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Urology & Nephrology
Despite significant progress regarding clinical detection/imaging evaluation modalities and genetic/molecular characterization of pathogenesis, advanced renal cell carcinoma (RCC) remains an incurable disease and overall RCC mortality has been steadily rising for decades. Concomitantly, clinical definitions have been greatly nuanced and refined. RCCs are viewed as a heterogeneous series of cancers, with the same anatomical origin, but fundamentally different metabolisms and clinical behaviors.
- renal cell carcinoma (RCC)
- immunohistochemistry (IHC)
- molecular pathology
- differential diagnosis
- subtyping
- prognosis
1. Introduction
Over the past decades, major technological and scientific breakthroughs have allowed for the development of important clinical tools for better RCC detection, evaluation, and therapeutic decision-making, while also reshaping medical understanding of RCC pathogenesis and progression-driving molecularities. Even so, RCC remains, to this day, one of the deadliest urological malignancies, accounting for ~3% of the total cancer burden in the global adult population [1].
The widespread integration of ultrasonography (US) and computer tomography (CT) in routine clinical practice has significantly improved RCC detection yields, reflected in the long-standing and still ongoing stable increase in RCC incidence rates globally [2]. Concurrently, a significant drop in RCC initial stage at diagnosis has also occurred, with most RCCs being detected incidentally currently, as asymptomatic, localized, small renal masses. Within this subgroup of RCC cases, 5-year survival rates have been significantly improved as a consequence of earlier curative intervention, i.e., partial/radical nephrectomy [3]. Conversely, despite important recent progress in systemic RCC therapeutic management, advanced RCC patients remain incurable, with persistently poor clinical outcomes. Thus, overall, RCC-specific mortality rates have been steadily increasing since the 1990s (~1.1%/year) [3,4].
To address these pervasive clinical limitations, regarding systemic/recurrent RCC therapeutic management and outcomes, contemporary RCC clinical definitions have been greatly nuanced and refined to better serve in RCC subtyping, prognosis assessment, and therapeutic response prediction. Classically, RCCs were simply defined as malignant parenchymatous renal neoplasms, spawning from tubular epithelial cellularity. However, in light of recent extensive RCC genomic profiling efforts and comprehensive metabolic molecular analyses, RCCs are currently viewed as an extremely heterogenic group of distinctive tumor subtypes, which only share an anatomical origin, while having different cellular progenitors, within the renal parenchyma. In fact, individual RCC subtypes demonstrate relatively specific, yet widely pleomorphic, tumor-driving molecular pathologies and pathognomonic genomics. This emerging intricate molecular amalgamation of novel RCC metabolic profiles, is firmly corroborated by the similarly extensive and well-documented clinical variability in RCC malignant behavior and therapeutic response, both between different RCC cases and disease phenotypes comparatively, but also during the natural evolution of individual RCCs (disease progression, systemic dissemination, and/or metastatic recurrence) [5,6].
In lack of a definitive systemic treatment modality for advanced RCCs, integration of the amounting fundamental medical knowledge regarding RCC molecular pathology into RCC clinical management has been paramount to obtaining improved risk stratification and evidence-based therapeutic decision-making. As a result, definitive pathological diagnosis and RCC subtyping have become more nuanced and cumbersome, with the latest guidelines (i.e., the World Health Organization—WHO’s 4th edition of the Urological Tumors Classification, 2016) identifying almost twenty distinct subtypes of malignant renal cell tumors, alongside mesenchymal, neuroendocrine, nephroblastic, and cystic variants [5,7]. Even so, these classifications will only become more comprehensive further on, as additional RCC entities, with distinguishing clinical, morphological, immunohistochemical, and/or molecular traits, have already been identified and are still awaiting formal acknowledgement [8,9,10,11,12,13,14,15].
To further complicate the already elaborate and dynamic landscape of RCC clinical management, systemic RCC therapy has been greatly diversified recently, with the advent of personalized oncological therapy and RCC tumor microenvironment molecular/genomic characterization initiatives. Notably, focused evaluation of the biologic implications of various inflammatory pathways regarding RCC metabolomics and proliferation, mainly the Von Hippel–Lindau (VHL), mechanistic target of rapamycin (mTOR), tumor necrosis factor (TNF), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) [16] pathways, has proven fruitful, providing multiple clinically relevant RCC-associated-antigen targeted molecules. Moreover, immunotherapy, a novel and promising type of oncotherapy, aiming to reactivate the cytotoxic immune response against tumor cells, has found important applications in advanced/recurrent RCC therapeutic management. Despite clinical difficulties regarding adequate immunotherapy response prediction for RCCs and the extensive therapy-associated costs, immunotherapy represents the only type of oncotherapy which allows, even theoretically, for the elaboration of a definitive cure for disseminated RCCs/cancer in general, as it is the only oncotherapy capable of targeting and annihilating non-dividing, quiescent, stem-like dysplastic cells [7,17].
Currently, as clinical definitions have become greatly nuanced, a complete RCC diagnosis requires careful multimodal evaluation, in a well-coordinated and phased approach, involving, at times, very difficult differentials.
2. Conventional Staining and RCC Microscopic Morphological Evaluation
Notwithstanding the long-held, central role of RCC morphological evaluation, using standard microscopy and conventional hematoxylin–eosin (HE) staining, in RCC definitive pathological diagnosis, essential for further therapeutic decision-making and predicting prognosis/risk stratification, in light of recent insights into RCC molecular pathology, the method, although still important, has become insufficient for adequate RCC subtyping. Even within the classical triad of conventional morphologies, namely clear cell (ccRCC), papillary (pRCC), and chromophobe (chRCC), which demonstrate relatively distinctive architectures and cellular features, microscopic evaluation of morphology remains subjective and user-dependent. Additionally, more often than not, meticulous evaluation will encounter, at least focally, overlapping patterns and/or atypical architectures, hindering definitive RCC subtyping. High-grade cellularity and severely dedifferentiated renal tumors lose characteristic morphological traits completely and cannot be subtyped using solely conventional staining microscopy. Determining metastatic cell origin, in advanced RCCs, with the additional difficulty of non-renal origin differentials, will most certainly require ancillary studies [6].
In fact, as a general consensus among pathologists, no truly reliable, objective, and validated histologic/ultrastructural criteria exist for distinguishing between benign and malignant renal epithelial tumors [30], with the very meek exception of oncocytomas and small low-grade papillary adenomas (≤5 mm) [30]. Furthermore, the amounting, emergent RCC clinical entities, have shown, for the most part, unspecific and poorly defined architectures, with heavily disputed morphological growth characteristics. Even within conventional RCC morphologies, absolute homogeneity is extremely rare and non-specific arrangement patterns (solid, papillary, tubular/cystic, sarcomatoid/rhabdoid) and cellular features (cytoplasmic clearing, eosinophilia/basophilia) are routinely identified, particularly in high-grade and/or poorly preserved tumor areas. Clearly, meticulous sampling of gross RCC specimens, especially in those which prove to be difficult to classify, will prove to be much more useful and cost-effective than additional staining. Transitional areas, from well-differentiated/low-grade patterns to more unusual, pleomorphic patterns, should be sought after and evaluated preferentially, as they usually offer the most useful diagnostic information [6,64].
All in all, conventional microscopic evaluation of RCC morphology remains an important initial step in tumor pathological analysis. It allows for an essential primary classification into RCC predominant morphological trait subgroups, simplifying the differential diagnosis and guiding further targeted analysis, while greatly reducing financial costs. Even so, it has become insufficient for an accurate definitive RCC diagnosis and prognosis assessment.
3. Definitions and Comprehensive Profiles for RCC Subtyping
Definitive RCC pathological diagnosis, meaning irrefutable demonstration of tumor renal cell origin and accurate RCC subtype identification, as well as the subsequent prognosis assessment, requires careful integration of clinical, pathological, and molecular characteristics, to allow for objective carcinogenic metabolic profiling and characterization of progression-driving tumor biology. Thus, as our understanding of RCC molecular pathology has become more nuanced, ancillary studies, mainly immunohistochemistry (IHC), has become integrated in clinical practice, as essential tools for routine RCC pathological evaluation. Despite the plethora of seemingly promising diagnostic and predictive applications reported, for the dozens of novel tumor-associated molecules and their corresponding IHC targeted-antibodies, due to the lack of validation studies, these data constitute no more than level 2/3 evidence. Moreover, heterogeneity within existing data regarding specific IHC methodology and antibody clone used (monoclonal/polyclonal) further encumbers comparisons between existing results [65,66,67,68,69,70,71]. In fact, as an investigative method, IHC has multiple inherent conceptual limitations, as well as significant technical (clone selection, titration, validation, false positives/negatives etc.) and interpretative (subcellular localization and pattern) weaknesses [64]. Table 1 provides a comprehensive summary of evidence regarding morphological, IHC, and genetic profiles for all currently accepted RCC subtypes.
A recurring issue is represented by the indiscriminate use of an inordinate number of antibodies, without a reasonable, structured, diagnostic approach, which only serves to generate additional confusion, while simultaneously wasting valuable resources. For this reason, in 2012, the International Society of Urological Pathology (ISUP) reviewed the use of IHC antibodies in adult renal tumors, in order to develop best practice recommendations regarding determining site of origin, typing, and prognosis, with the ultimate goal of establishing formal panels of biomarkers for specific diagnostic difficulties and, implicitly, establishing adequate guidelines for stewardship [6,64]. For a more systematic and practical approach to RCC subtyping, individual, differential diagnosis-driven IHC marker panels have been established (see Table 2), in order to differentiate among entities within the main RCC subgroups, manifesting specific morphological characteristics, namely predominantly clear cell (cc) population, significant papillary (p) component, extensive cytoplasmic eosinophilia, predominant sarcomatoid growth pattern, and architecture suggestive of distal nephron origin—collecting duct carcinoma (CDC) and renal medullary carcinoma (RMC). Quantitative and qualitative assessments of the staining results are equally important and continuous refinement of antibody panels, taking into account the proven value of new IHC markers and new clones of established markers, as they enter the market, is mandatory [6].
Overall, Paired box (PAX) 8, a transcription factor (415 aminoacids/48 kDa), involved in kidney, thyroid, and paramesonephric duct-derived, organogenesis, and homeostasis, represents the most useful IHC marker for establishing the diagnosis of metastatic RCC(mRCC) [72], being expressed in all RCC subtypes, including sarcomatoid RCC, mucinous tubular and spindle cell carcinoma (MTSC), and microphtalmia (MiT)-family translocation RCC, with a sensitivity of approximately 95% [73]. In healthy kidney tissue, PAX8 normally manifests diffuse staining in the, preferentially distal, renal tubules, and patchy staining of urothelium in the renal pelvis. In accordance with this pattern of developmental expression, in addition to RCCs, PAX8 consistently stains Műllerian neoplasms and thyroid neoplasms, and, in smaller subsets (~20%), urothelial carcinomas of the renal pelvis, Wolffian duct lesions, and thymic neoplasms [6].
Out of the PAX gene family for tissue specific transcription factors [74], PAX8 is generally the more sensitive marker [75]. PAX2 stains similarly to PAX8 [76,77]; with the possibly useful difference of negative PAX2 staining in thyroid neoplasms, admittedly only reported in small series [78]. When using older, polyclonal preparations, endocrine neoplasms (pancreatic islet cell tumors and gastrointestinal tract carcinoids) are often positive for PAX8; however, cross-reactivity with PAX6 is clarifying [79]. Additionally, B-cell lymphomas stain positive on polyclonal PAX8 preparation, while also manifesting cross-reaction with PAX5.
Table 1. Summary of evidence regarding RCC histological, IHC, and genetic profiles [5,6,64,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161].
| TYPE | MYCROSCOPY | IHC STAINING | GENETICS |
|---|---|---|---|
| Clear cell (cc) RCC | Hypercellularity: nests/sheets of cells, manifesting cytoplasmatic clearing and individualized membranes. Granular eosinophilic cytoplasm, seen in high-grade areas or near hemorrhage/necrosis. Rarely, intra/extracellular hyaline globules, basophilic cytoplasmic inclusions, abundant multinucleated giant cells. Architecture: solid, alveolar/nested, acinar/tubular, microcystic/macrocystic, rarely pseudopapillary. Stroma: ussually nonspecific, without desmoplasia or inflamatory modifications; may be fibromyxoid, with calcification/ossification. Hypervascular tumor: ramified network of small calibre, thin-walled vessels. High grade features: rhabdoid/sarcomatoid differentiation. |
POSITIVE: PAX8 (nuclear, ~100%); CAIX (diffuse membranous/box-like, 75–100%); proximal tubular antigens (also seen in normal cells): CD10 (membranous, 82–94%) and RCCm (cytoplasmic and membranous, 72–84%); epithelial markers (AE1/AE3, CAM 5.2, EMA); Vimentin (cytoplasmic and membranous). NEGATIVE: CK7 (focally positive or patchy in high grade areas or cystic components); CK20; AMACR; 34βE12; CD117; HMB-45; TFE3/TFEB; Cathepsin-K; GATA3; MelanA; Inhibin. |
SPORADIC: 3p loss/deletions or biallelic alteration of VHL gene (3p25) by mutation or hypermethylation (80–98%). ccRCC tumor suppressor genes harbored by 3p locus: KD-M6A (or UTX), KDM5C (or JARID1C), SETD2 and PBRM1. Loss of chromosome 4p, 8p, 9p, 14q. Gain of chromosome 5q. Mutations in BAP1 and PBRM1 genes. FAMILIAL: Von Hippel-Lindau disease; constitutional chromosome 3 translocations. |
| Papillary (p) RCC Type 1 | Hypovascular tumor. Cellularity: small cuboidal cells; uniform, diminished, pale/basophilic cytoplasm; hyperchromatic nuclei, absent nucleoli, lower grade than type 2; foamy macrophages and psammoma bodies are usually encountered. Architecture: single layer of of cells around fibrovascular cores (papillary), tubules and glomeruloid structures. |
POSITIVE: PAX8; CK7 (diffuse, but strong); EMA (polarized expression); AMACR; AE1/AE3; CD10. NEGATIVE: CAIX; GATA3; p63; 34βE12; TFE3/TFEB; Cathepsin-K. |
SPORADIC: Activating mutations or amplifications of MET proto-oncogene in >80% of sporadic cases. Gains in chromosomes 3, 7, and 17. FAMILIAL: Hereditary papillary renal carcinoma (HPRC). |
| pRCC Type 2 | Cellularity: abundant eosinophilic cytoplasm; frequent nuclear atypia with clearly visible nucleoli (usually ISUP grade ≥3) and nuclear grooves; intracytoplasmic hemosiderin; possible areas of clear cytoplasm; less frequent foamy macrophages and psammoma bodies than type 1. Architecture: papillary, with pseudostratified layers of large cells; variable necrosis. |
POSITIVE: PAX8; CD10; AMACR; Topoisomerase II alpha. NEGATIVE: CAIX; CK7 (or patchy positive); EMA (or focal positive); p63; HMB45; MelanA; Cathepsin-K; 34βE12; GATA3; TFE3/TFEB. |
SPORADIC: Less consistent than type 1. Heterogeneous, losses or gains of chromosomes 1, 3, 4, 5, 6, 8, 9, 10, 11, 15, 18, and 22. NRF-ARE2 pathway amplification. 8q gains and allelic imbalance of 9q13 (prognostic significance). In advanced stages, CDKN2A/B (18%), TERT (18%), NF2 (13%) and FH (13%) are commonly altered. FAMILIAL: FH gene (1q42–43) mutation in HLRCC. |
| Chromophobe (ch) RCC | Cellularity: sharply defined, pale cells; “plant-like” cell borders (“vegetable cells”); wrinkled and angulated, irregular nuclei, with coarse chromatin (“raisinoid”); frequent bi/multinucleation and perinuclear halo (koilocytic atypia), with rare mitotic figures; Fuhrman/WHO nuclear grading has no prognostic value and is discouraged. 2 types of cellular morphologies: Type 1—large, polygonal cells, hard cell borders, abundant cytoplasm with reticular pattern (classical variant); Type 2—smaller cells with finely granular eosinophilic cytoplasm (predominant in eosinophilic variant). Architecture: confluent, solid growth with nests, sheets or alveoli/trabeculae; minimal stroma, composed of incomplete fibro-vascular septae around solid sheets; minimal vasculature. |
POSITIVE: PAX8; Hale colloidal iron (histochemical stain; diffuse and strong, reticular); CK7 (diffuse and strong); CD117 (membranous); Ksp-cadherin (+/−); GATA3(+/−); E-cadherin; claudin7 (distal nephron marker); EMA (diffuse cytoplasmic); LMWCK (CK8/CK18); RCCm; CD10; Parvalbumin (calcium binding protein); Cytochrome c oxidase; DOG1; Progesteron Receptor (90% of cells); PDL1 22C3 (in a minority of cases). NEGATIVE: Vimentin (or weak); CAIX; AMACR; N-cadherin; Cathepsin-K; HMB-45; low Ki67 labeling index; Cyclin D1. |
SPORADIC: Multiple losses of whole chromosomes: 1, 2, 6, 10, 13, 17, 21 or Y. DNA rearrangement breakpoints within the TERT promoter region. Mutated TP53 and PTEN in 10–30% of cases (TCGA cohort) and NRAS, mTOR and TSC1/TSC2 in ~5%. Mutations in mitochondrial DNA most commonly affect MT-ND5, with increased expression of genes encoding Krebs cycle enzymes. FAMILIAL: Birt-Hogg-Dube (BHD) syndrome—FLCN gene mutation (17p11.2) |
| Clear Cell Papillary (ccp) RCC | Cellularity: clear cytoplasm; characteristic linear arrangement of nuclei away from the basal aspect of cells; low nuclear grade (Fuhrman grade 1–2). Architecture: variable mixture of cystic, branched tubular, solid and papillary components; papillae often tightly packed into anastomosing clear cell ribbons or projecting into cystic spaces; fibrous capsule and variable amounts of hyalinized or sclerotic stroma that may separate the tumor into nodules; never encountered: foamy macrophages, vascular invasion, oxalate crystals, necrosis. |
POSITIVE: PAX8; CK7 (strong and diffuse); CAIX (diffuse membranous or characteristic “cup shaped” distribution—lack of staining along luminal aspect); AE1/AE3; CAM 5.2; Vimentin; EMA; GATA3 (+/−); 34βE12. NEGATIVE: AMACR; CD10; RCCm; TFE3/TFEB; low PCNA; CD117; Cathepsin-K; HMB-45. |
No specific genomic imbalances. Lacks genetic modifications of classic pRCC (no +7/+17 or -Y) and/or classic ccRCC (no -3/-3p). Activation of hypoxia inducible factor (HIF) pathway, with underexpressed VHL transcripts, but without typical VHL mechanism (no VHL gene mutations/promoter hypermethylation). May appear in VHL disease. |
| Collecting duct carcinoma (CDC) | Cellularity: irregular channels, lined with high-grade cuboidal to hobnail cells, with eosinophilic cytoplasm; pleomorphic kariomegaly, with visible nucleoli and coarse chromatin; abundant mitosis. Architecture: the tumor is complex, infiltrative, and poorly circumscribed, composed of cords, tubules, tubulopapillary or tubulocystic structures, within an inflammatory-desmoplastic stroma; intracytoplasmic/intraluminal mucin, microcystic changes from dilation of the tubular structures and sarcomatoid transformation may be present. Tumor-adjacent tubular epithelium, lining collecting ducts, may appear dysplastic. |
POSITIVE: PAX8/PAX2; HMWCK (34βE12); LMWCK (CK7, CK8/18, CK19); Ulex europaeus lectins (Ulex-1); peanut lectin agglutinin (PNA); Mucin (strong); Vimentin; EMA; S100A1; INI-1/BAF47 retained. NEGATIVE: p63; Uroplakin II; CD10; AMACR; E-cadherin; CAIX; OCT3/4; CD117; GATA3. |
Lacking loss of 3p or trisomies 7 and 17. HER2/neu amplifications in 45% of cases. Genomic alterations in NF2 (29%), SETD2 (24%), SMARCB1 (18%) and CDKN2A (12%). Recurrent somatic single nucleotide variants in ATM, CREBBP, PRDM1, CBFB, FBXW7, IKZF1, KDR, KRAS, NACA, NF2, NUP98, SS18, TP53 and ZNF521. SLC7A11 (cisplatin resistance associated gene), overexpressed in 80% of cases. |
| Renal medullary carcinoma (RMC) | Cellularity: cohesive groups of pleomorphic tumor cells, with vacuolated eosinophilic cytoplasm, that often displaces or indents the hyperchromatic, enlarged nuclei; nuclear membranes are often irregular, with coarse or vesicular chromatin and prominent nucleoli; rhabdoid traits are frequent; abundant intratumoral neutrophils and lymphocytes at tumor rim; abundant sickled erythrocytes (drepanocytes) may be pathognomonic. Architecture: multiple distinct morphologic patterns—reticular; microcystic and adenoid cystic-like; tubular, glandular, tubulopapillary and solid (overlapping with CDC patterns); hemorrhagic and geographic necrosis; angiolymphatic invasion, desmoplastic stroma and infiltrative borders. |
POSITIVE: CAM 5.2; AE1/AE3; CK7/CK20 (may be variable); Vimentin; EMA and CEA; p53; PAX8; OCT3/4; Ulex-1 (focally positive in a minority of cases); vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF) may be strongly positive. NEGATIVE: Loss of INI-1/BAF47 expression; colloidal iron; PAS; desmin; 34βE12; GATA3 |
Prevalent loss of SMARCB1/INI1, a major driving feature, identifiable by IHC. Hypoxia inducible factor and VHL abnormalities. ALK rearrangement with vinculin (VCN) fusion. DNA topoisomerase II amplification. Rarely, vinculin—ALK fusion (young patients, less aggressive). Associated with sickle cell trait (monosomy 11—beta globin gene is at end of 11p). |
| Succinate dehydrogenase deficient renal (SDHD) RCC | Cellularity: flocculent, pale, eosinophilic, cytoplasmic vacuolation, with a wispy/bubbly appearance and low grade nuclei, with smooth nuclear contours and fine chromatin, with no nucleoli, represents a characteristic finding and must be present at least focally. Architecture: well circumscribed tumor or with a “pushing” border, commonly entrapping tubules; solid, nested or tubular growth pattern with scattered cysts containing eosinophilic material; necrosis, sarcomatoid change and areas with higher grade nuclei may also be present; variant morphologies have been rarely reported. Shares features with chRCC, oncocytoma, ccRCC and pRCC type 2. |
POSITIVE: PAX8; focal pancytokeratin and CAM 5.2; EMA. NEGATIVE: Loss of SDHB IHC staining (indicates disruption of the mitochondrial complex 2 for any reason, not just SDHB gene mutation and caution regarding overinterpretation of negativity in tumors with very clear cytoplasm is essential); CK7; CK20; AE1/AE3; CAIX; RCCm; CD117 (mast cells only); Vimentin; S100A1; TFE3/TFEB; neuroendocrine markers; minimal AMACR staining. |
Wild-type VHL, PIK3CA, AKT, mTOR, MET or TP53 genes. Genes for succinate dehydrogenase subunits (SDH-A, -B, -C, -D), encode protein components of mitochondrial complex II, linking the Krebs cycle with the electron transport chain, being involved in most cases of SDHD RCC. Germline mutations of SDH-A, SDH-B (1p36.13), SDH-C (1q23.3), SDH-D(11q23.1), SHDAF2, determining double hit inactivation, leads to dysfunction of mitochondrial complex II, increased oxidative stress, genomic injury and HIF1α stabilization. |
| Microphtalmia family translocation (MiT) RCC—Xp11/t(6;11) | Cellularity: voluminous cytoplasm and high-grade nuclei, with frequent psammoma bodies and occasional melanin pigment, similar to a pigmented perivascular epithelioid tumor—(PEC)coma. t(6:11) rearranged carcinomas are characteristically biphasic, with small cells clustered around basement membrane material (reminiscent of Call-Exner bodies in adult granulosa cell tumor) and larger epithelioid cells. Architecture: usually papillary and solid alveolar growth pattern, composed of clear to eosinophilic, discohesive pseudostratified cells. |
POSITIVE: TFE3/TFEB (strong nuclear staining, but difficult to standardize on automated platforms; FISH assays are more reliable)—weak TFE3 staining in adults may not be specific; PAX8; Cathepsin-K (~50%, cytoplasmic); CD10; AMACR; Vimentin; E-cadherin; melanocytic markers (HMB45 and MelanA), are common for t(6:11) carcinomas, but always focal, and infrequent for Xp11, which usually manifest variable CD117. NEGATIVE: Variable cytokeratin (only 30–50% positive, less than other RCC types) and EMA (50%, frequently only focal); CAIX usually negative except areas of necrosis; CK7; 34βE12; CD45; calretinin; smooth muscle actin. |
Fluorescence in situ hybridization (FISH) with a TFE3/TFEB breakapart probe is highly sensitive and specific, offering the final diagnosis when morphology and IHC are inconclusive. The TFE3 gene (on Xp11) has been reported to have multiple translocation gene partners, most commonly, ASPL (17q25) and PRCC (1q21), and less commonly NONO (Xq12), PSF/SFPQ (1p34), CLTC (17q23). t(6;11)(p21;q12), a translocation between TFEB and MALAT1 genes, results in overexpression of TFEB. t(X;17)(p11.2;q25), with balanced translocation of TFE3 gene at Xp11.2 and ASPL gene at 17q25, is present in renal neoplasms, whereas in alveolar soft part sarcoma, this translocation is unbalanced, der(17)t(X;17)(p11.2;q25). Melanotic Xp11 RCC and PSF/SFPQ-TFE3 (PEComa), may share the same genetic abnormalities. |
| Acquired cystic disease-associated (ACDA) RCC | Cellularity: moderately cellular, papillary clusters of polygonal to columnar cells with abundant eosinophilic granular cytoplasm, round and central nuclei, finely granular chromatin, prominent, central, grade 3 nucleoli; sometimes with prominent clear cell cytology. Architecture: cribriform, microcystic or sieve-like layout; intratumoral calcium oxalate crystals are very common, but not mandatory for diagnosis; nodules arising from cyst walls or masses separated from cysts may be encountered. |
POSITIVE: No specific IHC profile is required for diagnosis. CD10; AE1/AE3; AMACR; NEGATIVE: EMA; CK7 (but may be focally positive). |
Comparative genomic microarray and FISH studies reveal gains and losses of multiple chromosomes. Gains of sex chromosomes and gains of 3, 7, 16, 17, with a high prevalence of gains of Y, 3 and 16, distinguishing ACDA RCC from pRCC, which also has gains in chromosomes 7 and 17. VHL gene alterations. Chromosome 3p deletion. |
| Multilocular cystic clear cell renal neoplasm of low malignant potential (MCLMP) RCC | Cellularity: single layer of clear cells lining thin fibrous septae or in small clusters; low grade nuclei without nucleoli (ISUP grade 1–2); bland clear cells in septa may be mistaken for lymphocytes (vascularity is important). Architecture: cyst lining may be denuded and, in rare cases, cyst lining may be multilayered, with granular cytoplasm cells and small intracystic papillations; septa may contain calcification or ossification; no expansile growth of clear tumor cells/solid nodules; no necrosis, vascular invasion or sarcomatoid change. |
POSITIVE: PAX8/PAX2; CA IX; EMA; variable CK7. NEGATIVE: AMACR (negative in 80% of cases). |
Genetically related to ccRCC, with 74% of cases demonstrating 3p loss and VHL mutations identified in 25%. |
| Tubulocystic (TC) RCC |
Cellularity: tubules/cysts are lined by a single layer of flattened, cuboidal or columnar cells; hobnailing may be present, with modest to abundant amounts of eosinophilic cytoplasm, resembling oncocytoma cell; uniform, round nuclei, with distinct nucleoli (ISUP grade 3); minimal mitotic activity and atypia; very rare necrosis. Architecture: mixture of closely packed tubules and variably sized cysts, with overall low-grade morphology; cysts are separated by fibrous septa; no desmoplasia or cellular stroma; frequently associated with papillary cell neoplasms. |
POSITIVE: CK7; AMACR; Vimentin; EMA; PAX8; fumarate hydratase (FH); Mucin; keratins (AE1/AE3, Cam 5.2, CK8/18, CK19); variable 34βE12; CD10. NEGATIVE: 2 succino-cysteine, CA IX and CD117. |
Distinct molecular signature from other RCCs. Frequently, gains of chromosomes 7 and 17, and loss of Y, similar to pRCC. Mutations in 14 different genes have been documented by targeted, next generation sequencing, most frequently (60% of cases) in ABL1 and PDFGRA genes. |
| Mucinous tubular and spindle cell (MTSC) RCC | Cellularity: relatively uniform, bland, low-grade cuboidal cells, with round to oval nuclei and focally vacuolated, eosinophilic cytoplasm, within strands of metachromatic stromal tissue, which transition into anastomosing spindle cells; may present clear cells and focal clusters of foamy macrophages; rare high-grade nuclei may be present. Architecture: well circumscribed epithelial tumor, partially surrounded by a rim of compressed fibrous tissue; long tubular/cord-like growth pattern; myxoid and bubbly stroma, with abundant extracellular mucin, highlighted by Alcian blue (although some cases may be mucin poor); may manifest well-formed papillae, necrosis, rarely neuroendocrine differentiation or sarcomatoid change; usually, infiltrative growth, desmoplasia, inflammation, hobnail epithelium, and/or cysts are not encountered. |
POSTIVE: PAX2/PAX8; EMA (95%); AMACR (93%); AE1/AE3; E-cadherin; LMWCK: CK7 (81%); CK 8/18; CK19; Neuron Specific Enolase (NSE) and either Chromogranin or Synaptophysin. Histochemical stains: Periodic Acid-Schiff (PAS) (basal lamina around tubules), Alcian blue (mucin). Occasionally, HMWCK: 34βE12 (15%); vimentin; Ulex or CD10 (15%). NEGATIVE: CA IX (positive next to necrosis or focal cytoplasmatic in high-grade areas); GATA3; p63; RCCm (positive in 7%); Villin; Ki67 (<1%). |
Seemingly lacking the characteristic genetic modifications of classic pRCC (trisomies of chromosomes 7/17 or loss of chromosomes Y). Usually hypodiploid, with multiple chromosomal losses (-1, -4, -6, -8, -9, -13, -14, -15, -22), even hypertriploid in some cases, but with no identifiable pattern. |
| Hybrid oncocytic-chromophobe (HOCh) RCC | Cellularity: usually, dual population of eosinophilic cells—oncocytic (medium sized, round cell, with granular eosinophilic cytoplasm and concentric round nucleus, low nuclear:cytoplasmic ratios, prominent nucleolus) and chromophobe (large, polygonal, “plant-like” cell, with a distinct cell membrane, containing flaky eosinophilic cytoplasm, often with a perinuclear halo and an irregular “raisinoid” wrinkled nucleus), with a third cell subtype, manifesting overlapping cytonuclear features of both oncocytic and chromophobe morphology, being sometimes encountered; mitotic rate is very low. Architecture: well circumscribed, non-infiltrative, intrarenal tumor, with a solid alveolar and cystic architecture; may manifest vascular invasion and, rarely, necrosis. |
POSITIVE: CK7 (may be focal); AE1/AE3; Parvalbumin; Antimitochondrial Antigen; EMA; E-cadherin (most); CD117; S100A1; CD82; Vimentin (few cases); Hale colloidal iron (stains apical/luminal oncocytic cells and, often, intracytoplasmic in chromophobe cells. NEGATIVE: AMACR; CK20; CD10 and CA IX. |
SPORADIC: May present numerous molecular anomalies (both mono- and polysomies) of chromosomes 1, 2, 6, 9, 10, 13, 17, 21, and 22. Lack of mutations in the VHL, c-kit, PDGFRA, and FLCN genes. FAMILIAL: May be associated with BHD, autosomal dominant syndrome, characterized by a genetic abnormality on chromosome 17p11.2, leading to a mutation in the FLCN gene. |
| Predominant Morphological Trait |
Specific Panel | Entities Entering Differential Diagnosis with Corresponding IHC Profiles |
|---|---|---|
| Clear cell Population |
CAIX CK7 CD117 Cathepsin-K HMB-45 |
ccRCC: CAIX(+, diffuse membranous), CK7(−), CD117(−), Cathepsin-K(−), HMB-45(−) ccPRCC: CAIX(+, cup-like pattern), CK7(+), CD117(−), Cathepsin-K(−), HMB-45(−) classic chRCC: CAIX(−), CK7(+, cytoplasmic), CD117(+, membranous), Cathepsin-K(−), HMB-45(−) eAML: CAIX(−), CK7(−), CD117(−), Cathepsin-K(+, cytoplasmic), HMB-45(+, cytoplasmic) MiT-TFE RCC: - Xp11/TFE3: CAIX(+/−, focal), CK7(−), CD117(+/−), Cathepsin-K (+, 50%, cytoplasmic), HMB-45(−) - t[6;11]/TFEB: CAIX(+/−, focal), CK7(−), CD117(−), Cathepsin-K(+, cytoplasmic), HMB-45(+, focal) |
| Papillary Component |
CAIX CK7 AMACR Cathepsin-K 34βE12 TFE3/TFEB |
ccRCC with papillary growth: CAIX(+, membranous), CK7(−), AMACR(−), Cathepsin-K(−), 34βE12(−), TFE3/TFEB(−) pRCC “type I”: CAIX(−), CK7(+), AMACR(+), Cathepsin-K(−), 34βE12(−), TFE3/TFEB(−) pRCC “type II”: CAIX(−), CK7(+/variable), AMACR(+), Cathepsin-K(−), 34βE12(−), TFE3/TFEB(−) ccPRCC: CAIX(+, cup-like pattern), CK7(+, diffuse), AMACR(−), Cathepsin-K(−), 34βE12(−), TFE3/TFEB(−) MiT-TFE RCC: CAIX(variable, focal), CK7(−), AMACR(+), Cathepsin-K(+, 50%), 34βE12(−), TFE3/TFEB(+, but difficult to standardize on automated platforms, requires FISH assays) |
| Solid growth pattern | CK7 AMACR WT-1 CD57 |
Solid pRCC “type I”: CK7(+), AMACR(+), WT-1(−), CD57(−) Metanephric adenoma: CK7(−)/isolated cells, AMACR(−), WT-1(+, nuclear), CD57(+) Wilms’ Tumor: CK7(−)/isolated cells, AMACR(−), WT-1(+, nuclear), CD57(−) |
| Cytoplasmic Eosinophilia |
CD117 CK7 Ksp-cadherin HMB-45 Cathepsin-K |
Oncocytoma: CD117(+, membranous), CK7(−), Ksp-cadherin(+), HMB-45(−), Cathepsin-K(−) Eosinophilic chRCC: CD117(+, membranous), CK7(+, but variable), Ksp-cadherin (+, mostly), HMB-45(−), Cathepsin-K(−) Oncocytic PRCC: CD117(−), CK7(+, focal), Ksp-cadherin (unknown), HMB-45(−), Cathepsin-K (unknown) Oncocytic AML: CD117(−), CK7(−), Ksp-cadherin(−), HMB-45(+, focal), Cathepsin-K(−) |
| Sarcomatoid growth pattern |
Vimentin CAIX PAX8 CK7 34βE12 GATA3 p63 |
ccRCC: Vimentin(+), CAIX(+) membranous, PAX8(+), CK7(−), 34βE12(−), GATA3(−), p63(−) pRCC: Vimentin(+), CAIX(−), PAX8(+), CK7(focal/-), 34βE12(−), GATA3(−), p63(−) chRCC: Vimentin(+), CAIX(−), PAX8(+), CK7(+), 34βE12(−), GATA3(−), p63(−) MTSC: Vimentin(+), CAIX(−), PAX8(+), CK7(+), 34βE12(variable), GATA3(−), p63(−) Urothelial: Vimentin(+), CAIX variable/mostly(−), PAX8 mostly(−), CK7(+), 34βE12(+), GATA3(+), p63(+) Sarcoma: Vimentin(+), CAIX(−), PAX8(−), CK7(−), 34βE12(−), GATA3(−), p63(−) |
| Distal nephron origin | INI-1/BAF47 OCT4 GATA3 PAX8 |
CDC: INI-1/BAF47 (+, and—in 15%), OCT4(−), GATA3(−), PAX8(+) RMC: INI-1/BAF47 (−), OCT4(+), GATA3(−), PAX8(+) Urothelial: INI-1/BAF47 (+), OCT4(−), GATA3(+), PAX8(−, but + in 20%) |
Novel PAX8 antibodies (PAX8R1), to address the specificity issues of polyclonal PAX8 preparations, have been developed. They bind to the C-terminal of PAX8, targeting amino acids 318–426, which are highly divergent among PAX proteins, thus abolishing cross-reactivity with other PAX species [162].
Further nuancing of IHC differential diagnosis in mRCC involves markers almost always negative in RCC, such as pulmonary marker TTF-1, the intestinal marker homeobox protein CDX2, p63, prostate-specific antigen, and estrogen receptor, which will be useful in excluding other carcinomas that may manifest cross-positivity for PAX8. Positive staining with any of the aforementioned markers represents a strong argument against the diagnosis of mRCC [6]. Another useful distinction, between urothelial and renal epithelial origins, can be made using GATA3, a transcription factor involved in cell differentiation and proliferation in a variety of tissues and cell types, which will be expressed in most urothelial tumors, but not in RCCs [163,164].
Other supportive IHC markers of mRCC, currently in common practice—cluster of differentiation (CD)10, RCC marker antigen (RCCm), Kidney-Specific Cadherin (Ksp-cadherin)—manifest inadequate specificity and are not usually indicated or useful, outside of very specific, punctual, diagnostic subtleties.
RCCm stains a proximal tubular antigen and demonstrates focal labeling in approximately 80% of RCC [165,166], yet with notoriously poor specificity, seeing as it also labels many other carcinomas (breast, lung, colon, and of adrenal origin). It is useful in differentiating clear cell RCC (ccRCC) from ovarian clear cell carcinoma, as PAX8 would be positive in both tumors, whereas RCCm would be positive only in the renal neoplasm. Moreover, PAX8 is negative in adrenal cortical neoplasms, which preferentially stain for steroid factor-1 [167].
Another proximal tubular marker, CD10, also manifests high sensitivity, but again very low specificity for RCC, as lung, bladder, colon, and ovarian carcinomas all label for CD10 [168]. However, CD10 fairly consistently labels ccRCC, thus CD10 negative metastatic lesions represent an argument against this diagnosis for the primary tumor.
Lastly, Ksp-cadherin, a distal tubular marker, manifests high sensitivity for chromophobe RCC (chRCC), although it is not so useful in the metastatic diagnosis context, as the chromophobe variant rarely disseminates systemically [169]. Even so, at least focal staining can also be seen in other renal tumor variants, including high-grade ccRCC.
This entry is adapted from the peer-reviewed paper 10.3390/biomedicines10112926
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