Formalin-fixed Paraffin-embedded Melanoma Research Articles

Putative Cancer Stem Cell Markers are Frequently Expressed by Melanoma Cells in Vitro and in Situ but are also Present in Benign Differentiated Cells.

Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Abstract 2040: Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis

Abstract Anatomical pathology relies upon visual evaluation of clinical specimens using brightfield microscopy. This necessarily utilizes histological stains and immunohistochemical (IHC) chromogens that absorb visible light, between 430 and 690 nm. To complement the visible stains, we developed chromogens for use in IHC that absorb light outside the visible range, and imaging methods for detecting dyes absorbing from the ultraviolet (UV) through the near infrared (NIR). Invisible chromogens partnered with conventional visible stains on the same slide provides complementary information, enabling pathologists to gain deeper insights from a patient’s tissue. Methods: Invisible IHC was performed using conventional antibodies and antibody-enzyme conjugates utilizing covalently deposited chromogens absorbing in the UV and NIR. This was combined with IHC using visible chromogens or histological stains. Resulting stained specimens were evaluated interactively in real-time using a dual-camera microscope system comprising color and monochrome cameras, or by recording single microscope fields using multispectral imaging and the monochrome camera. Results: Multiplex IHC was performed with different combinations of visible and invisible chromogens, with the highest level of multiplexing - 7 biomarkers + hematoxylin counterstain (5 visible IHC chromogens plus a UV and an NIR chromogen) - demonstrated on a formalin-fixed paraffin-embedded (FFPE) prostate tumor. Images of each stain were recorded using multispectral imaging, and staining patterns were concordant with single stain DAB analysis on serial sections. In addition to increased IHC multiplexing capacity, multiplexing of IHC with histological stain was demonstrated by combining an NIR absorbing chromogen, identifying MART-1 expression, with H&E staining on FFPE melanoma specimens. The dual-camera microscope presented video of the visible H&E staining (color) next to the NIR IHC staining (monochrome) on the computer monitor as the specimen was manually scanned. Archiving was afforded by multispectral imaging which also enabled quantitative analysis. An additional advantage of the NIR chromogen was the suppression of melanin pigment, the absorbance of which was significantly reduced in the NIR. Other conventional stains multiplexed with invisible IHC on colon, lung, pancreas, and tonsil FFPE tissues also showed good separation of conventional and IHC staining. Conclusion: Taking advantage of invisible chromogens, IHC multiplexing capacity can be increased and conventional histological stains can be combined with IHC on a single slide. Clinical benefits include saving precious specimen, enabling differentiation of cell populations by simultaneous expression of multiple biomarkers, and providing biomarker expression within the cellular and morphological context of conventional stains. Citation Format: Larry E. Morrison, Mark R. Lefever, Lauren J. Behman, Monesh J. Kapadia, Daniel R. Bauer. Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2040.

Gene expression analysis in formalin fixed paraffin embedded melanomas is associated with density of corresponding immune cells in those tissues

Immune cell infiltrates in melanoma have important prognostic value. Gene expression analysis may simultaneously quantify numbers and function of multiple immune cell subtypes in formalin-fixed paraffin-embedded (FFPE) tissues. Prior studies report single gene expression can represent individual immune cell subtypes, but this has not been shown in FFPE melanomas. We hypothesized that gene expression profiling of human melanomas using a new RNA expression technology in FFPE tissue would correlate with the same immune cells identified by immunohistochemistry (IHC). This retrospective study included melanoma specimens analyzed by IHC on tumor tissue microarray (TMA) cores and by gene expression profiling with EdgeSeq Immuno-Oncology Assay using qNPA technology on the corresponding tumors. Standardized gene expression levels were analyzed relative to enumerated cells by IHC using Spearman rank test to calculate r-values. Multivariate analysis was performed by Kruskal–Wallis test. 119 melanoma specimens had both IHC and gene expression information available. There were significant associations between the level of gene expression and its quantified IHC cell marker for CD45+, CD3+, CD8+, CD4+, and CD20+ cells (all p < 0.001). There were also significant associations with exhaustion markers FoxP3+, PD-1+, and PD-L1+ (all p ≤ 0.0001). This new qNPA technology is useful to quantify intratumoral immune cells on FFPE specimens through RNA gene expression in metastatic melanoma. As previous studies have shown on other solid human tumors, we also confirm that the expression level of a single gene may be used to represent a single IHC immune cell marker in melanoma.

Identification of robust reference genes for studies of gene expression in FFPE melanoma samples and melanoma cell lines.

There is an urgent need for novel diagnostic melanoma biomarkers that can predict increased risk of metastasis at an early stage. Relative quantification of gene expression is the preferred method for quantitative validation of potential biomarkers. However, this approach relies on robust tissue-specific reference genes. In the melanoma field, this has been an obstacle due to lack of validated reference genes. Accordingly, we aimed to identify robust reference genes for normalization of gene expression in melanoma. The robustness of 24 candidate reference genes was evaluated across 80 formalin-fixed paraffin-embedded melanomas of different thickness, -/+ ulceration, -/+ reported cases of metastases and of different BRAF mutation status using quantitative real-time PCR. The expression of the same genes and their robustness as normalizers was furthermore evaluated across a number of melanoma cell lines. We show that housekeeping genes like GAPDH do not qualify as stand-alone normalizers of genes expression in melanoma. Instead, we have as the first identified a panel of robust reference genes for normalization of gene expression in melanoma tumors and cultured melanoma cells. We recommend using a geometric mean of the expression of CLTA, MRPL19 and ACTB for normalization of gene expression in melanomas and a geometric mean of the expression of CASC3 and RPS2 for normalization of gene expression in melanoma cell lines. Normalization, according to our recommendation will allow for quantitative validation of potential novel melanoma biomarkers by quantitative real-time PCR.

Association between microRNA-125b expression in formalin-fixed paraffin-embedded tumor tissues and prognosis in patients with melanoma.

Melanoma is an invasive and malignant type of tumor with unsatisfactory therapeutic outcomes. The present study aimed to detect the expression levels of microRNA (miR)-125b in formalin-fixed paraffin-embedded (FFPE) melanoma tissues and the association of its expression levels with the clinical features, diagnosis and prognosis of melanoma. Expression levels of miR-125b in 29 FFPE melanoma specimens (16 primary and 13 metastatic tumors), and 16 intradermal nevus (IDN) specimens as a control, were detected by reverse transcription-quantitative PCR. Associations among miR-125b expression and mortality, patient age and sex, tumor location and size, lymph node metastasis (LNM) and TNM stage were analyzed by t-test. The diagnostic value of miR-125b for melanoma was evaluated by receiver operating characteristic (ROC) curve analysis. Prognosis of patients in the microRNA-125b low- and high-expression groups was analyzed by Fisher's exact test. The association between miR-125b expression and the overall survival of patients with melanoma was assessed using Kaplan-Meier curve analysis and a Cox proportional hazards model. The results revealed that the expression levels of miR-125b in primary and metastatic melanomas were significantly lower than those in the IDN control group (P<0.05), and the expression levels of miR-125b in the metastatic group were significantly lower than those in the primary group (P<0.05). In addition, the expression levels of miR-125b were significantly associated with LNM (P=0.001) and TNM stage (P=0.004), but not with age, sex, tumor size or location (P>0.05). ROC curve analysis revealed that the area under the curve (AUC) was 0.880, with a 95% CI of 0.777–0.984 (P<0.05). The overall survival rate of the patients with a low expression level of miR-125b (20.0%) was lower than that of patients with a high expression level of miR-125b (64.3%) (P<0.05). miR-125b expression was an independent predictor of overall survival in patients with melanoma [hazard ratio (HR), 0.252; 95% CI, 0.087–0.729]. Overall, these findings indicated that a low expression level of miR-125b was associated with higher LNM and TNM stage in patients with melanoma, and that this has a certain diagnostic value. miR-125b may be used for the early screening of melanoma and determining the prognosis of patients with melanoma, and may be a potential target for the treatment of the disease.

Genetic profiling of melanoma in routine diagnostics: assay performance and molecular characteristics in a consecutive series of 274 cases

A deeper understanding of melanoma biology has opened up new avenues for mechanistically informed therapies. However, data on the prevalence of druggable genetic lesions in melanoma are still conflicting and real-world performance data on high-throughput genetic profiling of melanoma cases using formalin fixed, paraffin embedded (FFPE) tissue with variable tumour cellularity and quality are lacking. We retrospectively analysed targeted next-generation sequencing data of 274 consecutive melanoma samples obtained for routine diagnostics between December 2013 and July 2017. Actionable mutations were detected in 197 cases (71.9%), of which activating BRAF (mostly p.V600E/K) and RAS (mostly p.Q61R/K) mutations occurred in 40.5% (n=111) and 30.3% (n=83) of cases, respectively. We identified driver mutations of the Triple-WT subgroup in 10.6% of cases (n=29; 10 with activating KIT mutations). Median turnaround time was 5 working days with no dropouts. Tumour cellularity ranged from 5% to 95% and successful sequencing was possible at DNA concentrations as low as 0.03ng/μL (median 10.58ng/μL; range 0.03-209.05ng/μL). Fast, quality-controlled high-throughput genetic profiling of FFPE melanoma samples is feasible and provides a landscape of genetic aberrations in melanoma that is currently relevant in clinical practice and approximates TCGA subtypes.

A Prognostic Gene Signature Expressed in Primary Cutaneous Melanoma: Synergism With Conventional Staging.

BackgroundCurrent clinico-pathological American Joint Committee on Cancer (AJCC) staging of primary cutaneous melanoma is limited in its ability to determine clinical outcome, and complementary biomarkers are not available for routine prognostic assessment. We therefore adapted a gene signature, previously identified in fresh-frozen (FF) melanomas and adjacent stroma, to formalin-fixed paraffin-embedded (FFPE) melanomas. The aim was to develop a gene expression profiling (GEP) score to define patient survival probability at the time of first diagnosis.MethodsExpression of 11 FF melanoma signature genes was quantified by reverse transcription polymerase chain reaction in an FFPE melanoma training cohort (n = 125), corresponding to the combined FF melanoma training and validation cohorts. The resulting GEP score was validated technically and clinically in an independent FFPE melanoma cohort (n = 211). All statistical tests were two-sided.ResultsWe identified a prognostic eight-gene signature in the tumor area (tumor and adjacent tissue) of AJCC stage I–III melanomas. A signature-based GEP score correlated with melanoma-specific survival (MSS; Kaplan-Meier analysis: P < .0001) was independent of tumor stage (multivariable regression analysis: P = .0032) and stroma content (<5%–90%) and complemented conventional AJCC staging (receiver operating characteristic curve analysis: area under the curve = 0.91). In the clinical validation cohort, the GEP score remained statistically significant (P = .0131) in a multivariable analysis accounting for conventional staging. The GEP score was technically robust (reproducibility: 93%; n = 84) and clinically useful, as a binary as well as a continuous score, in predicting stage-specific patient MSS.ConclusionsThe GEP score is a clinically significant prognostic tool, contributes additional information regarding the MSS of melanoma patients, and complements conventional staging.

MiR-497-5p, miR-195-5p and miR-455-3p function as tumor suppressors by targeting hTERT in melanoma A375 cells.

BackgroundhTERT gene plays an important role in melanoma, although the specific mechanism involved is unclear. The aim of this study was to screen and identify the relative miRNAs with the regulation of hTERT in melanoma.Materials and methodsQuantitative real-time polymerase chain reaction (q-PCR) and immunohistochemistry were performed to detect hTERT mRNA and protein expression in 36 formalin-fixed paraffin-embedded melanoma tissues and 36 age- and sex-matched pigmented nevi cases, respectively. Bioinformatics analysis and custom miRNA polymerase chain reaction array were determined for predicting, screening and verifying miRNAs with the regulation of the hTERT gene. To investigate the biological functions, miRNAs mimics or inhibitors were transfected into melanoma A375 cells. The relative expression of miR-497-5p, miR-195-5p, miR-455-3p and hTERT mRNA was determined by q-PCR. The protein expression of hTERT was detected by Western blot. 3-(4,5-Dimethylthiazolyl-2-yl)-2,5-biphenyl tetrazolium bromide and flow cytometry were employed to detect cell proliferation ability, cell apoptosis and cell cycle. Transwell and wound healing assays were used to observe cell invasion and migration abilities. A direct target gene of miRNAs was analyzed by a dual luciferase reporter activity assay.ResultsMiR-497-5p, miR-195-5p, miR-455-3p were significantly downregulated, while hTERT was upregulated in melanoma tissues. hTERT expression level was inversely correlated with miR-497-5p, miR-195-5p and miR-455-3p. Overexpression of miR-497-5p, miR-195-5p and miR-455-3p inhibited A375 cell proliferation, migration and invasion, arrested the cell cycle, induced cell apoptosis and decreased hTERT expression at both mRNA and protein levels. Suppression of miR-497-5p, miR-195-5p and miR-455-3p partially reversed the inhibitory effects. Finally, hTERT was identified as a direct target of miR-497-5p, miR-195-5p and miR-455-3p.ConclusionsMiR-497-5p, miR-195-5p and miR-455-3p act as tumor suppressors by targeting hTERT in melanoma A375 cells. Therefore, miR-497-5p, miR-195-5p and miR-455-3p could be potential targeted therapeutic choice for melanoma.

Transcriptional response to hypoxic stress in melanoma and prognostic potential of GBE1 and BNIP3.

Gradients of hypoxia occur in most solid tumors and cells found in hypoxic regions are associated with the most aggressive and therapy-resistant fractions of the tumor. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia in melanoma. Using microarray technology, whole genome gene expression profiling was first performed on established melanoma cell lines. From gene set enrichment analyses, we derived a robust 35 probes signature (hypomel for HYPOxia MELanoma) associated with hypoxia-response pathways, including 26 genes up regulated, and 9 genes down regulated. The microarray data were validated by RT-qPCR for the 35 transcripts. We then validated the signature in hypoxic zones from 8 patient specimens using laser microdissection or macrodissection of Formalin fixed-paraffin-embedded (FFPE) material, followed with RT-qPCR. Moreover, a similar hypoxia-associated gene expression profile was observed using NanoString technology to analyze RNAs from FFPE melanoma tissues of a cohort of 19 patients treated with anti-PD1. Analysis of NanoString data from validation sets using Non-Negative Matrix Factorization (NMF) analysis (26 genes up regulated in hypoxia) and dual clustering (samples and genes) further revealed that the increased level of BNIP3 (Bcl-2 adenovirus E1B 19 kDa-interacting protein 3)/GBE1 (glycogen branching enzyme1) differential pair correlates with the lack of response of melanoma patients to anti-PD1 (pembrolizumab) immunotherapy. These studies suggest that through elevated glycogenic flux and induction of autophagy, hypoxia is a critical molecular program that could be considered as a prognostic factor for melanoma.

Abstract 3388: Copy number alteration in primary melanoma

Abstract The systematic analysis of the genomic characteristics of melanoma primaries has been infeasible given the limited size of these formalin-fixed paraffin embedded (FFPE) lesions. Further studying a clearly ascertained, deeply phenotyped patient population allows meaningful extrapolation of the prevalence of distinct genomic features and investigation of the association of these features with lifestyle exposures and germline profiles. For these reasons, we focused on a recently recruited patient cohort, the Leeds Melanoma Cohort (LMC) consisting of 2184 recruits from a distinct geographical region of Northern England; the only selection applied to recruitment was to only include those whose melanoma was less than 0.75 mm Breslow thickness in the early years of recruitment. Tumor samples from 703 cases have been analyzed for genome-wide gene expression (Pozniak et al, this meeting). For this study, we wanted to explore the prevalence of copy number alterations (CNA) and the mutation profile of tumors (reflecting the association of C&amp;gt;T mutations with UV exposure, the primary environmental risk factor) and so we have adopted a next-generation sequencing (NGS) approach. Tumor blocks were recovered from local pathology laboratories for those cases already deceased at the time that this CNA analysis was initiated plus those surviving more than 5 years post diagnosis at this same time. Blocks at risk of being compromised for clinical purposes by sampling for this research study were excluded from sampling; this amounted to almost 50% of identified samples. A total of 333 NGS libraries were sequenced on an Illumina GAII or HiSeq sequencer to produce &amp;gt;100bp paired-end reads (either 5 or 1 per lane). DNA reads were aligned and mapped achieving approximately 1.8x coverage (9.4x for those at 1 per lane); the number of reads falling within each 10kb window was adjusted for GC content and mappability and compared to a composite normal FFPE sample to determine local copy number. We adopted various approaches to assess the informativeness of the methodology. (i) Replicate samples showed high reproducibility (all p &amp;lt; 0.0001). (ii) Focused analysis of the CDKN2A region with segmentation analyses to determine CNA breakpoints yielded high quality, biologically plausible data revealing multiple copy number events. 67% of samples showed no CNA across the 4 Mb region covering CDKN2A while, as expected, only a small proportion of CNAs did not involve CDKN2A. Four common distinct patterns were observed. Copy number loss ranged from 20kb to &amp;gt; 4 Mb. (iii) A germline CNA could reliably be identified from the tumor data. (iv) CDKN2A expression levels correlated with estimated copy number. We have shown that small FFPE melanomas can be characterized for genomic alterations using NGS opening up the potential for studies associating epidemiological and germline profile with the resulting tumor. 1. Newton-Bishop, J.A., et al. (2009) J Clin Oncol, 27, 5439-5444. Funded by Worldwide Cancer Research and Cancer Research UK. Citation Format: D. Timothy Bishop, Anastasia Filia, Alastair Droop, Joey Diaz, Mark Harland, Jon Laye, Juliette Randerson-Moor, Julia A. Newton-Bishop. Copy number alteration in primary melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2017-3388

Expression of G protein‐coupled oestrogen receptor in melanoma and in pregnancy‐associated melanoma

The hormone sensitivity of melanoma and the role of 'classical' oestrogen receptor (ER) α and β in tumour progression have been intensively studied with rather contradictory results. The presence of 'non-classical' G protein-coupled oestrogen receptor (GPER) has not been investigated on human melanoma tissues. To analyse the expression of GPER, ERα and ERβ in pregnancy-associated (PAM) and in non-pregnancy-associated (NPAM) melanomas in correlation with traditional prognostic markers and disease-free survival (DFS). Receptor protein levels were tested using immunohistochemistry in 81 formalin-fixed paraffin-embedded melanoma tissues. PAMs (n = 38) were compared with age- and Breslow thickness-matched cases (n = 43) including non-pregnant women (NPAM-W) (n = 22) and men (NPAM-M) (n = 21). The association between receptor expression and DFS was analysed by uni- and multivariate Cox proportional hazards regression. G protein-coupled oestrogen receptor was detected both in PAMs and NPAMs. In 39 of the 41 (95.1%) GPER-positive melanomas, GPER and ERβ were co-expressed. GPER/ERβ-positive melanomas were significantly more common in PAM compared to NPAM (P = 0.0001) with no significant difference between genders (P = 0.4383). In PAMs, the distribution of GPER and ERβ was similar (78.4% vs. 81.6%; P = 0.8504), while in NPAM, ERβ was the representative ER (60.5% vs. 27.9%; P = 0.0010) without gender difference (59.1% vs. 61.9%). GPER-/ERβ-positive melanomas were associated with lower Breslow thickness, lower mitotic rate and higher presence of peritumoral lymphocyte infiltration (PLI) compared to GPER-/ERβ-negative cases (P = 0.0156, P = 0.0036 and P = 0.0001) predicting a better DFS (HR = 0.785, 95% CI 0.582-1.058). Despite the significantly higher frequency of GPER and ERβ expression in PAM, no significant difference was found in DFS between PAM and NPAM. All but one case failed to show ERα expression. The presence of GPER and its simultaneous expression with ERβ can serve as a new prognostic indicator in a significant subpopulation of melanoma patients.

Validation, in silico and in vitro, of a gene-signature based risk score in cutaneous melanoma.

9560 Background: Melanoma staging, as defined by the American Joint Committee on Cancer (AJCC), is limited in its ability to predict outcome. We have previously identified and validated a prognostic gene signature expressed in primary cutaneous melanoma and adjacent stroma. The signature comprises seven protective genes (down-regulated with tumor progression) and one risk-associated gene (up-regulated). A signature-based risk score independently predicts patient survival, across AJCC stages IA-IIIC, in formalin-fixed, paraffin-embedded (FFPE) melanomas (training cohort, n = 125; p = 0.0003, hazard ratio 1.85). The score has been validated in 206 melanomas, selected to be significantly mis-prognosticated by AJCC staging regarding patient survival (40.8% mis-prognostication). In this cohort, the score outperforms AJCC staging (p = 0.0005, hazard ratio 1.41 vs. p = n.s.), correcting 34.9% of AJCC-based mis-prognostications. Methods: Here, we report twofold external validation of the risk score, (i) prognostic performance in silico using the SurvExpress web tool (Aguirre-Gamboa et al., 2013), and (ii) technical performance in vitro(Dermatologikum Hamburg; IMGM Munich) . Results: (i) Kaplan Meier analysis and log-rank testing demonstrated that all signature genes combined predicted survival in four different cohorts of metastatic melanoma (from GEO Expression Omnibus or Cancer Genome Atlas; cohorts dichotomized at the median): see table. (ii) The risk score was re-analyzed in melanomas of the training cohort (n=69). The overall concordance of duplicate determinations was 90% (average scores of 1.12 ± 0.14 and 0.97 ± 0.14). Conclusions: In conclusion, we have validated a signature-based FFPE melanoma risk score, complementary to AJCC staging in predicting outcome: (i) Signature genes predicted patient survival in silico(n=449) (ii) The risk score proved to be reproducible and technically robust in vitro. The score improves risk stratification and decision making in melanoma, particularly regarding new adjuvant therapies. [Table: see text]

Abstract 853: Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients

Abstract While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents. FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n = 30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n = 21). Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n = 53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores. In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance. Citation Format: Jennifer Bordeaux, Douglas Johnson, Jeffrey Sosman, Ju Young Kim, Christine Vaupel, Bashar Dabbas, Justin Cates, Jeff Hall, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 853.

Differential Expression of Immune-Regulatory Genes Associated with PD-L1 Display in Melanoma: Implications for PD-1 Pathway Blockade.

Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1(+) versus PD-L1(-) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1(+) melanoma microenvironment coordinately contributing to immunosuppression. Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1(+) versus PD-L1(-) melanomas. Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1(+) melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1(+) melanomas, involved in CD8(+) T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells. These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies.

NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

BackgroundThe determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRASQ61R and BRAFV600E proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRASQ61R immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRASQ61R and VE1 anti-BRAFV600E antibodies in the theranostic mutation screening of melanomas.Methods142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry.Results23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRASQ61R and 23 BRAFV600E-mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRASQ61L, NRASQ61K, BRAFV600K and BRAFV600R mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity.ConclusionsIn our study, we showed that combining immunohistochemistry analysis targeting NRASQ61R and BRAFV600E proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-015-0359-0) contains supplementary material, which is available to authorized users.

Abstract 2861: Independent validation of a prognostic gene-signature based risk score in formalin-fixed paraffin-embedded melanomas

Abstract Current staging of melanoma, as defined in 2009 by the American Joint Committee on Cancer (AJCC), is based mainly on histopathological criteria but is limited in predicting outcome. Complementary molecular markers are not available for routine prognostic assessment. We have previously identified and validated a prognostic nine-gene signature expressed in fresh-frozen (FF) primary cutaneous melanomas (training cohort: n=91; validation cohort: n=44). A signature-based risk score predicts patient overall survival (OS) independently of AJCC staging (multivariate regression analysis: p = 0.0004; hazard ratio: 3.8). However, clinical application requires adaptation to formalin-fixed, paraffin-embedded (FFPE) melanomas. Therefore, we have transfered signature expression analysis onto FFPE melanomas. From FFPE melanomas matching the training and validation cohorts of the above FF melanoma study (n=125), RNA was prepared and transcribed into cDNA. Following cDNA pre-amplification, expression of the 9 signature genes, 2 additional candidate genes, and 3 housekeeping genes was quantified by real-time PCR. Correlation of gene expression with OS was evaluated using Cox regression analysis. Expression of a signature of 8 out of 11 genes (risk gene: KBTBD10; protective genes: DCD, GBP4, COL6A6, PIP, SCGB1D2, SCGB2A2, KRT9) was associated with OS in univariate regression and Kaplan Meier analysis. A signature-based risk score predicted OS independently of AJCC staging (multivariate analysis: p=0.0059, hazard ratio 3.09). The misclassification rates were 20% overall, 13.8% for low risk, and 5.7% for double low-risk (combined with AJCC staging). The risk score complemented and refined conventional AJCC staging. Thus, the FF melanoma risk score was successfully transfered onto FFPE melanomas. In order to validate the FFPE melanoma risk score, we analyzed signature expression in an independent cohort of 130 selected FFPE melanomas, which were particularly difficult to classify by AJCC staging (misclassification rate 40.8%), in order to stringently test the performance of the risk score. The misclassification rate of the FFPE melanoma risk score was comparable, even slightly better (39.2%) than that of AJCC staging, confirming its prognostic performance. The FFPE melanoma risk score was also externally validated in a Molecular Diagnostics Lab (Dermatologikum Hamburg). The concordance of melanoma classification exceeded 85%, demonstrating technical robustness of the risk score. We have established and independently as well as externally validated a quantitative, robust prognostic FFPE melanoma risk score that is complementary to AJCC staging in predicting outcome. This demonstrates clinical applicability and allows retrospective risk assessment of melanomas. The score identifies patients at low risk, not identified by AJCC staging, and defines high-risk patients in need of adjuvant therapy. Citation Format: Georg Brunner, Achim Heinecke, Ludwig Suter, Norbert Blödorn-Schlicht, Hans-Joachim Schulze, Jens Atzpodien. Independent validation of a prognostic gene-signature based risk score in formalin-fixed paraffin-embedded melanomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2014-2861

Association of TNFRSF10D DNA-methylation with the survival of melanoma patients.

In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012). In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 (95% CI: 2.0–11.0; p < 0.001), RR of relapse, 7.2 (95% CI: 2.8–18.3; p < 0.001)). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.

Abstract 3140: Using formalin-fixed paraffin-embedded melanoma tumors for the detection of copy number variation by next generation sequencing.

Abstract DNA copy number variation (CNV) has been identified in many cancers. Recent studies have shown that CNV data, in combination with other high-throughput techniques, can provide novel biological insight to tumor biology. Although a number of CNV whole-genome studies have been performed on melanoma using cell lines and frozen tumors, very few have used formalin fixed paraffin embedded (FFPE)-derived material. Melanoma tumors are small and rarely frozen, which means that FFPE tumor blocks would be a very valuable source of genomic material to be used for CNV analysis if the methodologies were adequately developed. It has been shown that reliable next generation sequencing data can be produced from FFPE-derived DNA in lung cancers (Wood et al., 2010). Here, we report a study showing that DNA from FFPE melanoma tumor blocks can be used for CNV detection on a next generation sequencing platform. DNA from 20 FFPE melanoma samples was used to prepare genomic libraries, which were sequenced on the Illumina GAIIx instrument. Successful library preparation occurred at DNA levels of at least 30ng, while current protocols generally suggest 50ng with high quality material and 1μg generally. The use of SPRIworks (Beckman Coulter) and Bravo (Agilent) robots was compared with the manual method for library preparation with input DNA mass of 30-150ng. Libraries prepared manually were always of better quality and yield. We also investigated means of improving library preparation. Melanin co-purifies with DNA and inhibits downstream reactions, and indeed lower coverage was seen in our samples if they were heavily pigmented. To overcome the adverse effects of melanin, 10 DNA samples (5 pairs) were used for library preparation with and without bovine serum albumin (BSA). BSA has been shown to bind to melanin, and as a result the melanin does not inhibit the DNA polymerase reaction. All 5 libraries with BSA were of higher quality and yield when compared to those prepared without BSA. All comparisons were made after running the libraries on an Agilent DNA chip. Currently available data from the GAIIx were used to develop the analysis pipeline. The first 10 samples were multiplexed together and run twice, while the remaining 10 were multiplexed at 5 samples per lane and run once. The average coverage obtained was 0.10x with a range of 0.03x-0.11x. We observed a strong effect on mapping quality based upon the end-cycle quality scores, leading us to adopt an aggressive data trimming procedure. More samples from the Leeds Melanoma Cohort will soon be prepared and sequenced on the Illumina HiSeq2000 instrument. We aim to identify if tumors with driver BRAF mutations have different genomic signatures when compared to those with NRAS mutations. Finally, genomic data will be combined with clinical data and we will check for the prognostic significance of CNV changes identified in our samples. Citation Format: Anastasia Filia, Alastair Droop, Mark Harland, Tim Bishop, Julia Newton-Bishop. Using formalin-fixed paraffin-embedded melanoma tumors for the detection of copy number variation by next generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2013-3140

A Reliable Method for the Selection of Exploitable Melanoma Archival Paraffin Embedded Tissues for Transcript Biomarker Profiling

The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of “good” samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series.

Molecular Diagnostics in Melanoma: Current Status and Perspectives

Abstract Context.—In the current “molecular” era, the advent of technology, such as array-based platforms, systems biology, and genome-wide approaches, has made it possible to examine human cancers, including melanoma, for genetic mutations, deletions, amplification, differentially regulated genes, and epigenetic changes. Advancement in current technologies is such that one can now examine ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein directly from the patient's own tumor. Objective.—To apply these new technologies in advancing molecular diagnostics in melanoma has historically suffered from a major obstacle, namely, the scarcity of fresh frozen, morphologically defined tumor banks, annotated with clinical information. Recently, some of the new platforms have advanced to permit utilization of formalin-fixed, paraffin-embedded (FFPE) tumor specimens as starting material. Data Sources.—This article reviews the latest technologies applied to FFPE melanoma sections, narrowing its focus on t...