Priya Rao

Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma.

Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib-treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line 786-O was chronically treated with sunitinib and assayed for AXL, MET, epithelial–mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by short hairpin RNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets vascular endothelial growth factor receptor, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT-associated gene expression changes, including upregulation of Snail and β-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/human umbilical vein endothelial cell co-culture models. The suppression of AXL or MET expression and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes prometastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

Everolimus Versus Sunitinib Prospective Evaluation in Metastatic Non–Clear Cell Renal Cell Carcinoma (ESPN): A Randomized Multicenter Phase 2 Trial

BACKGROUND Sunitinib and everolimus are standard first-line and second-line therapies, respectively, in clear cell renal cell carcinoma (ccRCC). OBJECTIVE To conduct a randomized phase 2 trial comparing sunitinib and everolimus in non-clear cell RCC (non-ccRCC). DESIGN, SETTING, AND PARTICIPANTS Patients with metastatic, non-ccRCC, or ccRCC with >20% sarcomatoid features (ccSRCC) were randomized to receive sunitinib or everolimus with crossover at disease progression. OUTCOME MEASUREMENT AND STATISTICAL ANALYSIS Primary end point was progression-free survival (PFS) in first-line therapy; 108 patients were needed to show improvement in median PFS (mPFS) from 12 wk with sunitinib to 20 wk with everolimus. RESULTS AND LIMITATIONS Interim analysis of 68 patients (papillary [27], chromophobe [12], unclassified [10], translocation [7], ccSRCC [12]) prompted early trial closure. The mPFS in first-line therapy was 6.1 mo with sunitinib and 4.1 mo with everolimus (p=0.6); median overall survival (mOS) was not reached with sunitinib and was 10.5 mo with everolimus, respectively (p=0.014). At final analysis, mOS was 16.2 and 14.9 mo with sunitinib and everolimus, respectively (p=0.18). There were four partial responses (PRs) in first-line therapy (sunitinib: 3 of 33 [9%]; everolimus, 1 of 35 [2.8%]) and four PRs in second-line therapy (sunitinib: 2 of 21 [9.5%]; everolimus, 2 of 23 [8.6%]), with mPFS of 1.8 mo and 2.8 mo, respectively. In patients without sarcomatoid features in their tumors (n=49), mOS was 31.6 mo with sunitinib and 10.5 mo with everolimus (p=0.075). Genomic profiling of a chromophobe RCC from a patient with a PR to first-line everolimus revealed a somatic TSC2 mutation. CONCLUSIONS In this trial, everolimus was not superior to sunitinib. Both agents demonstrated modest efficacy, underscoring the need for better therapies in non-ccRCC. PATIENT SUMMARY This randomized phase 2 trial provides the first head-to-head comparison of everolimus and sunitinib in patients with metastatic non-clear cell renal cell carcinoma (non-ccRCC). The observed very modest efficacy underscores the need to develop more effective therapies for non-ccRCC.

Primary vascular tumors and tumor-like lesions of the kidney: a clinicopathologic analysis of 25 cases.

Vascular tumors of the kidney are distinctly rare, and to date no large series have been reported. We analyzed a series of primary vascular tumors of the kidney to further delineate their clinicopathologic features and identify organ-specific morphologic features, if present. Twenty-five renal cases previously coded as “arteriovenous malformation,” “hemangioma,” and “angiosarcoma” were retrieved from the archives of 4 collaborating institutions and were reevaluated histologically. Tumors were classified according to the 2002 World Health Organization classification of tumors of soft tissue and bone. There were 18 males and 7 females (M:F=2.6:1) ranging from 21 to 95 years (mean 56.7 y). Lesions ranged from “microscopic” to 30 cm (mean 6.0 cm) and were tan-brown, cystic, and hemorrhagic. On re-review, cases were classified as arteriovenous malformation (n=3), capillary hemangioma (n=14), and angiosarcoma (n=8). Arteriovenous malformations were identical to their somatic soft tissue counterparts. Renal capillary hemangiomas often lacked a well-formed lobular pattern and 5 cases showed a “sieve-like” arrangement reminiscent of splenic sinusoids, a pattern previously noted by others (anastomosing hemangioma). All hemangiomas were noninfiltrative and lacked cytologic atypia and mitotic activity. GLUT-1, D2-40, and CD8 were performed in 3 anastomosing hemangiomas and were all negative. Angiosarcomas were diffusely infiltrative with extensive parenchymal destruction; all showed at least small areas of conventional vasoformative growth, but were frequently dominated by spindled and epithelioid histology. All cases were positive for some combination of vascular tumor-associated markers (CD31, CD34, and FLI-1). Cytokeratin expression was absent in all angiosarcomas. Follow-up was available for 15 cases: all patients with arteriovenous malformation and hemangioma with follow-up were disease free after complete excision; 4 cases of angiosarcoma died of the disease at 1, 1, 6, and 11 months. Our review shows that many capillary hemangiomas of the kidney are morphologically distinctive tumors, which often show “spleen-like” or “anastomosing” features. Angiosarcomas of the kidney are highly aggressive tumors with poor outcome and may have morphologic features (spindling and epithelioid change), which could result in confusion with sarcomatoid carcinomas and other renal mesenchymal tumors.

Resistance to Antiangiogenic Therapy Is Associated with an Immunosuppressive Tumor Microenvironment in Metastatic Renal Cell Carcinoma

Therapeutic PD-1/PD-L1 blockade requires preexisting tumor-infiltrating T cells. In a subset of metastatic RCC patients, antiangiogenic therapy increased T-cell infiltration and PD-L1 upregulation, increasing the likelihood that they may uniquely benefit from combination checkpoint and antiangiogenic therapy. Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer, and antiangiogenic therapy is the current mainstay of treatment. Patients with RCC develop innate or adaptive resistance to antiangiogenic therapy. There is a need to identify biomarkers that predict therapeutic resistance and guide combination therapy. We assessed the interaction between antiangiogenic therapy and the tumor immune microenvironment and determined their impact on clinical outcome. We found that antiangiogenic therapy–treated RCC primary tumors showed increased infiltration of CD4+ and CD8+ T lymphocytes, which was inversely related to patient overall survival and progression-free survival. Furthermore, specimens from patients treated with antiangiogenic therapy showed higher infiltration of CD4+FOXP3+ regulatory T cells and enhanced expression of checkpoint ligand programed death-ligand 1 (PD-L1). Both immunosuppressive features were correlated with T-lymphocyte infiltration and were negatively related to patient survival. Treatment of RCC cell lines and RCC xenografts in immunodeficient mice with sunitinib also increased tumor PD-L1 expression. Results from this study indicate that antiangiogenic treatment may both positively and negatively regulate the tumor immune microenvironment. These findings generate hypotheses on resistance mechanisms to antiangiogenic therapy and will guide the development of combination therapy with PD-1/PD-L1–blocking agents. Cancer Immunol Res; 3(9); 1017–29. ©2015 AACR.

Can MDM2 analytical tests performed on core needle biopsy be relied upon to diagnose well-differentiated liposarcoma?

Well-differentiated liposarcoma/atypical lipomatous tumor can be difficult to differentiate from benign lipomatous tumors, especially on limited biopsy material. Adjunctive tests for MDM2 (murine double minute 2) have proven useful in whole-tissue sections; however, their utility has not been determined within the increasingly popular core needle biopsy. Herein, we compare the ability of MDM2 immunohistochemistry and MDM2 fluorescence in situ hybridization (FISH) to discriminate benign lipomatous tumors from well-differentiated liposarcoma on core needle biopsies. Well-differentiated liposarcoma (n=17) and an assortment of benign lipomatous tumors (n=37), which had concurrent or previous core needle biopsies, and resection specimens were subjected to both MDM2 immunohistochemistry and MDM2 FISH on both whole-tissue sections and corresponding core needle biopsy sections. Percentage tumor cells positive for MDM2 by immunohistochemistry and an MDM2:CEP12 FISH ratio was calculated in each biopsy and resection specimen pair and the results were compared. MDM2 FISH had a higher sensitivity (100%) and specificity (100%) compared with MDM2 immunohistochemistry (65 and 89%) in core needle biopsies, respectively. In addition, MDM2 immunohistochemistry had a false-positive rate of 11%, compared to 0% with FISH. The average MDM2:CEP12 ratio was similar in the biopsy material compared with the whole-tissue sections in both well-differentiated liposarcoma and the benign lipomatous tumor group of neoplasms. Detection of MDM2 amplification by FISH is a more sensitive and specific adjunctive test than MDM2 immunohistochemistry to differentiate well-differentiated liposarcoma from various benign lipomatous tumors, especially on limited tissue samples.

Collecting duct carcinoma versus renal medullary carcinoma: An appeal for nosologic and biological clarity

Mahul B. Amin, MD,* Steven C. Smith, MD, PhD,* Abbas Agaimy, MD,w Pedram Argani, MD,z Eva Marie Compérat, MD, PhD,y Brett Delahunt, MD, FRCPA, FRCPath,8 Jonathan I. Epstein, MD,z John N. Eble, MD,z David J. Grignon, MD,z Arndt Hartmann, MD,w Ondřej Hes, MD, PhD,# Michelle S. Hirsch, MD, PhD,** Rafael E. Jimenez, MD,ww Lakshmi P. Kunju, MD,zz Guido Martignoni, MD,yy Jesse K. McKenney, MD,88 Holger Moch, MD,zz Rodolfo Montironi, MD,## Gladell P. Paner, MD,*** Priya Rao, MD,www John R. Srigley, MD,zzz Satish K. Tickoo, MD,yyy and Victor E. Reuter, MDyyy

Immunohistochemical evaluation of novel and traditional markers associated with urothelial differentiation in a spectrum of variants of urothelial carcinoma of the urinary bladder

Data on immunohistochemical expression of novel and traditional urothelial markers in the wide range of urothelial carcinoma variants have so far been very limited. In this study, whole tissue sections from 130 bladder urothelial carcinoma and variants were stained with a panel of novel and traditional immunomarkers supportive of urothelial lineage. The positivity rates were as follows: (a) urothelial carcinomas with or without divergent differentiation: GATA3 (50%), S-100P (86%), uroplakin III (20%), thrombomodulin (40%), cytokeratin 7 (CK7) (80%), CK20 (55%), p63 (87%), and high molecular weight cytokeratin (HMCK) (89%); (b) urothelial carcinoma variants (micropapillary, plasmacytoid, nested, clear cell, and microcystic): GATA3 (88%), S-100P (96%), uroplakin III (33%), thrombomodulin (49%), CK7 (95%), CK20 (61%), p63 (69%), and HMCK (96%); and (c) undifferentiated carcinomas (lymphoepithelioma-like carcinoma, small cell carcinoma, sarcomatoid carcinoma and carcinoma with rhabdoid and giant cells): GATA3 (28%), S-100P (31%), uroplakin III (0%), thrombomodulin (22%), CK7 (50%), CK20 (3%), p63 (50%), and HMCK (49%). In urothelial carcinoma with squamous differentiation, GATA3 expression was lower (20%) in contrast to p63 and S-100P. In urothelial carcinoma with glandular differentiation, GATA3 (50%) and p63 (60%) expression was lower than S-100P (100%). p63 expression was relatively lower in micropapillary (54%) and plasmacytoid (50%) variants compared with the other urothelial carcinoma variants. This study provides comprehensive data for novel and traditionally used markers to support urothelial lineage in urothelial carcinoma variants. Our findings show that GATA3, S-100P, CK7, CK20, HMCK, and p63, in the appropriate differential diagnostic setting, are useful to support urothelial lineage of variant morphologies.

Protocol for the Examination of Specimens From Patients With Merkel Cell Carcinoma of the Skin

The College of American Pathologists offers these protocols to assist pathologists in providing clinically useful and relevant information when reporting results of surgical specimen examinations. The College regards the reporting elements in the ‘‘Surgical Pathology Cancer Case Summary (Checklist)’’ portion of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice. The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with these documents. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of these documents. PROTOCOL FOR THE EXAMINATION OF SPECIMENS FROM PATIENTS WITH MERKEL CELL CARCINOMA OF THE SKIN This protocol applies to Merkel cell carcinoma of cutaneous surfaces only. The seventh edition TNM staging system for Merkel cell carcinoma of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) is recommended.

Clear cell-papillary renal cell carcinoma of the kidney not associated with end-stage renal disease: Clinicopathologic correlation with expanded immunophenotypic and molecular characterization of a large cohort with emphasis on relationship with renal angiomyoadenomatous tumor

Clear cell-papillary renal cell carcinoma (CC-Pap RCC) is a recently described renal tumor initially reported in the setting of end-stage renal disease (ESRD). It has unique morphologic and immunohistochemical features that differentiate it from the more common clear cell RCC and papillary RCC. Recently, these tumors have also been described in a sporadic setting. We studied 64 cases of CC-Pap RCC not associated with ESRD (57 CC-Pap RCCs and 7 cases with features of renal angiomyoadenomatous tumors [RAT] including 5 initially diagnosed as such). The morphologic features of all cases and the immunohistochemical profile of 59 cases were studied along with the clinical and molecular features of 30 and 12 cases, respectively. All the tumors were well circumscribed with a mean tumor size of 2.6 cm and showed a wide array of architectural patterns, usually mixed, including tubular (77%), papillary (62%), tubulocystic (52%), and compact nested (21%). Seventy-three percent of the cases showed areas in which the tumor nuclei had a distinct orientation away from the basement membrane. Ninety-two percent of the cases had a low Fuhrman nuclear grade (nuclear grade 2%‐86%, and nuclear grade 1%‐6%); however, 8% cases showed foci of Fuhrman nuclear grade 3. In 4 cases, epithelial tumor comprised <5% of the tumor; >95% of the tumor was cystic or hyalinized. The stroma varied from being minimal to occasionally prominent myxoid to hyalinized and rarely with organized amianthoid fibers or well-defined smooth muscle bundles. Pathologic stage was reliably assigned in 60 cases, of which 93.3% (56 cases) were pT1, 3.3% (2 cases) were pT2, and 3.3% (2 cases) were pT3a with extension into the perinephric fat. One case had coagulative necrosis; sarcomatoid change and vascular invasion was not identified. The tumors showed a fairly typical immunoprofile characterized by positivity for CK7 (100%), HMCK (96%), CAIX (94%), and vimentin (100%) with negativity for AMACR, RCC, and TFE3; CD10 was positive in 24%. None of the cases tested showed recurrent chromosomal imbalances by virtual karyotyping, fluorescence in situ hybridization, or 3p loss of heterozygosity analysis. VHL gene mutations were, however, noted in 3 cases (2 in exon 1 and 1 in exon 3). Clinical follow-up information was available in 47% of the patients, with a mean and median follow-up of 47 and 37 months, respectively (range, 18 to 108 mo). One case occurred in the setting of VHL syndrome and multiple benign cysts. None of the cases showed local recurrence, metastasis, or death due to disease. Morphology, immunophenotype, and molecular studies did not vary between typical cases, those with prominent smooth muscle (so-called RAT), and historically published data on cases occurring in ESRD. Our analysis confirms that CC-Pap RCC is a unique subtype of adult renal epithelial neoplasia in which tumors are frequently small, are of low nuclear grade and pathologic stage, and have extremely favorable short to intermediate range prognosis. Tumors occurring sporadically, with prominent smooth muscle stroma (so-called RAT), and occurring in ESRD are in the spectrum of the same category of tumors.

Angiosarcoma: A tissue microarray study with diagnostic implications

Background:Angiosarcoma (AS) is a rare soft tissue sarcoma showing endothelial differentiation as indicated by morphology and expression of CD31 (blood), D2-40 (lymphatic), factor VIII, and CD34 (both). We sought to examine the pattern of immunohistochemical markers of differentiation in AS and correlate these with outcome. Design:An AS tissue microarray (n = 70 specimens) was constructed for immunohistochemical analysis of CD31, CD34, factor VIII, D2-40, and pan-cytokeratin. Samples on this array were linked to clinicopathologic and outcome data for these patients. Univariate analyses were used to explore disease-specific survival (DSS) factors. Results:Nine metastatic, 23 localized, and 4 recurrent cases were included. Information about the tissue status (ie, primary or metastasis) was unavailable in 4 patients. Primary sites for the tumor included bone (n = 1), breast parenchyma (n = 11), breast skin (n = 4), heart (n = 5), skin (n = 8), soft tissue (n = 7), and unknown (n = 3). Three patients presented with multifocal disease (primary sites in these patients included breast, skin, and soft tissue). Metastatic sites included lung, bone, lymph nodes, brain, liver, and parotid. Of the 40 cases, 8 (20%) showed a pure or predominant epithelioid histology. Of the biomarkers evaluated by tissue microarray, 92% of tumors expressed at least one endothelial marker (factor VIII = 83%, CD31 = 80%, CD34 = 63%, and D2-40 = 43%) with 88% expressing 2 or more markers. Eighty-eight percent of tumors expressing D2-40 coexpressed CD31, an unusual combination in normal vessels. No endothelial marker clearly associated with disease-specific survival. Fifty percent (4/8) of epithelioid cases and 9% (3/32) of nonepithelioid cases showed keratin expression. Conclusions:Unusual patterns and loss of endothelial markers are common in AS, suggesting use of multiple markers in challenging cases and perhaps indicating important biologic characteristics.