was detected only in papillary renal cell carcinomas. Other nonrandom karyotype changes occurred with the same incidence in both types of tumor. Thus, some karyotype alterations in renal cell carcinomas couldperhaps be regarded as a genetic mechanism responsible for the phenotype of conversed tubular cells. (Am J Pathol 1989, 134: 27-34) Renal Cell Carcinomas (RCC) are known to be characterized by recurrent deletion of the short arm of one of the two homologous chromosomes 3.1-4 The breakpoint was localized in most cases at 3p13-14. This chromosomal finding was corroborated by recent molecular genetical observations concerning the loss of constitutional heterozygosity for 3p specific DNA probes using restriction fragment length polymorphism analysis.5-7 Furthermore, constitutional translocations involving the short arm of chromosome 3 were found in two families with predisposition for bilateral or multifocal RCC development (Kovacs G, unpublished data).e The chromosomal and molecular genetical data on sporadic as well as familial RCCs provide strong evidence that a particular location on 3p should be regarded as the site of tumor suppressor gene responsible for the growth regulation of tubular cells of the kidney.
Papillary RCC is a rare form of human renal cancer and not widely recognized.9 This histologic variant of RCC was also left out of consideration in most previous cytogenetic and moleculargenetic studies. Recently, rearrangement of chromosome 3p was described in six of seven RCCs with clear cell histology, but the two papillary RCCs of this study failed to show any change of chromosome 3.31 analyzed the chromosomal pattern of 100 renal tumors including 4 oncocytomas and found a recurrent loss of 3p segment in 81 tumors. In one of the last cases with multiple papillary tumors could not find any rearrangement of the 3p segment. This finding called attention to the unique karyotype of papillary RCCs. reviewed the histology of 100 cases and attempted to separate the papillary tumors from nonpapillary carcinomas. This paper describes the morphologic characteristics and detailed cytogenetic analysis of 11 RCCs of the papillary variant found in 8 nephrectomy specimens.

Materials and Methods
Tumor specimens were obtained from patients at the time of nephrectomy before any chemotherapy at the Hannover Medical School and Siloah Hospital Hannover. A macroscopically homogeneous area free of fibrosis and necrosis was excised for this study. One part of this tumor sample was fixed in 4% buffered formaldehyde and used for histologic examination as a reference slide for tissue cultures and chromosome analysis. Cell cultures and karyotype analysis were performed as described previously.4 Briefly, tumor as well as normal kidney tissues were minced, washed in culture medium, and the small tissue fragments were then incubated in 0.1 % collagenase (Worthington CSL ll) dissolved in RPMI 1640 medium supplemented with 10% fetal calf serum for 30 to 60 min- utes at 37 C. The small fragments were then washed twice in medium, resuspended, and dispersed vigorously in 3-4 ml medium. The cell clusters released from tumor and normal tissues were maintained in Falcon flasks containing culture medium at 37 C in a humidified atmosphere with 5% C02. Chromosome analysis of tumor and normal kidney cells was carried out on days 3 to 7 of primary cultures by adding 0.1 ,ug/ml colchicine for 30-60 minutes. The cells were then treated with 0.075 M potassium chloride solution for 10-20 minutes and fixed 2 or 3 times with cold methanol-acetic acid (3:1). Chromosome analysis was routinely performed using Gand C-banding techniques, and for identification of the Y chromosome Q-banding technique. Each well-banded metaphase was karyotyped and the stemline and sidelines were determined according to the International System for Human Cytogenetic Nomenclature.11 All of the 100 renal tumors were classified on routine histologic examination. Three to 15 paraffin blocks, according to the size of tumor, were analyzed in each case with exception of the very small tumors. Clear or granular cell, oncocytic or pleomorphic RCCs with solid, acinar, tubular, or cystic pattern and also tumors with occasionally papillary areas were classified among the nonpapillary RCCs. Tumors were considered to be papillary if papillary structures comprised at least 75% of the tumor while the rest consisted of tubulopapillary structures.

Morphologic Study
Among the 100 renal cell tumors, 1 1 with papillary growth pattern were found. The main clinical and morphologic data on these tumors are summarized in Table 1. Seven patients were male, one was female, ranging in age from 47 to 77 years. The tumor size ranged from 7 mm to 19 cm in diameter. In two cases multiple tumor development was noted, one of them (HA245) was described in detail earlier. 11 In the case of HA455, the main 3 cm tumor was encased by a thin fibrous capsule, and was separated in two parts by a fibrous septum (HA455A1 and A2). An additional small subcapsular tumor 7 mm in diameter (HA455B) was located 4 mm from the main tumor, and another subcapsular lesion 8 mm in diameter (HA455C) was located on the other pole of the kidney.
The larger tumors were encased by a fibrous capsule of varying thickness. All but one of the small tumors (<1 cm) were without a sign of encapsulation; only HA245 had a wrinkled, thick fibrous capsule suggesting regressive changes in a previously larger tumor. The most remarkable macroscopic and microscopic feature of papillary RCCs was the presence of massive necrosis. Tumors larger than 3 cm in diameter showed extensive necrosis, and also one of the smaller tumors (HA455A1) was characterized by small microscopic necrosis. Extensive or focal, old or fresh hemorrhage showing scattered inflammatory cells and cholesterol clefts was seen in larger tumors. An accumulation of hemosiderin pigment in macrophages and tumor cells was noted in areas of old hemorrhage.

Chromosome Study
Cytogenetic data of 11 tumors are summarized in Table  2. The tumors HA221 and HA245 have been described elsewhere.4'1 Cells from cultured non-neoplastic kidney tissue showed normal karyotype. The modal chromosome number of the tumors was found to be in the diploid range. Two RCCs showed a pseudodiploid karyotype, two others a hypodiploid one, and in the remaining tumors a hyperdiploid mode was found. Five hyperdiploid tumors (HA221, HA393, HA396, HA455B, and HA455C) showed only numerical chromo-some aberrations, whereas the others were characterized by numerical and structural karyotype changes. Tumor HA394 had five aberrant chromosomes in the stemline as well as in the six sidelines in addition to the loss or gain of whole chromosomes. A balanced translocation between chromosomes 10 and 22, a rearranged chromosome 15, and a long marker of unknown origin was found in HA425 ( Figure 2). An interesting chromosome pattern was observed in case HA455. One part of the main tumor, HA455A1, showed a complex karyotype change. The stemline cells were characterized by tetrasomy 7, trisomy 17 and 20, and translocation 1 1;16. Additionally, the long arm of chromosome 3 was duplicated in the first sideline, and this duplicated chromosome segment was then translocated to chromosome 22 in the second sideline and to chromosome 14 in the third sideline. Cytogenetic analysis of the other part of this tumor, HA455A2, showed a karyotype with marker chromosomes other than those observed in HA455A1. Rearrangement of chromosome 8 and X chromosome and a marker carrying the 5q22-qter segment was found in each cell analysed (Figure 3). The two  other tumors, HA455B and HA455C, showed only numeric chromosome changes (Figure 4). In the karyotype of HA456 six markers of unknown origin and three markers with rearranged chromosomes 1, 15, and 19 were noted. Whole or partial (long arm) trisomy of chromosome 17 was noted in 7 of 11 papillary renal tumors, while trisomy or tetrasomy 7 was found in 5 cases. The chromosome 16 was involved 5 times in karyotype changes, while chromosome 14 only 4 times. Loss of the 3p segment or gain of the 5q segment was not found.

Discussion
RCC is commonly a slowly growing tumor composed of clear or granular cells arranged in acinar, tubular, cystic, solid, and sometimes papillary structures. Some tumors show focally or completely a sarcomatous pattern. Because of the various microscopic features of RCC, many authors have attempted to classify this neoplasm on the basis of cell type and histologic configuration,12 14 with controversial results. The purely papillary variant of RCC was ignored or mentioned only superficially in many previous studies dealing with this subject.
Mancilla-Jimenez, Stanly, and Blath9 showed a striking morphologic and clinical difference between papillary and nonpapillary variants of RCC. In a retrospective study of 224 RCCs, they compared the follow-up data of the 2 variants of RCC and found a significantly higher survival for the 34 papillary RCC than that for nonpapillary tumors. They found that papillary RCC is densely infiltrated by macrophages and lymphocytes and has a remarkable strong tendency for massive necrosis. Therefore, they discussed a possible role of host mechanism in the destruction of papillary RCCs. In the present study, 1 1 of 100 RCCs were of papillary histology. This incidence does not differ from that reported by other authors.9 15 These results confirmed the findings of Mancilla-Jimenez, Stanley, and Blath:9 the larger tumors were massively necrotic, and each tumor showed an extensive foam cell and lymphocyte infiltration independently of the size of tumors or the extent of necrosis. Contrary to papillary RCCs, the most common nonpapillary variant has a tendency for diffuse or central fibrosis, and necrosis occurs with some exceptions only in the sarcomatous form of RCC or in tumors of grade I l I l malignancy. Papillary RCC differs from the more common nonpapillary form of renal cancer not only in morphologic character and biologic behavior9 but also in karyotype changes observed. All of the 11 papillary RCCs of this study were characterized by clonal chromosome aberrations, but failed to show any rearrangement of the critical chromo-some 3p segment. From previous studies it is well known, that RCCs are marked by a recurrent loss of 3p segment.1`4 In our series of cytogenetical analysis of renal tumors, 81 of 85 nonpapillary RCCs showed a rearrangement of the 3p segment or the loss of the whole chromosome 3 (unpublished data). This is significant because other studies did not separate karyotype changes corresponding to a distinct histologic type of RCC. The only report using this morphologic discrimination described a difference in the karyotype between papillary and nonpapillary renal carcinomas. 3 A frequent karyotype change of nonpapillary RCCs is a trisomy of the 5q22-qter segment (unpublished data),4 which was often found to be translocated to chromosome 3p. This polysomy of the 5q segment was observed in about 50% of nonpapillary RCCs (unpublished data). In contrary to these data, all but one papillary RCC showed two microscopically normal chromosomes 5. In this case chromosome 5 was rearranged, but the 5q22-qter segment was maintained in the karyotype.
A gain of chromosome 17 was noted in 7 of the 11 papillary RCCs, but has never been found in nonpapillary tumors with near-diploid karyotype. Because the trisomy 17 was found in small papillary tumors, its appearance should be an early event in the development of this type of tumor. Chromosome 17 was also affected in one of the two papillary carcinomas of the kidney described by Carrol et al. 3 The involvement of chromosome 7, 14, and Y chromosome occurred with the same incidence as found in nonpapillary RCCs (unpublished data).4 The present cytogenetic data suggest that the papillary variant of RCC is marked by karyotype changes different from those found in nonpapillary tumors. The most remarkable differences are the lack of rearrangement of the chromosome 3p segment, the absence of trisomy 5q, and the gain of the 17q segment in papillary RCCs. The role of these chromosome aberrations in the development, progression, and phenotypic differentiation of the two variants of RCC is not yet clear. Genetic mechanisms controlling the malignant proliferation and also the phenotypic alteration of conversed tubular cells of the kidney are unknown. Recently, a retinoblastomalike mecha-nism was proposed for the development of RCCs regarding the recurrent loss of the 3p segment from the karyotype.56 According to this hypothesis, originally suggested by Knudson16 for the development of retinoblastoma, the tumorigenicity may arise through elimination of both alleles of a tumor supressor gene. The first event may be a mutation or submicroscopic deletion of one of the two alleles at the tumor loci, followed by the elimination of the wild type allele by different mitotic mechanisms. The breakpoint on chromosome 3 in familial RCCs (unpublished data)8 is consistent with the most common and more distal breakpoint found in sporadic, nonpapillary RCCs. If the rearrangement or small deletion within a specific chromosomal site predisposes to the development of tumor in families carrying such karyotype alteration, and this site is also involved in initial karyotype changes of sporadic RCCs, one may suspect that this region is the location of a recessive cancer gene responsible for the regulation of normal growth of tubular cells of kidney.
Theoretically, both papillary and nonpapillary variants of sporadic RCC may arise subsequently to a somatic Ai6k.. C. mutation at the putative RCC locus on one of the chromosome 3 in a common progenitor cell. For the malignant proliferation of this cell the loss of the second allele is required. This may be a result of different mitotic mechanisms leading to visible chromosome aberrations of the second chromosome 3 to monosomy 3, deletion 3p, or translocation as found in nonpapillary RCCs.1-46 Because of the absence of microscopic rearrangements of chromosome 3 in papillary RCC, other mitotic events, such as mitotic recombination, gene conversion, submicroscopic deletion, or a second mutation, would be expected for the homozygosity of the mutant allele at the relevant locus (a nondisjunction with duplication of the mutant chromosome 3 could be excluded17). This would be consistent with the cytogenetic pattern of RCCs and suggests that variable mitotic mechanisms affecting an identical RCC locus become effective in the initiation of RCCs with different phenotype and biology. How can the constant loss of chromosome 3p segment in nonpapillary and the maintainance of two microscopically normal chromosome 3 in papillary RCC be explained? One could speculate that there may be same clustering of differentiation genes at the 3p segment distal of the putative RCC locus, and the loss of these genes or the loss of one allele of erbA2 and raf-1 proto-oncogenes could be responsible for the phenotypic and biologic difference between the papillary and nonpapillary variant of RCC. The action of other genes localized to chromosome 5q or 1 7q, or of other genetic mechanisms for the phenotype alteration of RCCs can not be excluded. Alternatively, one could speculate that papillary RCC is a distinct type of renal epithelial tumor, the development of which is initiated by the inactivation of other supressor genes. Cloning of the putative RCC gene and further molecular genetic and cytogenetic studies on the common and rare variants of RCC are required to evaluate the genetic mechanisms responsible for the phenotype of the varying form of RCC.