Histopathologic regression in patients with primary cutaneous melanoma undergoing sentinel lymph node biopsy is associated with favorable survival and, after metastasis, with improved progression-free survival on immune checkpoint inhibitor therapy: A single-institutional cohort study.

BACKGROUND: Histopathologic regression of cutaneous melanoma is considered a favorable prognostic factor, but its significance in clinical practice remains controversial. OBJECTIVE: To investigate the prognostic importance of regression in patients with primary cutaneous melanoma undergoing sentinel lymph node (SLN) biopsy and to assess its significance in patients progressing to an unresectable stage requiring systemic therapy. METHODS: We retrospectively reviewed patients with newly diagnosed melanoma undergoing SLN biopsy between 2010 and 2015 and available information on histopathologic regression (n = 1179). Survival data and associations of clinical variables with SLN status were assessed. RESULTS: Patients with regressive melanoma showed favorable relapse-free (hazard ratio [HR], 0.52; P = .00013), distant metastasis-free (HR, 0.56; P = .0020), and melanoma-specific survival (HR, 0.35; P = .00053). Regression was associated with negative SLN (odds ratio, 0.48; P = .0077). In patients who progressed to an unresectable stage, regression was associated with favorable progression-free survival under immune checkpoint inhibition (HR, 0.43; P = .031) but not under targeted therapy (HR, 1.14; P = .73) or chemotherapy (HR, 3.65; P = .0095). LIMITATIONS: Retrospective, single-institutional design. CONCLUSIONS: Regression of cutaneous melanoma is associated with improved prognosis in patients eligible for SLN biopsy as well as in patients with unresectable disease receiving systemic therapy with immune checkpoint inhibitors.

Diagnostic and prognostic classification of atypical spitzoid tumours based on histology and genomic aberrations: A prospective cohort study with long-term follow-up.

BACKGROUND: Histological classification of atypical spitzoid tumours (ASTs) is unreliable, and categorisation of these lesions into benign and malignant is poorly reproducible. Here, we classified ASTs based on histology and chromosomal aberrations and explored the prognostic significance of genomic aberrations in a prospective cohort with a long-term follow-up. PATIENTS AND METHODS: Histologically equivocal ASTs from 76 patients were analysed by array comparative genomic hybridisation (aCGH). Tumours were histologically assessed by a panel of dermatopathologist before and after aCGH and classified as benign, ambiguous or malignant. Chromosomal aberrations were correlated with an outcome. RESULTS: Chromosomal aberrations were detected in 45 (59%) of 76 ASTs (median age: 16 years, range: 0-74; median follow-up: 90 months, range: 13-153). The initial histological diagnosis was changed upon presentation of aCGH results in 36 of 76 cases (47%). The final diagnostic interpretation classified 61% of the lesions as benign, 18% as ambiguous and 21% as malignant. Positive sentinel lymph node biopsies (6+/29) occurred at similar rates in all diagnostic groups (P = 0.83) and were not associated with an unfavourable outcome. Two patients had local recurrences, but none of the patients developed metastasis beyond the sentinel lymph node. CONCLUSIONS: All ASTs had an excellent prognosis, even in cases with worrisome morphology and chromosomal aberrations. With no distant metastasis or death in long-term follow-up of 76 patients, no correlation between chromosomal aberrations and prognosis was possible. However, it seems likely that in larger cohorts, metastases would arise in cases with complex aberrations and these patients should undergo clinical follow-up.

Micrographic Surgery Allows Fascia Preservation in Dermatofibro-sarcoma Protuberans.

Removal of the deep fascia is recommended in therapy for dermatofibrosarcoma protuberans, but its necessity in the context of micrographic surgery is unclear. A retrospective clinicopathological analysis of 48 patients with dermatofibrosarcoma protuberans treated by micrographic surgery was performed, to determine in which tumours fascia preservation was feasible and safe. Histologically, 93% of tumours on the trunk and extremities and 14% of tumours in the head and neck region were fully located above the fascia. Localization on the head and neck was the only significant risk factor for tumour extension beyond the subcutis (p<0.001). Overall, 44% of tumours were completely excised above the fascia and 56% with deeper excisions. Two deeply infiltrating tumours (4%) on the head recurred, but in none of these lesions was the fascia spared. These results show that micrographic surgery allows fascia preservation in superficial tumours outside the head and neck region.

Diffuse PRAME Expression Is Highly Specific for Thin Melanomas in the Distinction from Severely Dysplastic Nevi but Does Not Distinguish Metastasizing from Non-Metastasizing Thin Melanomas.

BACKGROUND: PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemistry is increasingly used as diagnostic adjunct in the evaluation of melanocytic tumors. The expression and prognostic significance of PRAME in melanomas ≤1.0 mm and its diagnostic utility in the distinction from severely dysplastic compound nevi (SDN) have not been studied. METHODS: We investigated and compared the immunohistochemical PRAME expression in 70 matched thin metastasizing and non-metastasizing melanomas and 45 nevi from patients with long-term follow-up (35 SDN and 10 unequivocally benign compound nevi). RESULTS: Diffuse PRAME staining in >75% of lesional epidermal and dermal melanocytes identified 58.6% of thin melanomas but did not distinguish metastasizing from non-metastasizing melanomas (p = 0.81). A superficial atypical melanocytic proliferation of uncertain significance, in which the final diagnostic interpretation favored a SDN was the only nevus with diffuse PRAME expression (1/45). Melanomas and SDN with PRAME immunoreactivity exhibited different staining patterns. Most melanomas (67.6%) showed uniform PRAME expression in the in situ and invasive component, whereas most SDN (81.0%) showed a decreasing gradient with depth. CONCLUSION: Diffuse intraepidermal and dermal PRAME staining is highly specific for melanomas in the distinction from SDN. PRAME expression is not a prognostic biomarker in melanomas ≤1.0 mm.

Pretreatment metastatic growth rate determines clinical outcome of advanced melanoma patients treated with anti-PD-1 antibodies: a multicenter cohort study.

BACKGROUND: Checkpoint inhibitors revolutionized the treatment of metastatic melanoma patients. Although tumor burden and lactate dehydrogenase (LDH) are associated with overall survival (OS), the impact of tumor growth kinetics remains elusive and in part contradictory. The aims of this study were to develop a novel simple and rapid method that estimates pretreatment metastatic growth rate (MGR) and to investigate its prognostic impact in melanoma patients treated with antiprogrammed death receptor-1 (PD-1) antibodies. METHODS: MGR was assessed in three independent cohorts of a total of 337 unselected consecutive metastasized stage IIIB-IV melanoma patients (discovery cohort: n=53, confirmation cohort: n=126, independent multicenter validation cohort: n=158). MGR was computed during the pretreatment period before initiation of therapy with anti-PD-1 antibodies nivolumab or pembrolizumab by measuring the increase of the longest diameter of the largest target lesion. Tumor doubling time served as quality control. Kaplan-Meier analysis and univariable as well as multivariable Cox regression were used to examine the prognostic impact of MGR. RESULTS: Pretreatment MGR >3.9 mm/month was associated with impaired OS in the discovery cohort (HR 6.19, 95% CI 2.92 to 13.10, p<0.0001), in the confirmation cohort (HR 3.62, 95% CI 2.19 to 5.98, p<0.0001) and in the independent validation cohort (HR 2.57, 95% CI 1.56 to 4.25, p=0.00023). Prior lines of systemic treatment did not influence the significance of MGR. Importantly, the prognostic impact of MGR was independent of total tumor burden, diameter of the largest metastasis, number of prior lines of systemic treatment, LDH, as well as liver and brain metastasis (discovery and confirmation cohorts: both p<0.0001). Superiority of MGR compared with these variables was confirmed in the independent multicenter validation cohort (HR 2.92, 95% CI 1.62 to 5.26, p=0.00036). CONCLUSIONS: High pretreatment MGR is an independent strong prognostic biomarker associated with unfavorable survival of melanoma patients receiving anti-PD-1 antibodies. Further investigations are warranted to assess the predictive impact of MGR in distinct systemic therapeutic regimens.

Outcome of melanoma patients with elevated LDH treated with first-line targeted therapy or PD-1-based immune checkpoint inhibition.

BACKGROUND: Elevated lactate dehydrogenase (LDH) is a known predictive and prognostic factor for a poor outcome in patients with metastatic melanoma. It is unclear whether first-line targeted therapy (TT) or immune checkpoint inhibition (ICI) is more beneficial in melanoma patients with elevated LDH because prospective studies in this area are lacking. METHODS: This multicentre retrospective cohort study was conducted at 25 melanoma centres worldwide to analyse progression-free survival (PFS) and overall survival (OS) among melanoma patients with elevated LDH. The role of confounders was addressed by using inverse probability of treatment weighting. RESULTS: Among 173 BRAFV600-mutant patients, PFS at 12 months in the TT group was 22% compared with 52% in the combined anti-PD-1 and anti-CTLA-4 group (HR 0.6, 95% CI 0.4-1.0, p = 0.07) and 18% in the anti-PD-1 monotherapy group (HR 1.8, 95% CI 1.2-2.8, p = 0.003). Twelve months' OS was 48% in the TT group compared with 83% in the combined anti-PD-1 and anti-CTLA-4 group (HR 0.5, 95% CI 0.3-1.0, p = 0.03) and 50% in the anti-PD-1 monotherapy group (HR 1.2, 95% CI 0.8-2.0, p = 0.37). The ORR in the TT group was 63%, compared with 55% and 20% in the combined anti-PD-1 and anti-CTLA-4 and anti-PD-1 monotherapy group, respectively. Among 314 patients receiving ICI first-line, PFS at 12 months was 33% in the anti-PD-1 group versus 38% in the combined anti-PD-1 and anti-CTLA-4 group (HR 0.8, 95% CI 0.6-1.0; p = 0.07). OS at 12 months was 54% in the anti-PD-1 group versus 66% in the combined ICI group (HR 0.7, 95% CI 0.5-1.0; p = 0.03). The ORR was 30% in the anti-PD-1 monotherapy group and 43% in the combined anti-PD-1 and anti-CTLA-4 group. Results from multivariate analysis confirmed the absence of qualitative confounding. CONCLUSIONS: Among BRAF-mutant patients with elevated LDH, combined anti-PD-1 and anti-CTLA-4 blockade seems to be associated with prolonged OS compared with first-line TT. Among patients receiving ICI as a first-line treatment, OS appears to be longer for the combination of anti-PD-1 and anti-CTLA-4 than for anti-PD-1 alone.

Serum S100B and LDH at Baseline and During Therapy Predict the Outcome of Metastatic Melanoma Patients Treated with BRAF Inhibitors.

BACKGROUND: Despite impressive response rates, most patients with advanced melanoma ultimately progress following therapy with B-Raf proto-oncogene (BRAF) inhibitors (BRAFi). Therefore, frequent radiologic assessments are necessary, and reliable serum biomarkers would be beneficial for disease monitoring. OBJECTIVE: This study investigated the ability of lactate dehydrogenase (LDH) and S100 calcium-binding protein B (S100B) to detect response and disease progression during treatment with BRAFi. PATIENTS AND METHODS: Baseline levels of LDH and S100B and repeated measurements during therapy were recorded retrospectively in 191 patients with metastatic melanoma. LDH and S100B levels were compared between distinct time points (baseline, first follow-up visit [FV], best objective response [BR], and progressive disease [PD]). The prognostic ability of the serum biomarkers in relation to disease-specific survival (DSS) was assessed with univariable and multivariable Cox regression analysis. RESULTS: Elevated baseline LDH and S100B correlated with impaired DSS. In contrast with LDH (P = 0.12), S100B levels at FV correlated with response (P = 0.0030). Both markers significantly decreased during the first weeks of BRAFi treatment (LDH, P = 0.00034; S100B, P < 0.0001) and increased between BR and PD (LDH, P = 0.016; S100B, P < 0.0001). Patients with elevated S100B (P = 0.00062) but not with elevated LDH (P = 0.067) at the time point of radiologically confirmed PD showed significantly impaired DSS after PD. Interestingly, DSS after PD differed significantly according to S100B levels determined as early as 8 weeks (median) before PD (P = 0.0024). CONCLUSIONS: LDH and S100B are suitable serum biomarkers during therapy with BRAFi. S100B shows stronger correlation with response and exhibits more accuracy in predicting PD. Close biomarker monitoring with S100B is recommended during treatment with BRAFi to detect PD early.

Histological and Immunohistochemical Changes of Congenital Melanocytic Nevi With Age.

ABSTRACT: Clinical but not histological changes of congenital melanocytic nevi (CMN) with age are well characterized. Our objective was to analyze histological changes of CMN with age and discuss possible clinical implications of our findings. We investigated serial excisions of 21 patients with CMN and compared histological and immunohistochemical features over time. The median number of serial excisions was 6 [interquartile range (IQR) 5-7], the median age at the first excision was 12 months (IQR 5-98), and the median time between the first and last analyzed excision was 53 months (IQR 45-64). The projected adult size of the excised CMN was "large" or "giant" in 14 of the 21 CMN (67%) and "medium" in the remaining lesions (33%). Nineteen CMN (90%) involved the subcutaneous fat, and 16 of the 21 CMN (76%) reached the lower surgical margin. The histological pattern and depth did not change over time but the cellularity and HMB-45 expression of dermal melanocytes decreased in 16 of the 21 patients (76%) and in 15 of the 21 patients (71%), respectively (both P < 0.001). Patients with decreasing HMB-45 expression were significantly younger at the first excision (median 6 months, IQR 4-28) than patients with unchanged HMB-45 expression (median 176 months, IQR 12-186; P = 0.018). The expression of Ki-67 and p16 did not change significantly with age. Our study demonstrates that (1) the cellularity and pigment production of CMN decreases with age, (2) the histological pattern and extension in depth remain stable, and (3) clear resection margins can rarely be achieved in larger CMN.

Prognostic role of gamma-glutamyl transferase in metastatic melanoma patients treated with immune checkpoint inhibitors.

BACKGROUND: Hepatic immune-related adverse events (irAE) including elevated liver function tests (transaminases) occur in 1.4-22.3% of melanoma patients receiving immune checkpoint inhibitors (ICPI) and constitute a potentially serious toxicity that is challenging to treat. In contrast to the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST), only little is known about the frequency and impact of gamma-glutamyl transferase (GGT) elevations. METHODS: GGT determined prior to and during therapy of metastatic melanoma patients treated with ICPI were retrospectively assessed in two independent cohorts (PD-1: n = 218, Ipi + Nivo: n = 148). Overall survival (OS) and best objective response were analyzed according to baseline and immune-related GGT (irGGT) elevations during treatment. RESULTS: In multivariate analysis, OS was reduced in patients with elevated baseline GGT (PD-1 group: hazard ratio [HR] 1.76, p = .0073; Ipi + Nivo group: HR 1.77, p = .032). Immune-related GGT elevation was recorded in 17% (PD-1 group) and 38.5% (Ipi + Nivo group). Of these patients, the majority (81 and 68%, respectively) had normal ALT and AST and showed no clinical signs of hepatotoxicity. Patients who experienced irGGT elevation had superior response (PD-1 group: odds ratio [OR] 3.57, p = .00072; Ipi + Nivo group: OR 1.74, p = .12) and OS (PD-1 group: HR 0.37, p = .0016; Ipi + Nivo group: HR 0.33, p = .00050). CONCLUSIONS: The frequency of hepatic irAE is currently underestimated. The addition of the sensitive enzyme GGT to the laboratory panel before and during therapy with ICPI allows to detect two to three times more patients developing hepatic or hepatobiliary toxicity than known so far. Immune-related GGT elevations correlate with response and favorable survival. Precis for use in the Table of Contents The frequency of hepatotoxicity under immune checkpoint blockade is currently underestimated. We suggest the addition of gamma-glutamyl transferase to the laboratory panel in checkpoint inhibitor patients for the detection of hepatobiliary toxicity.

Expression of DNA Methyltransferase 1 Is a Hallmark of Melanoma, Correlating with Proliferation and Response to B-Raf and Mitogen-Activated Protein Kinase Inhibition in Melanocytic Tumors.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy.

Absolute and relative differential blood count predicts survival of AJCC stage I-II melanoma patients scheduled for sentinel lymph node biopsy.

BACKGROUND/OBJECTIVES: Elevated neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is associated with poor overall survival (OS) in metastatic melanoma patients receiving immunotherapy. However, the impact of peripheral blood cells in patients undergoing sentinel lymph node biopsy (SLNB) is still unclear. This study was intended to characterize the impact of peripheral blood leukocytic cells on overall survival (OS) in melanoma patients undergoing SLNB. METHODS: A total of 1412 AJCC stage I-II melanoma patients scheduled for SLNB at a single institution in the period 2010-2015 with available perioperative blood tests were randomly assigned to two independent cohorts. Associations of peripheral blood leukocytes with OS were analysed using Kaplan-Meier estimator and multivariate Cox proportional hazards model. RESULTS: NLR >4.26, absolute neutrophil count >5800/µL, relative neutrophil count >69.7% and relative lymphocyte count ≤ 17.5% were significantly associated with reduced OS in both cohorts. Absolute monocytes >810/µL, absolute eosinophils ≤200/µL, relative monocytes >6.6%, relative eosinophils ≤2.7% and relative basophils ≤0.6% were significantly associated with reduced OS in one cohort each. On multivariate analysis, a combined score including absolute levels of neutrophils, lymphocytes, monocytes and eosinophils was significantly associated with OS in both cohorts. The hazard ratio of patients with a risk score of 3-4 was 5.42 (95% confidence interval: 1.52-19.42, P = 0.0094) in cohort 1 and 9.42 (2.06-43.06, P = 0.0038) in cohort 2, respectively. CONCLUSIONS: We conclude that peripheral blood leukocytes are independently associated with OS in stage I-II melanoma patients and should be considered as prognostic markers in these patients. Eosinophils and basophils deserve more attention in future investigations.

Aplasia cutis congenita of the scalp: Histopathologic features and clinicopathologic correlation in a case series.

BACKGROUND: Aplasia cutis congenita (ACC) is a rare and heterogeneous disorder characterized by congenital absence of skin. The scalp is the most commonly affected site and lesions may overlie deeper ectodermal abnormalities. The exact etiology is still unknown, and histopathologic features are poorly defined. METHODS: A series of 10 cases from nine patients was analyzed to characterize the clinicopathologic spectrum and age-related changes of ACC of the scalp. Hematoxylin and eosin, S100, Elastica van Gieson, and Weigert elastic stains were performed, and clinical information was retrieved from archived medical files. RESULTS: Patient ages ranged from 1 day to 39 years (median 57 months). All cases resembled deep-reaching scars with almost complete loss of all adnexal structures. Isolated residual hair follicles were present in 8/10 and sweat glands and ducts in 2/10 cases. The subcutis was thinned or absent. Elastic fibers were always more fragmented than in normal tissue, and the thickness and density increased over time. There was no gain of adnexal structures with increasing age. CONCLUSIONS: ACC represents a congenital scarring alopecia with permanent loss of skin appendages. Histopathologic changes resemble a deep-reaching scar with fragmented elastic fibers and differentiate ACC from all other forms of non-traumatic congenital alopecias.

Age as key factor for pattern, timing, and extent of distant metastasis in patients with cutaneous melanoma: A study of the German Central Malignant Melanoma Registry.

BACKGROUND: Melanoma incidence rates rise as people age, but the impact of aging on distant metastasis is unclear. OBJECTIVE: To investigate how timing, pattern, and extent of distant metastasis is influenced by age. METHODS: Analysis of a single-center cohort of 1457 patients of the German Central Malignant Melanoma Registry with prospectively documented follow-up. Findings were compared with those for 1682 patients from 5 different institutions. All patients presented initially with stage IA to IIC and developed distant metastasis in their further disease course. RESULTS: The number of metastatic sites decreased with increasing age at melanoma diagnosis (P < .001). The rate of stage M1d disease decreased from 50.2% in patients aged 50 years or younger to 30.1% in patients older than 70 years, and the rate of stage M1b disease increased from 5.8% to 21.5%. The rate of lung metastases remained stable in all investigated age groups (P = .54). Distant metastases occurred earlier and were more synchronized in patients older than 70 years than in patients aged 50 years or younger. An age-dependent decrease in metastatic sites and stable rate of lung metastasis were found and confirmed by data on the multi-institutional cohort. LIMITATIONS: The study was not population based. CONCLUSION: Pattern, timing, and extent of distant metastasis change as people age. These findings may be considered when treating patients with melanoma of different ages.

Safety of shortened infusion times for combined ipilimumab and nivolumab.

BACKGROUND: Combined ipilimumab and nivolumab induces encouraging response rates in patients with unresectable or metastatic melanoma. However, the approved protocol for dual checkpoint inhibition (3 mg/kg ipilimumab over 90 min and 1 mg/kg nivolumab over 60 min) is time-intensive and several trials have shown that both single agents can be safely administered at faster infusion rates. AIM: To investigate whether combined checkpoint inhibition with 3 mg/kg ipilimumab and 1 mg/kg nivolumab can be safely administered over 30 min per agent. PATIENTS AND METHODS: We reviewed the rate of infusion-related reactions (IRRs) in the first 12 months of our single-institution experience using shortened infusion times for combined checkpoint inhibition with ipilimumab and nivolumab. RESULTS: Between May 24, 2016 and June 10, 2017, a total of 46 melanoma patients received 100 shortened cycles of combined 3 mg/kg ipilimumab and 1 mg/kg nivolumab. One patient (2.2%; 1/46) had a questionable reaction after administration of 1 mg/kg nivolumab over 30 min, but none of the other patients had a bona fide IRR. CONCLUSIONS: Shortened infusion times for combined ipilimumab and nivolumab treatment are safe, thereby facilitating a more efficient use of outpatient facilities and enhancing patient's convenience.

Serial or Parallel Metastasis of Cutaneous Melanoma? A Study of the German Central Malignant Melanoma Registry.

For more than a century the Halstedian hypothesis of contiguous metastasis from the primary tumor through the lymphatics to distant sites shaped lymph node surgery for melanoma. We challenge this dogma of serial metastatic dissemination. A single-center series of 2,299 patients with cutaneous metastatic melanoma was investigated to analyze overall survival and distant metastasis-free survival of stage IV patients with or without primary lymphatic metastasis. Results were then compared with those of 2,134 patients from three independent centers of the German Central Malignant Melanoma Registry. A multivariate binary logistic regression model was used to identify risk factors for the initial metastatic pathway. Distant metastasis-free survival (hazard ratio = 1.02; 95% confidence interval = 0.91-1.14; P = 0.76) and overall survival (HR = 1.09; 95% CI = 0.96-1.23; P = 0.177) did not differ between stage IV patients with primary hematogenous or primary lymphatic metastasis. Melanoma localization was the only significant risk factor for the initial metastatic pathway. These findings indicate that regional and distant metastases originate from the primary tumor itself in a rather parallel than serial fashion and could explain the lack of survival benefit associated with immediate complete lymph node dissection in sentinel lymph node-positive melanoma patients.

Expression and prognostic significance of a comprehensive epithelial-mesenchymal transition gene set in renal cell carcinoma.

PURPOSE: Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance. MATERIALS AND METHODS: We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis. RESULTS: The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma. CONCLUSIONS: Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.

CXC chemokine receptor 4 is essential for maintenance of renal cell carcinoma-initiating cells and predicts metastasis.

In many solid tumors, cancer stem cells (CSC) represent a population with tumor-initiating, self-renewal, and differentiation potential, which can be identified by surface protein markers. No generally applicable markers are yet known for renal cell carcinoma (RCC). Two RCC cell lines (RCC-26, RCC-53) were found to differ widely in their capacity to form spheres in vitro and to establish tumors in mice, potentially reflecting differences in CSC content. A subpopulation expressing the CXC chemokine receptor 4 (CXCR4) was present only in the more tumorigenic cell line RCC-53. When grown as spheres, most of the RCC-53 cells were CXCR4-positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Sorted CXCR4-positive cells exhibited greater capacity for sphere formation and tumor growth-inducing potential in vivo than CXCR4-negative cells. Significantly, higher CXCR4 mRNA levels in primary RCC tumors from patients with localized but not disseminated disease predicted shorter survival. Downregulation of CXCR4 expression by small interfering RNA (siRNA) or pharmacological inhibition by AMD3100 compromised tumor sphere formation, viability of CXCR4-positive cells, and increased their responsiveness toward tyrosine kinase inhibitors. In conclusion, CXCR4 identifies a subpopulation of tumor-initiating cells in RCC cell lines and plays a role in their maintenance. The relative insensitivity of such cells to tyrosine kinase inhibitors might contribute to the development of therapy resistance in RCC patients. Future therapies therefore could combine blockade of the CXCR4 signaling pathway with standard therapies for more effective treatments of metastatic RCC.