CME/SAM. Significant Variation of Immunohistochemical Marker Expression in Paired Primary and Metastatic Clear Cell Renal Cell Carcinomas

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1 AJCP / Original Article Significant Variation of Immunohistochemical Marker Expression in Paired Primary and Metastatic Clear Cell Renal Cell Carcinomas Zenggang Pan, MD, PhD, 1,2 William Grizzle, MD, PhD, 2 and Omar Hameed, MD 2,3,4,5 From the 1 Department of Pathology, University of Colorado Medical Center, Aurora; 2 Department of Pathology, University of Alabama at Birmingham; 3 Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN; 4 Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, TN; and 5 Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN. CME/SAM Key Words: Immunohistochemistry; Renal markers; Metastatic clear cell renal cell carcinoma; RCC antigen; Kidney-specific cadherin; Carbonic anhydrase IX; Paired box gene 2; Paired box gene 8 ABSTRACT Objectives: To compare the immunohistochemical expression of diagnostic markers in primary clear cell renal cell carcinomas (RCCs) and their matched metastases. Methods: Tissue microarrays were constructed from 15 pairs of primary and metastatic clear cell RCCs and then evaluated for the immunohistochemical expression of renal cell carcinoma antigen (RCCA), kidney-specific cadherin, carbonic anhydrase IX (CAIX), and paired box genes 2 (PAX2) and 8 (PAX8). Results: There was significantly higher overall marker expression in metastatic tumors compared to their matched primaries (P <.001). Individually, there was greater CAIX, PAX2, and PAX8 expression and lower RCCA expression in metastatic tumors. Most importantly, a significant proportion of originally RCCA-positive tumors lost such expression in metastases. Conclusions: Metastatic RCCs have significantly higher expression of PAX2 and PAX8 compared to primary RCCs. RCCA is not very reliable in this diagnostic setting, both because of its lower overall sensitivity and loss of expression in metastatic RCCs. Upon completion of this activity you will be able to: list immunohistochemical markers used for the evaluation of clear cell and other subtypes of renal cell carcinoma (RCC). describe the limitations of these markers, especially RCC antigen, in the evaluation of clear cell RCC, both in the primary and metastatic settings. appropriately select and interpret immunohistochemical markers in the diagnostic evaluation of potential metastatic clear cell RCC. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 442. Exam is located at Renal cell carcinomas (RCCs) account for approximately 2% to 3% of new cancers annually in the United States, and the American Cancer Society estimated that approximately 65,150 new cases of kidney cancer (40,430 in men and 24,720 in women) would occur in Patients with RCCs develop metastatic spread in approximately 33% of cases, in which pathologists often have to make that diagnosis. In most cases, this diagnosis is based almost entirely on the presence of classic morphologic features along with a prior clinical history of RCC. On the other hand, in some situations, the diagnosis is less straightforward, such as when there is no documented history of a previous RCC (eg, occult primary), limited sampling (eg, needle core biopsy material), and/or unusual morphology. In these situations, immunohistochemical staining becomes invaluable to confirm the diagnosis of metastatic RCC and/or to differentiate it from its numerous mimics. Although CD Am J Clin Pathol 2013;140: Downloaded 410 from Pan_ indd 410

2 AJCP / Original Article and vimentin are often used as RCC markers, 2,3 they generally lack sufficient sensitivity or specificity. More renal-specific markers that have greater utility in this area include RCC antigen (RCCA), kidney-specific cadherin (KSC), carbonic anhydrase IX (CAIX), and paired box gene 2 (PAX2) and paired box gene 8 (PAX8). 2,4-6 Interestingly, recent studies have shown that, with disease progression, metastatic RCCs may upregulate certain genes and proteins (MMP16, B7-H1, BCL2L2, FRA2, CD44, Annexin II, phosphorylated AKT, phosphorylated S6, 4E-binding protein 1 [4EBP1], c-myc, and p27) and downregulate others (CD151, IKBA, and HNF-1B) It is unclear, however, whether disease progression in RCC is associated with any significant changes in the expression of the renal diagnostic markers noted above. Such changes, if present, could potentially affect how these markers are used in the diagnosis of metastatic RCCs. In this study, we specifically address this question by comparing the expression of these selected diagnostic markers in matched metastatic and primary clear cell RCCs. Materials and Methods Case Selection and Construction of Tissue Microarray After approval by the University of Alabama at Birmingham Institutional Review Board, the surgical pathology files from 2005 through 2010 at our institution were retrospectively reviewed to identify cases of clear cell RCC with matched metastatic and primary specimens. H&E-stained sections were then reviewed to confirm the diagnosis and to select paraffin blocks with adequate tissue for tissue microarray (TMA) construction. After extensive selection, 15 pairs of clear cell RCC cases were eventually included in this study, including 11 cases of metachronous metastases and 4 synchronous metastases. For each case, the primary and metastatic tumors were punched in triplicate (1.0 mm) and separately plated onto 2 TMAs (one for primary tumor and the other for the metastatic tumor). Triplicate sampling was performed to include different tumor morphologies and/or grades when present and account for potential heterogeneous marker expression in these tumors. Immunohistochemical Staining The formalin-fixed, paraffin-embedded TMA blocks were sectioned at 4 mm thick. Immunohistochemical staining was then performed on the sections according to the manufacturer s manual. The antibodies used in this study included RCCA, KSC, CAIX, PAX2, and PAX8. The antibodies used, as well as their clones, dilutions, antigen retrieval methods, and manufacturers, are summarized in Table 1. Immunostains for RCCA, KSC, and PAX2 were performed on a Lab Vision autostainer (Thermo Fisher Scientific, Kalamazoo, MI), and immunostains for CAIX and PAX8 were performed by Clarient (Aliso Viejo, CA). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Antigen retrieval for RCCA, KSC, and PAX2 was carried out in citrate buffer (258 mg/dl [10 mmol/l], ph 6.0) at 100 C in a microwave oven for 15 minutes, and for CAIX and PAX8, it was performed in EDTA buffer (ph 9.0) at 100 C for 20 minutes. Appropriate positive and negative control slides for each antibody were prepared. The negative control slides consisted of tissue sections of each case processed without incubation with the primary antibody. Evaluation of Immunohistochemical Staining and Statistical Analysis Immunohistochemical staining for all 5 markers was evaluated in a semiquantitative method by scoring the intensity of each immunostain on a scale from 0 to 3 (0, negative; 1, weak; 2, moderate; and 3, strong). The intensity (0-3) and percentage of positive cells (0%-100%) were then used to calculate an H-score for each core, and the average H-score from the 3 tumor cores was used for statistical analysis. Complete absence of expression or weak expression in less than 10% of cells (H-score <10) was considered negative. Expression of the 5 selected markers in metastatic and primary RCCs was compared using a paired t test and paired analysis of variance (ANOVA), while categorical tests were compared using c 2. A P value of.05 or less was considered statistically significant. Results Clinical and Histologic Information of the Matched RCCs There were 15 pairs of primary and metastatic clear cell RCCs for which representative blocks were available Table 1 Immunohistochemical Stains Used in This Study Antibody Clone Dilution Antigen Retrieval Manufacturer RCCA PN15 1:40 Citrate, ph 6.0, 100 C, 15 min Cell Marque, Rocklin, CA KSC MRQ33 1:50 Citrate, ph 6.0, 100 C, 15 min Cell Marque CAIX TH22 1:100 EDTA, ph 9, 100 C, 20 min Leica, Buffalo Grove, IL PAX2 Polyclonal Predilute Citrate, ph 6.0, 100 C, 30 min Cell Marque PAX8 Polyclonal 1:60 EDTA, ph 9, 100 C, 20 min Cell Marque CAIX, carbonic anhydrase IX; PAX2, paired box gene 2; PAX8, paired box gene 8; KSC, kidney-specific cadherin; RCCA, renal cell carcinoma antigen. Downloaded from Am J Clin Pathol 2013;140: Pan_ indd 411

3 Pan et al / Renal Markers in Paired Metastatic and Primary RCCs for TMA production that formed the basis of the study. Of these 15 cases, 2 had focal sarcomatoid change in the primary tumor, including 1 in which the metastatic focus was predominantly sarcomatoid also. Other than that, there was no significant morphologic progression or transformation between the metastatic and the primary tumor within each pair. The metastatic sites included the lung (n = 5), lymph node (n = 3), bone (n = 2), adrenal gland (n = 1), thyroid gland (n = 1), brain (n = 1), abdominal soft tissue (n = 1), and multiple sites (n = 1). All 3 lymph node metastases and the 1 metastasis with multiple sites were synchronous, whereas the metastases in the remaining cases were metachronous. Immunohistochemical Findings Immunohistochemistry was performed for 5 selected renal cell markers: RCCA, KSC, CAIX, PAX2, and PAX8. The pattern of staining for RCCA, KSC, and CAIX was predominantly membranous, whereas that for PAX2 and PAX8 was nuclear Image 1. Except for RCCA, in which there was variable expression in different cores, individual marker Image 1 Immunohistochemical staining of the tissue microarray with 5 selected renal markers. The intensity of the immunostain was scored on a scale from 0 to 3: 0, negative; 1, weak; 2, moderate; and 3, strong. The intensity (0-3) and percentage of positive cells (0%-100%) were used to calculate the H-score for each marker. CAIX, carbonic anhydrase IX; KSC, kidney-specific cadherin; PAX2, paired box gene 2; PAX8, paired box gene 8; RCCA, renal cell carcinoma antigen ( 100). 412 Am J Clin Pathol 2013;140: Downloaded 412 from Pan_ indd 412

4 AJCP / Original Article expression was similar among the 3 cores sampling different areas of primary tumor or metastasis (data not shown). The combined average H-score for all 5 markers in the metastatic lesions was significantly higher than that observed in the primary tumors (112 vs 86, P =.003, paired t test) Figure 1. There was also significant variation (P <.001; ANOVA) in marker expression when metastatic and primary tumors were grouped together Figure 2, with the lowest and highest average H-scores observed with RCCA and CAIX, respectively. H-Score The expression of each marker was compared in metastatic clear cell RCCs with matched primary renal tumors. Metastatic clear cell RCCs had significantly higher PAX2 (P =.01, paired t test) and PAX8 (P =.009, paired t test) H-scores than those in the matched primary cases Figure 3 and Image 2. Metastatic tumors also showed higher expression of CAIX, although this was not statistically significant (P =.11, paired t test). The average expression levels of KSC and RCCA were slightly lower in the metastatic tumors than those in the primary lesions, but these differences also did not reach statistical significance (Figure 3). Besides the variation in protein expression levels, there was complete loss of marker expression (no expression or weak expression in <10% of cells; H-score <10) with the development of metastasis in 6 cases. This was significantly more frequent with RCCA Image 3 than with the other antibodies (4/8 originally positive cases vs 0/15, 1/14, 1/12, and 0/14 for KSP, CAIX, PAX2, and PAX8, respectively; P <.001). 0 Primary Metastasis Figure 1 Combined antigen expression levels of all 5 markers in metastatic and primary renal cell carcinomas (RCCs). The overall combined H-score for all 5 markers in metastatic RCCs was significantly higher than that observed in the primary tumors (P =.003). Discussion Despite the small nature of this study, we have shown that there is significant variation in renal marker expression in metastatic vs primary clear cell RCCs, most evident with higher PAX2 and PAX8 expression and lower RCCA expression in the metastases. From a diagnostic standpoint, however, only loss of RCCA expression seen in a significant P =.01 P = H-Score 100 H-Score RCCA KSC CAIX PAX2 PAX8 Figure 2 Comparison of individual marker expression with metastatic and primary renal cell carcinomas grouped together. There was significant variation (P <.001) in marker expression, with the lowest and highest average H-scores observed with RCCA and CAIX, respectively. CAIX, carbonic anhydrase IX; KSC, kidney-specific cadherin; PAX2, paired box gene 2; PAX8, paired box gene 8; RCCA, renal cell carcinoma antigen. 0 RCCA KSC CAIX PAX2 PAX8 Figure 3 Comparison of the expression of each marker in paired metastatic and primary renal cell carcinomas (RCCs). The metastatic RCCs had significantly higher expression of PAX2 (P =.01) and PAX8 (P =.009) than that in the matched primary cases. CAIX expression was also higher in metastatic RCCs but did not reach statistical significance (P =.11). CAIX, carbonic anhydrase IX; KSC, kidney-specific cadherin; PAX2, paired box gene 2; PAX8, paired box gene 8; RCCA, renal cell carcinoma antigen. Downloaded from Am J Clin Pathol 2013;140: Pan_ indd 413

5 Pan et al / Renal Markers in Paired Metastatic and Primary RCCs proportion of cases appears to be of clinical significance. These findings are in support of using KSC, CAIX, PAX2, and PAX8, but not necessarily RCCA, as an aid to confirm the renal origin of a metastatic carcinoma. The diagnosis of metastatic clear cell RCC can occasionally be challenging. This may be due to it presenting as an occult metastatic tumor initially without any known clinical history of a renal mass or many years after the initial diagnosis of a primary renal neoplasm. It may also be related to it presenting in an unusual site such as the skin, salivary glands, thyroid gland, and stomach. Metastatic clear cell RCCs may also display morphologic features that are different from the primary tumors, whereas many nonrenal neoplasms can have a clear cell component mimicking metastatic clear cell RCCs. All this is compounded by the limited sampling imposed by the increasing use of needle core biopsy and fine-needle aspiration procedures to arrive at the diagnosis. Because of these factors, immunohistochemistry has become an invaluable tool to reach an accurate diagnosis of metastatic clear cell RCCs. Many renal markers can be used for the diagnosis of metastatic clear cell RCCs, including the ones evaluated in this study. Renal cell carcinoma antigen, a terminally differentiated molecule, is typically localized to the brush border of proximal tubular cells. It is a specific marker for primary A B C D Image 2 Examples of increased paired box gene 2 (PAX2) and paired box gene 8 (PAX8) expression in metastatic renal cell carcinomas (RCCs). Two primary RCCs (A and C) with greater PAX2 expression in the matched metastatic tumors (B and D, respectively) and another 2 primary RCCs (E and G) with greater PAX8 expression in the matched metastatic tumors (F and H, respectively) ( 200). 414 Am J Clin Pathol 2013;140: Downloaded 414 from Pan_ indd 414

6 AJCP / Original Article renal neoplasms, particularly clear cell and papillary RCCs, both of which are derived from proximal tubules. However, RCCA has been less sensitive than other renal markers due to its low intensity and small percentage of positive cells.12 Kidney-specific cadherin is expressed in the basolateral infolding membrane of normal distal tubular and collecting duct cells and corresponding renal neoplasms, including chromophobe RCC and oncocytoma5,6,13; 2 studies5,14 also found it to be expressed in clear cell RCCs, which was one reason why we chose to evaluate it in this study. Carbonic anhydrase IX, a tumor-associated metalloenzyme, has a major role in regulating hydrogen ion (H+) flux. Most clear cell RCCs show diffuse and strong expression of CAIX; in addition to RCC, CAIX is also expressed in solid tumors from the breast, lung, colon, and brain. Expression of CAIX is generally associated with disease progression and unfavorable prognosis Therapeutic agents targeting CAIX by immunotherapy have shown excellent potential as candidates in cancer chemotherapy.19,20 Paired box gene 2 is a transcriptional regulator of the paired box family that controls the development of the kidney and the organs related to the Wolffian and Müllerian ducts. It is a sensitive and specific marker for tumors of renal or Müllerian origin,4,21 and its expression in RCCs is correlated with the proliferation index and is significantly higher in patients with metastatic disease.4 Paired box gene 2 is a useful marker for distinguishing metastatic clear cell RCC from its potential morphologic mimics, and it seems to be more sensitive than RCCA and KSC.14,22 Studies of PAX2 in metastatic clear cell RCCs have shown moderate sensitivity (47%-85%) and high overall specificity (90%-97%).21,22 E F G H Downloaded from Pan_ indd Am J Clin Pathol 2013;140: /1/13 12:06 PM

7 Pan et al / Renal Markers in Paired Metastatic and Primary RCCs Paired box gene 8, another member of the family of paired box-containing genes similar to PAX2, is essential in the development of the kidney, Müllerian organs, and thyroid,23 and it is a sensitive and specific marker for the tumors of these organs in both primary and metastatic sites.23,24 Although immunohistochemistry has become an invaluable tool in confirming metastatic RCCs, recent studies have found that metastatic RCCs may upregulate or downregulate the expression of certain genes and proteins. For example, gene expression profiling has determined a significant decrease in expression of CD151 and IKBA in metastatic RCCs compared with nonmetastatic tumors, whereas the expression of MMP16, B7-H1, BCL2L2, and FRA2 was significantly increased in metastatic tumors.7 Downregulation of the HNF-1B gene was associated with RCC tumor progression and metastasis, and patients with high HNF-1B messenger RNA levels had a significantly better prognosis.8 Expression of CD44 was significantly higher in metastatic RCCs compared with primary RCCs, and CD44 expression in primary RCCs was an independent predictor of progression-free survival.9 Annexin II was significantly higher in metastatic RCCs, and its expression in the primary tumors showed a strong association with a higher nuclear grade and a higher clinical stage.10 More recently, Schultz et al11 noticed that metastatic clear cell RCCs had significantly higher levels of phosphorylated AKT, phosphorylated S6, 4EBP1, c-myc, and p27 than those detected in the primary tumors. A B C D Image 3 Examples of loss of renal cell carcinoma antigen (RCCA) expression in metastatic renal cell carcinomas (RCCs). Two primary RCCs with strong RCCA expression (A and C) and no expression in the matched metastatic tumors (B and D, respectively) ( 200). 416 Am J Clin Pathol 2013;140: Downloaded 416 from Pan_ indd 416 8/1/13 12:06 PM

8 AJCP / Original Article Given the differences in the immunohistochemical expression patterns of primary and metastatic renal carcinomas for the markers noted above, one wonders if such differences exist for the markers more frequently used for diagnostic purposes and how that might affect their utility for the diagnosis of metastatic RCCs. Several studies have evaluated the expression of such renal markers in primary and metastatic sites but not in matched cases. One such study has shown that RCCA was expressed in 80% of primary vs 67% of metastatic RCCs, 12 whereas Ozcan et al 24 found that PAX8 was similarly expressed in both primary (89%) and metastatic RCCs (88%). Another study with unmatched RCCs found that PAX2 expression was more frequent in patients with metastatic disease than in those without disease. 4 These data suggest that there may be potential differences in the expression of diagnostic biomarkers in primary and metastatic RCCs, but because these studies did not look at matched cases (a self-criticism noted in some of these studies), it is unclear how such differences might affect their diagnostic utility. Accordingly, we specifically designed this study comparing primary and matched metastatic RCCs to systematically address this issue. We did in fact demonstrate that such significant differences do exist, since the combined average H-score for all markers in metastatic RCCs was significantly higher than that observed in primary tumors. This appeared to be secondary to higher expression levels of PAX2, PAX8, and CAIX, as well as lower expression of KSC and RCCA in metastatic lesions. Compared with other markers, there was weaker RCCA expression in both metastatic and primary tumors, which is in concert with prior studies. 2,14 Although the proportion of primary and metastatic tumors that expressed RCCA was not significantly different (50% vs 44%), loss of RCCA expression in metastatic tumors was seen in 4 (50%) of 8 of those expressing this marker in the primary neoplasm, highlighting the value of using matched cases for comparison. Apart from an apparently more heterogeneous pattern of expression, loss of RCCA expression in metastatic tumors was significantly more frequent than what was observed with the remaining antibodies, confirming its comparatively lower sensitivity for the diagnosis of metastatic RCCs. On the other hand, there was at least similar, if not greater, expression frequency of PAX2, PAX8, and CAIX in metastatic RCCs, which, despite the small size of our study, still supports their superior value in this diagnostic setting. An important finding in our study was that there was significant upregulation of PAX2 and PAX8 expression in metastatic RCCs. Previous studies have shown that downregulation of PAX2 expression in RCC cell lines resulted in growth inhibition, 25,26 and PAX8 positively regulated telomerase expression. 27 Although these data provide some insight as to why there is higher expression of PAX2 and PAX8 in metastatic disease, the detailed mechanism(s) for upregulation in metastatic disease and full biological significance are yet to be determined. A somewhat unexpected finding in our study was that KSC expression in clear cell RCCs was higher than that reported in the literature for this tumor type (17%- 31%). 5,14 These 2 studies used a different antibody clone than we did, which could certainly explain the higher expression seen herein, as might other methodologic differences, including antigen retrieval methods and scoring criteria. It may also be speculated that clear cell RCCs that have already developed metastasis (all cases in this series) have higher KSC expression levels than localized tumors, on which the other studies appear to focus. Although further studies will be needed to investigate this and other possibilities, our data suggest that immunohistochemistry for KSC, at least when using the MRQ33 clone, may have a role in evaluating potential metastasis of RCCs. Aside from the small study size, one might argue that inclusion of metastatic cases with morphologies more divergent than the primary tumors could have potentially resulted in greater immunohistochemical variation and potentially more informative results. This is certainly a possibility but is a caveat of most reports evaluating immunohistochemical stains in which unequivocal cases are used to define the characteristic immunoprofile of a particular lesion or tumor, which then might be extrapolated to less classic or straightforward cases in clinical practice. Of note, our study was not selective and included all consecutive cases within the study period that had available material. Another argument is that we used H-scores to compare the expression in the 2 groups when such scoring is rarely used in clinical practice. That might be the case for most of the statistical analysis; however, we also used a clinical cutoff to confirm the inferior diagnostic utility of RCCA compared with the other markers. In summary, we have shown that the metastatic RCCs have significantly higher expression of PAX2 and PAX8 than the paired primary RCCs. Although this may have minimal diagnostic implications for the immunohistochemical workup of suspected metastatic RCCs, it is certainly worthy of further study to explore the biological significance in tumor progression and metastasis. On the other hand, our findings confirmed that RCCA is not very reliable in this diagnostic setting because of its lower overall sensitivity and loss of expression in metastatic RCCs. Address reprint requests to Dr Hameed: Dept. of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, C-3321A MCN, st Ave S, Nashville, TN ; omar.hameed@vanderbilt.edu. References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, CA Cancer J Clin. 2013;63: Downloaded from Am J Clin Pathol 2013;140: Pan_ indd 417 8/1/13 12:06 PM

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