An acinar epithelioid appearance (Fig. 8) occurred when the cord cells aligned their nuclei against the septa and their prominent eosinophilic cytoplasm toward the center of the cord cell clusters. In contrast to the true tubules of a Wilms tumor, such structures did not feature well-defined luminal borders.
The palisaded “verocay-body” pattern resulted from the alignment of spindled cord cells in parallel linear arrays with nuclei perpendicular to the septa (Fig. 9). The septa were usually surrounded by collagenous material in these formations, creating a pink contrast to the aligned blue nuclei. The appearance raised the differential diagnosis of schwannoma, particularly in soft tissue metastasis.
Spindle cell patterns resulted in a variety of appearances that suggested the diagnosis of other sarcomas. Spindled transformation of both cord and septal cells produced a storiform pattern of growth that mimicked fibrohistiocytic neoplasms (Fig. 10). More commonly, the cord cell/septal cell demarcation was completely lost, resulting in a monomorphic high-grade sarcomatous appearance. In a spindled nucleus the fine chromatin characteristic of CCSK was more difficult to appreciate. In several cases, most of the sections showed a high-grade spindle cell sarcoma appearance, with the diagnosis of CCSK becoming evident only in small characteristic foci or in recurrences (Fig. 11).
Anaplasia, defined by nuclear hyperchromasia, nuclear gigantism, and atypical mitoses, was identified in nine tumors (Fig. 12). Six of these tumors were untreated; in one the anaplasia was diffuse, whereas the other five showed only discrete foci in anaplasia. Anaplasia was identified in three of 53 histologically examined posttreatment specimens. In two of these, the anaplasia was identified in a nephrectomy specimen obtained after preoperative dactinomycin/vincristine therapy. The anaplasia was focal and no preoperative biopsy had been performed. In the other treated case, anaplasia appeared in a brain metastasis of a non-anaplastic primary tumor following intensive cranial radiation therapy.
No one histologic pattern was overrepresented in lymph node metastases at presentation. Occasionally, a pattern not seen in the primary tumor was evident in its metastasis. However, the entrapped renal tubules and the epithelioid patterns seen in primary tumors were not encountered in a metastasis.
Posttreatment relapses generally displayed the same range of patterns seen in primary tumors, with the notable absence of the epitheliod pattern. However, a trend toward more sclerotic, less cellular lesions was seen, possibly reflecting treatment effect. These non-pleomorphic, sclerotic relapses often entrapped native epithelial structures (bile ducts, alveoli) in their metastatic sites, mimicking benign bile duct or pulmonary hamartomas. Two unique patterns were identified among the metastases. The first was a hypocellular spindle cell pattern that simulated fibromatosis (Fig. 13). On diligent search, small residual foci of epithelioid cord cells could be identified, allowing a diagnosis of metastatic CCSK to be made with confidence. The second was a paucicellular myxoid pattern that featured pools of myxoid material bearing only rare free-floating tumor cells. 9 Confusion with myxoma was heightened by a focal absence of the characteristic CCSK vasculature.
A paraffin-embedded tissue block was available for immunohistochemical staining from a total of 45 cases of CCSK. Vimentin was immunoreactive in all cases, with staining often accentuated in a perinuclear dot-like pattern. This consistent staining served as a control for antigen preservation in each of the blocks studied, making negative results with other immunostains believable even in the absence of internal controls on the slide.
In 13 of 31 cases studied, the cord cells showed weak cytoplasmic staining for muscle specific actin. However, the reactivity was always less intense than that of the pericytic cells surrounding internal capillaries. All 30 cases tested were negative for desmin, including all of the ones that showed weak actin staining.
Tumor cells were not immunoreactive for either cytokeratin AE1/AE3 or CAM5.2 in any of the 41 cases studied. Stains for EMA were similarly negative in 31 cases. Nonetheless, these stains highlighted entrapped native renal tubules that easily could be missed on routine hematoxylin and eosin-stained sections, emphasizing the diagnostically helpful infiltrative nature of these tumors. Of note, the cytokeratin-negative cases included 13 in which distinct epithelioid foci were present: 4 showed trabecular epithelioid patterns, 4 contained microcystic foci, and 5 showed acinar formation simulating true glands. One of these acinar cases was previously reported as a case of CCSK showing epithelial differentiation. 31 The authors illustrated a silver stain which had converted from a single-cell (sarcomatous) pattern to a nested (epithelial) pattern in the acinar foci, although all their immunohistochemical stains had been unsuccessful. By destaining a stained slide and performing antigen retrieval before cytokeratin staining, we found the acinar focus to be cytokeratin-negative with positive staining of adjacent renal pelvic urothelial mucosa serving as an internal control.
Immunohistochemical stains for the MIC2 antigen failed to reveal the characteristic membranous pattern in any of 24 cases studied, including four with cellular foci that raised the differential diagnosis of PNET. Some cases did show blush cytoplasmic staining, a pattern generally considered to be a negative result with this antibody. Other negative stains included S100 protein (23 cases), Factor 13a (18 cases), synaptophysin (20 cases), GFAP (20 cases), LCA (20 cases), NSE (23 cases), c-kit (9 cases), and polyclonal CEA (23 cases). CD34, Factor 8, and Ulex immunostains highlighted thin capillaries within the tumor, but neither antibody-stained tumor cells in 20 cases studied.
To assess proliferative activity, immunohistochemical stains for the MIB-1 antigen were performed on 22 cases. One hundred tumor nuclei were counted from four separate areas on each slide to obtain the percentage of nuclei stained (staining index) for each case. A mean staining index of 21% was obtained (range among cases: 8%–32% of cells staining). No obvious correlation with histologic pattern was noted in the sections available for staining.
A similar scoring system was used to evaluate immunohistochemical stains of p53. Among 29 non-anaplastic CCSKs, 25 showed minimal (less than 5% of nuclei) staining. The four other non-anaplastic CCSKs had intermediate staining indices of 11%, 18%, 25%, and 40%. Of note, the lesion showing 40% staining was a posttreatment metastasis. In contrast, two of three anaplastic CCSKs available for staining showed intense, diffuse (>75% of nuclei) staining for p53. Non-anaplastic foci present on the slides in both of these cases showed the minimal p53 staining pattern usually seen in non-anaplastic CCSK, so that a sharp transition to the anaplastic foci could be appreciated (Fig. 12).
Multivariate analysis revealed four independent significant prognostic factors for survival: treatment with doxorubicin, revised stage, patient age, and the presence of tumor necrosis (Table 3). The significance of tumor necrosis was lost when relapse-free survival was the end point. Variables lacking statistical significance for either survival or relapse-free survival included patient gender, tumor diameter, specimen weight, lymph node status, and any single histologic pattern other than necrosis.
The addition of doxorubicin to therapeutic regimens significantly improved outcome for children with CCSK (Fig. 2;Tables 4, 5, and 6). This beneficial effect accounts for the progressive improvement in both relapse-free survival and overall survival that was noted from NTWS 1 through 4. Once the progressively increased use of doxorubicin over these trials was taken into account, the survival differences across the trials lost their significance.
The remarkable 98% overall survival rate among revised stage 1 patients was significantly better than those of the more advanced stages (Table 4;Fig. 14). The survival rates for stages 2 and 3 were diminished, but rose from approximately 30% to 75% with the addition of doxorubicin to therapeutic regimens. The few stage 4 patients had a poorer prognosis.
Patients within 1 year of the mean age of diagnosis (ages 2–4 yrs) survived more frequently than those younger (less than 2 years of age) or older (greater than 4 years) than the mean (Table 5). The survival for patients in the latter two categories was improved when doxorubicin was added to therapy.
Necrosis proved to be an adverse prognostic factor for survival. The minority of tumors lacking gross or histologic evidence of necrosis were associated with a high (97%) survival rate (Table 6).
Among 68 patients with tumor relapse, both doxorubicin therapy and tumor necrosis significantly affected tumor-specific survival, whereas age and stage at diagnosis did not. Doxorubicin therapy was associated with a 66% reduction in tumor-related mortality (p = 0.02), whereas necrosis was associated with a 5.8-fold increased risk of death resulting from tumor (p = 0.004).
This study is the largest series of CCSK ever reported. We have confirmed several of the previous concepts of this tumor originating from smaller studies and reached several new conclusions. We confirmed the beneficial effect of doxorubicin therapy by multivariate analysis for both survival and relapse-free survival. Prior studies had demonstrated the trend toward better outcomes with doxorubicin, but the results were not statistically significant. 10 The larger number of patients in the prognostic group in this study and the requirement for central pathologic review likely permitted statistical significance to be attained. With this review, 22 CCSKs were excluded from analysis, leaving a more homogeneous population of tumors. This study also confirmed the striking propensity of this tumor to metastasize to bone, which we documented as its most common site of recurrence. In contrast, Wilms tumor classically spreads to lymph nodes, lung, and liver but rarely metastasizes to bone. We have also documented a large number of unusual sites of metastasis, particularly in the soft tissue, brain, and orbit, that could potentially fool the unwary oncologist. These findings emphasize the need for complete physical examinations and bone imaging 8 in clinical follow up of these patients. Prolonged vigilance is also required, because we found that approximately 20% of documented CCSK metastases occurred 3 years or more after diagnosis and some as long as 10 years later. In contrast, recurrences of Wilms tumor almost always present within 2 years. Doxorubicin therapy tended to prolong the interval to relapse among NWTS cases, but therapy alone cannot account for the propensity to late recurrence that CCSK exhibits. Indeed, the two patients with the longest intervals to relapse (9 and 10 years) did not receive doxorubicin.
An unexpected finding was the high (29%) frequency of lymph node metastases identified at presentation. This figure is extraordinarily high for sarcomas that classically spread through hematogenous routes. This finding is concordant with the high frequency of vascular invasion noted in this study, and together they suggest that CCSK has a propensity to permeate the renal and perirenal lymphovascular system. This concept may explain the high number of local (abdominal) recurrences that we have documented. Complete nodal dissections may be required to remove all tumor in such cases, as is suggested by the cases in which we found positive periaortic lymph nodes despite histologically confirmed benign hilar nodes. Whether such more extensive surgery would decrease the chances of local recurrence or impact on survival remains to be proven.
Another unexpected but likely related finding was the high survival rate (98%) for patients with revised stage 1 disease, results that are at variance with previous studies which have shown a high mortality among all stages. 23,32 Such reports have fostered the view that CCSK disseminates early so that apparent low-stage patients have micrometastases at presentation. Such studies also have shaped NWTSG therapeutic protocols under which CCSKs are treated similarly regardless of stage.
The difference in the staging system we used compared with previous studies likely accounts for the differences in our results. Under updated NWTS 5 criteria, invasion of renal sinus vasculature, not gross protrusion beyond the hilar plane, is a basis of upstaging from stage 1 to stage 2. This criterion makes more biologic sense, because the sinus vessels represent the primary route of lymphatic and venous outflow and hence tumor exit from the kidney. Twenty-three of the NWTS cases previously classified as stage 1 became stage 2 tumors on our review, usually on the basis of renal sinus vascular invasion. These tumors, having access to the systemic circulation, would be expected to have a diminished prognosis. Once this aggressive subset was excluded, a more pristine population of stage 1 tumors was obtained which proved to have an excellent prognosis. The long-held concept that stage 1 CCSKs disseminate early seems at variance with their slow growth rate, as reflected by the relatively low MIB-1 staining indices we obtained, the documented long intervals to recurrence, and the low percentage of patients who initially presented with stage 4 disease. Furthermore, the four revised stage 1 patients in this study who did not receive the benefit of doxorubicin therapy all survived, suggesting that these tumors were not disseminated and may have been cured by surgical excision. Instead, it is likely that stage 2 tumors disseminate microscopically in this fashion, as is reflected in their diminished prognosis compared with stage 1, and account for the previously cited results with “low-stage” tumors.
The only histologic variable that independently correlated with survival was the presence of necrosis. Because necrosis is typically a feature of aggressive high-grade sarcomas, a correlation of the absence of necrosis with favorable outcome is not unexpected. This concept is supported by the magnified effect of tumor necrosis on the outcome of relapsed tumors in which surgery is unlikely to be curative but instead response to chemotherapy is required. As the effects of stage and age are both mediated through relapse, these variables may reflect the probability of complete excision. Regardless of the potential explanations, it is somewhat comforting that we can identify subsets of CCSK (that is, stage 1, ages 2–4 yrs, no tumor necrosis) with a superior prognosis; such patients may not need the intensive therapy given for patients with advanced disease. The need to minimize unnecessary therapy is underscored by the fact that treatment-related deaths (2) outnumbered tumor-related deaths (1) among our revised stage 1 patients.
Whereas the variant histopathologic patterns identified in CCSK do not appear to carry prognostic import, they do highlight differential diagnostic dilemmas. Of particular difficulty is the distinction of cellular CCSK from blastemal Wilms tumor and PNET, both of which have a prominent vascular pattern. Useful differential diagnostic criteria are presented in Table 7. Both blastemal Wilms tumor and PNET are more aggressively invasive than CCSK, entrapping whole islands of native renal parenchyma as opposed to the single tubules entrapped by CCSK. Both feature coarser chromatin than CCSK. Of particular use is our finding of consistent MIC2 negativity in CCSK. Hence, in a small biopsy or other challenging case, true membranous MIC2 staining strongly argues against CCSK. An equally challenging diagnostic problem is the distinction of epithelioid CCSK patterns, particularly acinar types, from the true tubular differentiation of a Wilms tumor. We show that stains for cytokeratin can reliably make this distinction; no CCSK in our study expressed cytokeratin regardless of how epithelioid it appeared. Finally, predominantly spindled CCSKs can be difficult to distinguish from the plump cell variant of cellular congenital mesoblastic nephroma, metanephric stromal tumor, and other sarcomas. This is particularly problematic because the characteristic fine chromatin of CCSK is more difficult to appreciate in a narrow spindled nucleus. Besides its chromatin, the best clue to CCSK is the presence of foci of its classic pattern, which may be absent in small biopsy samples of large tumors. Given that all CCSKs in our study stained negatively for desmin and S100 protein, positivity for either of these markers can be used as strong evidence against CCSK. In contrast, a significant percentage of congenital mesoblastic nephromas stain with desmin. 24
This study also clearly defines the existence of an anaplastic subset of CCSKs. This subtype is slightly less common (3%) among CCSKs than anaplasia among Wilms tumors (5%). Our results refute our previous assumption that anaplasia in CCSK was usually related to treatment effect. First, anaplasia was not overwhelmingly more frequent in post-therapy specimens examined (3 of 53 [6%]). Second, in only one case did we document that a non-anaplastic tumor became anaplastic after therapy. Hence, anaplasia in CCSK, as with Wilms tumor, appears to arise de novo. Of particular interest was the dramatic p53 overexpression in the anaplastic foci of two tumors in which the non-anaplastic regions did not overexpress p53. The low frequency of p53 positivity in non-anaplastic CCSK likely represents native p53 expressed by proliferating cells; it is unlikely to represent mutated p53, which usually accumulates enough to be detected in a higher percentage of cells. These results are consistent with a model set by other pediatric tumors, such as Wilms tumor and rhabdomyosarcoma, in which only anaplastic tumors show p53 protein overexpression that results from gene mutation. 1,26 The absence of genetic losses at 17p in a recently completed comparative genomic hybridization study of CCSK further argues against p53 alterations being a primary genetic event in CCSK. 28
This study more sharply defines the clinical behavior and pathologic features of CCSK but the histogenesis of this tumor remains an enigma. While its nearly exclusive occurrence in the kidney suggests an origin from the developing renal mesenchyme, no viable candidate cell of origin is apparent. The presence of verified extrarenal tumors in this and other studies 14,33 that are indistinguishable from CCSK perhaps favors origin from a non-organ-specific mesenchymal cell. The eventual delineation of this cell of origin will no doubt further our understanding of this tumor.
The National Wilms Tumor Study Group is supported primarily by United States Public Health Service Grant CA-42386. Additional funding for this study was provided by a generous award from the Children's Cancer Foundation of Maryland. The authors thank the many institutional pathologists who have over the years provided the case material on which this study is based. They also thank those pathologists who sent blocks specifically for use in this study: Dr. David Kelly (Birmingham, AL) and Dr. Philip Faught (Indianapolis, IN); the staff of the NWTSG Data and Statistical Center, particularly Dr. Weiva Sieh, Tracy Bergemann, and Janice Takashima, for their tireless work; Mary North, Josephine Geh, and George Pettis for performing the immunohistochemical stains; Lisa Madden for typing the manuscript and creating Figure 1; and Pete Lund for photographic assistance.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
Clear cell sarcoma; Kidney; Childhood cancer