ABCC2 brush-border expression predicts outcome in papillary renal cell carcinoma: a multi-institutional study of 254 cases.

AIMS: Papillary renal cell carcinoma (PRCC) histologic subtyping is no longer recommended in the 2022 WHO classification. Currently, WHO/ISUP nucleolar grade is the only accepted prognostic histologic parameter for PRCC. ABCC2, a renal drug transporter, has been shown to significantly predict outcomes in PRCC. In this study we evaluated the prognostic significance of ABCC2 IHC staining patterns in a large, multi-institutional PRCC cohort and assessed the association of these patterns with ABCC2 mRNA expression. METHODS AND RESULTS: We assessed 254 PRCCs for ABCC2 IHC reactivity patterns that were stratified into negative, cytoplasmic, brush-border <50%, and brush-border ≥50%. RNA in situ hybridization (ISH) was used to determine the transcript level of each group. Survival analysis was performed with SPSS and GraphPad software. RNA-ISH showed that the ABCC2 group with any brush-border staining was associated with a significant increase in the transcript level, when compared to the negative/cytoplasmic group (P = 0.034). Both ABCC2 groups with brush-border <50% (P = 0.024) and brush-border ≥50% (P < 0.001) were also associated with worse disease-free survival (DFS) in univariate analysis. Multivariate analysis showed that only ABCC2 IHC brush-border (<50% and ≥50%) reactivity groups (P = 0.037 and P = 0.003, respectively), and high-stage disease (P < 0.001) had a DFS of prognostic significance. In addition, ABCC2 brush-border showed significantly worse DFS in pT1a (P = 0.014), pT1 (P = 0.013), ≤4 cm tumour (P = 0.041) and high stage (P = 0.014) groups, while a similar analysis with high WHO/ISUP grade in these groups was not significant. CONCLUSION: ABCC2 IHC brush-border expression in PRCC correlates with significantly higher gene expression and also independently predicts survival outcomes.

Recurrent KRAS mutations are early events in the development of papillary renal neoplasm with reverse polarity.

We evaluated the clinicopathologic and molecular characteristics of mostly incidentally detected, small, papillary renal neoplasms with reverse polarity (PRNRP). The cohort comprised 50 PRNRP from 46 patients, divided into 2 groups. The clinically undetected (<5 mm) neoplasms (n = 34; 68%) had a median size of 1.1 mm (range 0.2-4.3 mm; mean 1.4 mm), and the clinically detected (≥5 mm) neoplasms (n = 16; 32%) which had a median size of 13 mm (range 9-30 mm; mean 16 mm). Neoplasms were positive for GATA3 (n = 47; 100%) and L1CAM (n = 34/38; 89%) and were negative for vimentin (n = 0/44; 0%) and, to a lesser extent, AMACR [(n = 12/46; 26%; weak = 9, weak/moderate = 3)]. KRAS mutations were found in 44% (n = 15/34) of the clinically undetected PRNRP and 88% of the clinically detected PRNRP (n = 14/16). The two clinically detected PRNRP with wild-type KRAS gene were markedly cystic and contained microscopic intracystic tumors. In the clinically undetected PRNRP, the detected KRAS mutations rate was higher in those measuring ≥1 mm vs <1 mm [n = 14/19 (74%) vs n = 1/15 (7%)]. Overall, the KRAS mutations were present in exon 2-codon 12: c.35 G > T (n = 21), c.34 G > T (n = 3), c.35 G > A (n = 2), c.34 G > C (n = 2) resulting in p.Gly12Val, p. Gly12Asp, p.Gly12Cys and p.Gly12Arg, respectively. One PRNRP had a G12A/V/D complex mutation. Twenty-six PRNRP were concurrently present with other tumors of different histologic subtypes in the ipsilateral kidney; molecular testing of 8 of the latter showed wild-type KRAS gene despite the presence of KRAS mutations in 5 concurrent PRNRP. On follow up, no adverse pathologic events were seen (range 1-160 months; mean 44 months). In conclusion, the presence of KRAS mutations in small, clinically undetected PRNRP provides a unique finding to this entity and supports its being an early event in the development of these neoplasms.

New developments in existing WHO entities and evolving molecular concepts: The Genitourinary Pathology Society (GUPS) update on renal neoplasia.

The Genitourinary Pathology Society (GUPS) reviewed recent advances in renal neoplasia, particularly post-2016 World Health Organization (WHO) classification, to provide an update on existing entities, including diagnostic criteria, molecular correlates, and updated nomenclature. Key prognostic features for clear cell renal cell carcinoma (RCC) remain WHO/ISUP grade, AJCC/pTNM stage, coagulative necrosis, and rhabdoid and sarcomatoid differentiation. Accrual of subclonal genetic alterations in clear cell RCC including SETD2, PBRM1, BAP1, loss of chromosome 14q and 9p are associated with variable prognosis, patterns of metastasis, and vulnerability to therapies. Recent National Comprehensive Cancer Network (NCCN) guidelines increasingly adopt immunotherapeutic agents in advanced RCC, including RCC with rhabdoid and sarcomatoid changes. Papillary RCC subtyping is no longer recommended, as WHO/ISUP grade and tumor architecture better predict outcome. New papillary RCC variants/patterns include biphasic, solid, Warthin-like, and papillary renal neoplasm with reverse polarity. For tumors with 'borderline' features between oncocytoma and chromophobe RCC, a term "oncocytic renal neoplasm of low malignant potential, not further classified" is proposed. Clear cell papillary RCC may warrant reclassification as a tumor of low malignant potential. Tubulocystic RCC should only be diagnosed when morphologically pure. MiTF family translocation RCCs exhibit varied morphologic patterns and fusion partners. TFEB-amplified RCC occurs in older patients and is associated with more aggressive behavior. Acquired cystic disease (ACD) RCC-like cysts are likely precursors of ACD-RCC. The diagnosis of renal medullary carcinoma requires a negative SMARCB1 (INI-1) expression and sickle cell trait/disease. Mucinous tubular and spindle cell carcinoma (MTSCC) can be distinguished from papillary RCC with overlapping morphology by losses of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22. MTSCC with adverse histologic features shows frequent CDKN2A/2B (9p) deletions. BRAF mutations unify the metanephric family of tumors. The term "fumarate hydratase deficient RCC" ("FH-deficient RCC") is preferred over "hereditary leiomyomatosis and RCC syndrome-associated RCC". A low threshold for FH, 2SC, and SDHB immunohistochemistry is recommended in difficult to classify RCCs, particularly those with eosinophilic morphology, occurring in younger patients. Current evidence does not support existence of a unique tumor subtype occurring after chemotherapy/radiation in early childhood.

Novel, emerging and provisional renal entities: The Genitourinary Pathology Society (GUPS) update on renal neoplasia.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in renal neoplasia, particularly focusing on the newly accumulated evidence post-2016 World Health Organization (WHO) classification. In the era of evolving histo-molecular classification of renal neoplasia, morphology is still key. However, entities (or groups of entities) are increasingly characterized by specific molecular features, often associated either with recognizable, specific morphologies or constellations of morphologies and corresponding immunohistochemical profiles. The correct diagnosis has clinical implications leading to better prognosis, potential clinical management with targeted therapies, may identify hereditary or syndromic associations, which may necessitate appropriate genetic testing. We hope that this undertaking will further facilitate the identification of these entities in practice. We also hope that this update will bring more clarity regarding the evolving classification of renal neoplasia and will further reduce the category of "unclassifiable renal carcinomas/tumors". We propose three categories of novel entities: (1) "Novel entity", validated by multiple independent studies; (2) "Emerging entity", good compelling data available from at least two or more independent studies, but additional validation is needed; and (3) "Provisional entity", limited data available from one or two studies, with more work required to validate them. For some entities initially described using different names, we propose new terminologies, to facilitate their recognition and to avoid further diagnostic dilemmas. Following these criteria, we propose as novel entities: eosinophilic solid and cystic renal cell carcinoma (ESC RCC), renal cell carcinoma with fibromyomatous stroma (RCC FMS) (formerly RCC with leiomyomatous or smooth muscle stroma), and anaplastic lymphoma kinase rearrangement-associated renal cell carcinoma (ALK-RCC). Emerging entities include: eosinophilic vacuolated tumor (EVT) and thyroid-like follicular renal cell carcinoma (TLFRCC). Finally, as provisional entities, we propose low-grade oncocytic tumor (LOT), atrophic kidney-like lesion (AKLL), and biphasic hyalinizing psammomatous renal cell carcinoma (BHP RCC).

Integrated Molecular Analysis of Papillary Renal Cell Carcinoma and Precursor Lesions Unfolds Evolutionary Process from Kidney Progenitor-Like Cells.

Papillary renal cell carcinoma (PRCC) is the most common type of RCC in end-stage kidney disease (ESKD). Papillary adenoma (PA) is a small benign lesion morphologically similar to PRCC and is suggested to be its precursor. PA is also prevalent in ESKD. The evolution of PAs to PRCCs and their relationship to ESKD are poorly understood. A total of 140 PAs, normal kidneys, ESKDs, and PRCCs were analyzed. Previously described markers of renal tubular progenitor cells were analyzed using immunohistochemistry and quantified with digital analysis. Progenitor cells were significantly increased in ESKD (P < 0.0001) and PAs (P = 0.02) in comparison with the normal kidney. Pathway analysis using global miRNA and chromosomal copy number variations revealed a common developmental theme between PA and the PRCCs. Whole exome sequencing showed a KMT2C-specific pathogenic mutation among all PAs and PRCCs. KMT2C is a chromosome 7 epigenetic regulator implicated in development and oncogenesis. Collectively, results show possible connection of PRCCs to PA and the progenitor-like cell population, which are increased in response to renal tubular injury. In addition, each PRCC histologic subtype had its own set of mutational changes, indicating divergence from a common precursor. The study reports previously unknown biological aspects of PRCC development and could influence current surveillance criteria and early detection strategies of PRCC tumors.

A Novel Approach to Detect IDH Point Mutations in Gliomas Using Nanopore Sequencing: Test Validation for the Clinical Laboratory.

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n = 66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

ABCC2 expression in papillary renal cell carcinoma provides better prognostic stratification than WHO/ISUP nucleolar grade.

Papillary renal cell carcinoma (PRCC) classification has traditionally been divided into two histologic types, type 1 and type 2. A new biological stratification system has recently been proposed based on comprehensive morphologic and genomic analysis. The predominant molecular marker in this 4-tiered stratification is the renal drug transporter ABCC2. In this study, we assessed and validated the value of the biological grouping in a PRCC cohort of 176 patients and provided a comprehensive assessment of clinicopathological variables. Tissue microarrays (TMAs) were constructed from nephrectomy specimens. The TMAs were stained with ABCC2 and GATA3 antibodies, and the PRCC cohort was stratified into four groups PRCC1-PRCC4: PRCC1 25%, PRCC2 37%, PRCC3 36%, and PRCC4 2%. PRCC1 demonstrated lower disease stage (p = 0.041) than PRCC2 and PRCC3. The biological stratification was significant on univariate analysis when analyzing both overall survival (p = 0.039) and disease-free survival (p = 0.011). The biological groups maintained the significance of predicting overall survival after adjusting for WHO/ISUP grade, age, pathological stage, and necrosis (p = 0.049, hazard ratio: 5.008, 95% confidence interval: 1.007 to 24.909). In contrast, WHO/ISUP grade did not maintain its significance on multivariate survival analysis. ABCC2 expression profile also separated cases ≤ 4 cm, based on disease-free survival (p = 0.038). None of the patients in the PRCC1 group died of disease during the follow-up period. The proposed biologic stratification adds molecular markers to the traditional morphologic assessment to better stratify patients' prognosis. ABCC2 expression can also potentially serve as a predictive biomarker owing to its known implication in cancer biology and drug resistance.

Integrated Phenotypic/Genotypic Analysis of Papillary Renal Cell Carcinoma Subtypes: Identification of Prognostic Markers, Cancer-related Pathways, and Implications for Therapy.

BACKGROUND: Two histologic subtypes are recognized for papillary renal cell carcinoma (PRCC). Studies have shown that the subtypes differ in characteristic genetic alterations and clinical behavior. Clinically, the subtypes are managed similarly. OBJECTIVES: To analyze the biological differences between the two PRCC histological subtypes, in order to further guide their clinical management. DESIGN, SETTING, AND PARTICIPANTS: PRCC cohort consisting of 317 patients from the Cancer Genome Atlas database and our institution. Patients were stratified according to histologic criteria as type 1, type 2, or not otherwise specified (NOS). Gene and miRNA expression data for the cohort were examined via unsupervised and supervised clustering. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Significant molecular signatures for each subtype were used to unravel the implicated molecular pathways via bioinformatics analysis. Survival was compared between the subtypes. Newly discovered biomarkers were used to further stratify survival of patients in the NOS category. RESULTS AND LIMITATIONS: Tumor genotyping revealed two distinct PRCC subtypes. The top molecular pathways enriched in PRCC1 were WNT, Hedgehog, and Notch signaling (p=0.001-0.01); highlighting an embryonic developmental theme to the pathogenesis of this subtype. PRCC2 showed enrichment in the mTOR, VEGF (p=7.49E-09) and HIF (p=7.63E-05) signaling pathways. Overall survival and disease-free survival significantly differed between the types. ABCC2 expression was identified as a significant prognostic biomarker for the NOS group in univariate (log rank p<0.0001; hazard ratio [HR] >11.63) and multivariate analysis (p=0.003; HR >2.12). ABCC2 expression and its effect on survival should be further validated at the protein level. CONCLUSIONS: The classical PRCC types 1 and 2 have two distinct genotypes. We unraveled pathways that indicate that the two types could potentially respond differently to current therapies. We also identified biomarkers that stratify tumors within the PRCC NOS category into prognostic subgroups. Our findings highlight the need for molecular markers to accurately subtype PRCC and guide clinical management. PATIENT SUMMARY: The two types of papillary renal cancer are treated similarly. We show that the two types have a different genetic makeup, and hence they should be considered two different tumors. There is a different biology underlying each tumor type that can potentially affect the way they respond to treatment. We uncovered genes that can be tested for to guide therapy in some problematic cases for which it hard to define the tumor type.

Modulating ATP binding cassette transporters in papillary renal cell carcinoma type 2 enhances its response to targeted molecular therapy.

Papillary renal cell carcinoma (PRCC) is the most common nonclear cell RCCs and is known to comprise two histological subtypes. PRCC2 is more aggressive and is molecularly distinct from the other subtypes. Despite this, PRCCs are treated together as one entity, and they show poor response to the current therapies that do not target pathways implicated in their pathogenesis. We have previously detected ABCC2 (an ABC transporter), VEGF, and mTOR pathways to be enriched in PRCC2. In this study, we assess the therapeutic potential of targeting these pathways in PRCC2. Twenty RCC cell lines from the Cancer Cell Encyclopedia were compared to the Cancer Genome Atlas PRCC cohort (290), to identify representative PRCC2 cell lines. Cell lines were further validated in xenograft models. Selected cell lines were treated in vitro and in vivo (mice models) under five different conditions, untreated, anti-VEGF (sunitinib), ABCC2 blocker (MK571), mTOR inhibitor (everolimus) and sunitinib + MK571. Sunitinib +ABCC2 blocker group showed a significant response to therapy compared to the other treatment groups both in vitro (P ≤ 0.0001) and in vivo (P = 0.0132). ABCC2 blockage resulted in higher sunitinib uptake, both in vitro (P = 0.0016) and in vivo (P = 0.0031). Everolimus group demonstrated the second best response in vivo. The double-treatment group showed the highest apoptotic rate and lowest proliferation rate. There is an urgent need for individualized therapies of RCC subtypes that take into account their specific biology. Our results demonstrate that combined targeted therapy with sunitinib and ABCC2 blocker in PRCC2 has therapeutic potential. The results are likewise potentially significant for other ABCC2 high tumors. However, the results are preliminary and clinical trials are needed to confirm these effects in PRCC2 patients.

Melanotic MiT family translocation neoplasms: Expanding the clinical and molecular spectrum of this unique entity of tumors.

MiT family translocation tumors are a group of neoplasms characterized by translocations involving MiT family transcription factors. The translocation renal cell carcinomas, TFE3 (Xp11.2) and TFEB (t6;11) are known members of this family. Melanotic Xp11 translocation renal cancer is a more recently described entity. To date only 14 cases have been described. It is characterized by a distinct set of features including a nested epithelioid morphology, melanin pigmentation, labeling for markers of melanocytic differentiation, lack of labeling for markers of renal tubular differentiation, predominance in a younger age population and association with aggressive clinical behavior. There are noted similarities between that entity and TFE3 associated PEComas. There are no cases reported of equivalent melanotic TFEB translocation renal cancer. We report 2 rare cases of melanotic translocation renal neoplasms. The first is a melanotic TFE3 translocation renal cancer with an indolent clinical course, occurring in a patient more than 3-decades older than the usual average age in which such tumors have been described. The other case is, to our knowledge, the first reported melanotic TFEB translocation cancer of the kidney. Both cases exhibit the same H&E morphology as previously reported in melanotic translocation renal cancers and label accordingly with HMB45 and Melan-A. While the TFE3 melanotic tumor lacked any evidence of renal tubular differentiation, the TFEB melanotic cancer exhibited some staining for renal tubular markers. Based on the unique features noted above, these two cases expand the clinical and molecular spectrum of the melanotic translocation renal cancers.

Toward Biological Subtyping of Papillary Renal Cell Carcinoma With Clinical Implications Through Histologic, Immunohistochemical, and Molecular Analysis.

Papillary renal cell carcinoma (PRCC) has 2 histologic subtypes. Almost half of the cases fail to meet all morphologic criteria for either type, hence are characterized as PRCC not otherwise specified (NOS). There are yet no markers to resolve the PRCC NOS category. Accurate classification can better guide the management of these patients. In our previous PRCC study we identified markers that can distinguish between the subtypes. A PRCC patient cohort of 108 cases was selected for the current study. A panel of potentially distinguishing markers was chosen from our previous genomic analysis, and assessed by immunohistochemistry. The panel exhibited distinct staining patterns between the 2 classic PRCC subtypes; and successfully reclassified the NOS (45%) cases. Moreover, these immunomarkers revealed a third subtype, PRCC3 (35% of the cohort). Molecular testing using miRNA expression and copy number variation analysis confirmed the presence of 3 distinct molecular signatures corresponding to the 3 subtypes. Disease-free survival was significantly enhanced in PRCC1 versus 2 and 3 (P=0.047) on univariate analysis. The subtypes stratification was also significant on multivariate analysis (P=0.025; hazard ratio, 6; 95% confidence interval, 1.25-32.2). We propose a new classification system of PRCC integrating morphologic, immunophenotypical, and molecular analysis. The newly described PRCC3 has overlapping morphology between PRCC1 and PRCC2, hence would be subtyped as NOS in the current classification. Molecularly PRCC3 has a distinct signature and clinically it behaves similar to PRCC2. The new classification stratifies PRCC patients into clinically relevant subgroups and has significant implications on the management of PRCC.