Background: Current 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. Methods: Expression 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. Results: We 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. Conclusions: The GEP score is a clinically significant prognostic tool, contributes additional information regarding the MSS of melanoma patients, and complements conventional staging.

Abstract Background: Melanoma is the most aggressive skin cancer and, despite recent advances in therapy, about 20% of the patients die of their disease. Early relapse detection and monitoring of therapy response are crucial for efficient treatment of advanced melanoma. Thus, there is a need for blood-based biomarkers in melanoma management. Serum-derived U2 small nuclear RNA fragments (RNU2-1f) were previously shown to be blood-based biomarkers for gastrointestinal and gynecologic malignancies. Here we examined whether RNU2-1f may also serve as diagnostic biomarker in advanced melanoma. Methods: Circulating RNU2-1f levels were quantified by comparative reverse transcription PCR in a training cohort of patients with metastatic melanoma (n=33, thereof regionally metastasized to skin and lymph nodes, n=23, and distantly metastasized, n=10) vs. patients with benign naevi (n=16) vs. healthy controls (n=39). Results were validated in an independent patient cohort with distant metastasis (n=16) vs. controls (n=18). Results: Circulating RNU2-1f levels in the training cohort were significantly increased in serum of regionally and distantly metastatic patients, compared with patients with benign naevi or healthy controls (p<0.0001) and allowed accurate detection of regional (AUC 0.80) as well as distant (AUC 0.84) metastasis. In the validation cohort, increased RNU2-1f levels were confirmed and enabled highly specific detection of distant metastasis (sensitivity 81%, specificity 100%, AUC 0.94). Conclusions: This is the first report to suggest a blood-based snRNA serving as a diagnostic biomarker for melanoma metastasis. Our data provide a rationale for further defining clinical utility of circulating RNU2-1f in metastasis detection in the management of melanoma patients at risk of relapse and/or with advanced disease.

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

Purpose Current histopathological staging of cutaneous melanoma is limited in predicting outcome, and complementary molecular markers are not available for prognostic assessment. The purpose of this study was to identify a quantitative gene expression score in primary melanoma and adjacent stroma that can be used in clinical routine to define, at the time of diagnosis, patient risk and need for therapy. Methods Expression of 92 candidate genes was quantified by RT-PCR in a training subset of 38 fresh-frozen melanomas. Correlation of gene expression with overall survival (OS) was evaluated using univariate regression analysis. Expression analysis of 11 prognostically significant genes in the complete training cohort of 91 melanomas yielded nine genes predicting outcome. Results were confirmed in a validation cohort of 44 melanomas. Results We identified a nine-gene signature associated with OS and distant metastasis-free survival. The signature comprised risk and protective genes and was applicable to melanoma samples across all AJCC stages in the presence of adjacent stroma. A signature-based risk score predicted OS in both the training cohort (multivariate regression analysis: p = 0.0004, hazard ratio 3.83) and the validation cohort, independently of AJCC staging. Consequently, when combining risk score and AJCC staging, patients in the AJCC intermediate-risk stages, IIA/B or IIIA, were re-classified either to low or high risk. Conclusions Our gene score defines patient risk and need for therapy in melanoma. The score has the potential to be utilized in clinical routine, since it is quantitative, robust, simple, and independent of AJCC stage and sample purity.

Abstract Melanoma incidence is rapidly increasing – with a doubling rate of 10-20 years. Precision and reliability of conventional histological and clinical staging, however, remain limited in predicting clinical outcome. On the other hand, complementary molecular prognostic markers are not yet available. We have recently identified, for the first time, a prognostic gene signature expressed in fresh-frozen primary melanomas (n = 135), which is associated with overall survival (multivariate Cox regression analysis: p = 0.0004, hazard ratio 3.83). The clinical value of a signature-based risk score is its ability to identify patients at low risk, not identified by conventional AJCC staging, and to define risk patients in need of adjuvant therapy. The purpose of the present study was to establish analysis of the signature genes in formalin-fixed, paraffin-embedded (FFPE) melanoma tissue and to validate prognostic significance. We developed a sensitive and robust methodology to analyze and normalize gene expression in FFPE tissue samples (some of them more than 20 years old): Total RNA was prepared from FFPE sections matching the above fresh-frozen primary melanomas (131 out of 135), quality-controled, and transcribed into cDNA. Human reference RNA was included as an internal standard. Following pre-amplification of the cDNA, expression of the nine signature genes (KRT9, KBTBD10, DCD, ECG2/SPINK7, PIP, SCGB1D2, SCGB2A2, COL6A6, HES6) and of four house-keeping genes (18S rRNA, GAPDH, GUSB, BPNT1) was quantified by real-time PCR using TaqMan assays specific for short amplicons. Gene expression data were normalized, in two steps, to correct for inter-assay technical variability (based on the reference RNA data) and inter-sample variability of RNA quality (based on the data for the house-keeping genes). Significance of correlation of FFPE gene expression data (CT values or estimated mRNA copy numbers) with data from matched fresh-frozen tissue samples (two-sided t-test) or with patient overall survival (univariate Cox regression analysis; clinical follow-up data up to 273 months) was evaluated. The majority of FFPE primary melanomas (125 out of 131) yielded mRNA of sufficient quality. Expression of all nine signature genes in FFPE melanomas correlated with that in matched fresh-frozen samples. Significance of correlation was higher with CT values (r = 0.58 – 0.19; p = 0.001 – 0.05) than with estimated mRNA copy numbers. Expression of 7 out of the 9 genes (dichotomized CT values) in FFPE melanomas was significantly associated with patient overall survival (p = 0.0001 – 0.0335). Thus, our prognostic melanoma gene signature was successfully transfered from fresh-frozen onto FFPE tissue samples. This facilitates clinical use of a gene-signature based prognostic risk score and, in addition, allows the retrospective prognostic analysis of primary melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2011-5070

Incidence of malignant melanoma is rapidly increasing - with a doubling rate of 10-20 years. Conventional histopathological and clinical staging (based on Breslow tumor thickness, lymph node status, and ulceration) is largely inadequate for predicting clinical outcome of malignant melanoma. On the other hand, molecular prognostic markers are not yet available. Here, we identified a nine-gene signature which is closely associated with overall survival of melanoma patients. To identify prognostic genes we correlated gene expression profiles of 136 primary melanomas with patient overall survival using Cox regression analysis: Initially, a comparative analysis of 20 high-risk vs. 20 low-risk primary melanomas with a clinical follow-up of more than 20 years (training cohort) was performed using whole-genome DNA microarray analysis and yielded 92 prognostically relevant candidate genes. Technical and statistical validation using TaqMan Array real-time RT-PCR and correlation of gene expression with patient overall survival reduced candidate gene number to 11. Expression of these 11 genes was further analyzed in an extended group of 91 primary melanomas (study cohort), yielding a nine-gene signature with prognostic significance. This novel prognostic melanoma gene signature was successfully validated using an independent set of 45 primary melanomas (validation cohort). A risk score, based on the expression of the nine genes of the signature (KRT9, SPINK7/ECG2, KBTBD10, DCD, HES6, COL6A6, PIP, SCGB1D2, SCGB2A2), or any subgroup thereof, predicted patient overall survival in the study cohort (p = 0.0004, hazard ratio 3.83), independently of conventional AJCC 2002 staging. When combining gene expression score and AJCC staging, approximately two thirds (29/45, 64%) of patients with AJCC intermediate prognosis (i.e. stages IIA, IIB, and IIIA) were reclassified into good prognosis, exhibiting a long-term overall survival probability of 95%. Misclassification rate of all patients classified into good prognosis (low-risk gene score combined with AJCC stages I IIA/B, or IIIA) was very low at 4.6% and 6.25% in the training and validation cohorts, respectively. The prognostic value of this novel signature-based risk score is its ability to identify patients at low risk, not identified by AJCC staging. This re-classification may allow these patients to stay treatment-free while experiencing excellent long-term survival. The remaining patients are risk patients and are in need of adjuvant therapies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4652.

Experimental investigations were carried out on non-azeotropic refrigerant mixtures, named M1A (mass fraction of 20%R152a and 80%R245fa), M1B (mass fraction of 37% R152a and 63%R245fa) and M1C (mass fraction of 50%R152a and 50%R245fa), based on a water-to-water heat pump system in the condensing temperature range of 70–90 °C with a cycle temperature lift of 45 °C. Performance of R245fa was tested for comparison. Unfair factors in experimental comparative evaluation research with the same apparatus were identified and corrected. Experimental cycle performance of the mixtures were tested and compared with improved experimental assessment methodology. The results show that all of the mixtures deliver higher discharge temperature, higher heating capacity, higher COP and higher εh,c than R245fa. M1B presents the most excellent cycle performance and is recommended as working fluid for moderate/high temperature heat pump.

Promyelocytic leukemia zinc finger (PLZF) is a transcriptional repressor and tumor suppressor inhibiting melanoma cell growth in vitro and in vivo in animal models. In this study, we analyzed the impact of in vivo primary tumor gene expression of PLZF on the long-term survival of malignant melanoma patients. PLZF expression was assessed by using DNA microarray and real-time polymerase chain reaction analysis of 41 primary malignant melanomas from patients with a defined histology and a close to 20-year clinical follow-up, of 29 melanoma metastases, and of 6 different melanoma cell lines. Kaplan-Meier survival analyses, log-rank statistics and Cox regression analysis were employed to identify the impact of PLZF expression on long-term survival. We detected PLZF expression in 92% of primary melanoma tumors in vivo but not in melanoma cell lines in vitro. By univariate analysis, we identified: (1) PLZF mRNA expression < or = 10,000 mRNA copies/mug total tumor RNA, (2) Breslow tumor thickness >4 mm, and (3) American Joint Committee on Cancer stages IIC, IIIB, IIIC, and IV as statistically significant pretreatment risk factors. We defined a continuous prognostic index (i.e., risk score) for primary melanoma patients based on the regression coefficient of PLZF mRNA expression. Applying a cutpoint to the prognostic index at - 1.65, patients were assigned to one of two risk groups: low-risk patients (n = 28) with a median overall survival of 79 months (5-year survival of 61%) and high-risk patients (n = 13) with a median overall survival of 32 months (5-year survival of 23%) (p < 0.05). This is the first time that PLZF mRNA expression has been linked to a prognostic model for primary malignant melanoma patients to derive prognostic groups for clinical purposes (e.g., improved melanoma immunotherapies).

Background Cosmetic changes are to be expected after radiotherapy for skin tumours.Objectives This study aimed to answer the questions: How frequent are cosmetic changes after soft X-ray therapy? Do treatment parameters, tumour thickness, localization and size of the irradiated field have a major influence? Were patients irritated by the visual appearance of the irradiated field?Methods In total, 2474 examinations of 1149 irradiated fields were performed.Results Hypopigmentation was found in 64.7% of examinations more than 90 days after therapy, teleangiectases in 43.1%, erythema in 24.8%, and hyperpigmentation in 16.8%. The frequency of hypopigmentation, teleangiectases and hyperpigmentation increased with time from X-ray exposure; more than 4 years after therapy hypopigmentation was diagnosed in 91.8% and teleangiectases in 82.2% of examinations. Total dose, the time-dose-fractionation factor (TDF), field size and dose per fraction were significantly related to the frequency of cosmetic changes. Incidence rates of cosmetic changes differed by less than 15% if different treatment conditions were compared: thicker vs. thinner tumours, larger vs. smaller fields, higher vs. lower total doses, doses per fraction, and TDF. Frequencies of hypopigmentation, teleangiectases, erythema and hyperpigmentation differed by more than 15% between some localizations on the head. Women reported irritation by the visual appearance of the irradiated field in 12.6% of 1116 interviews, and men in 4.4% of 1284 interviews.Conclusions Cosmetic changes after soft X-ray therapy are relatively frequent. Treatment parameters, tumour thickness and field size have only a minor influence. Few patients, but more women than men, were irritated by the visual appearance of the irradiated field.

BACKGROUND:Pruritus, burning, epiphora and insufficient occlusion of the mouth have been less extensively studied than cosmetic changes in irradiated fields.OBJECTIVES:How frequent are these late adverse effects? Do they usually occur permanently? Are they influenced by treatment and tumour parameters, sex and age of the patients?METHODS:Patients were interviewed at least once later than 90 days after soft X-ray therapy.RESULTS:Pruritus has been reported in 18.5% of the interviews, burning in 7.7%, epiphora in 36.2% and insufficient occlusion of the mouth in 11.5%. Patients were usually not permanently troubled and irritated by these symptoms: pruritus more than once per week was reported in every interview for 0.6% of the fields, burning for 0.2%, epiphora for 6.4% and insufficient occlusion for 0%. Irritation by these symptoms has been stated in every interview for 5.1% of fields around the eye and for 1.4% of fields at other sites. Late pruritus, burning and epiphora were less frequently reported after irradiation with lower total doses, lower time-dose-fractionation factor (TDF) and by men. Patients older than 70 years of age experienced pruritus and burning less frequently. The largest diameter of the irradiated field influenced pruritus and the half value depth of the X-rays influenced burning and epiphora.CONCLUSIONS:Late pruritus, burning, epiphora and insufficient occlusion of the mouth do not considerably reduce the value of soft X-ray therapy because these adverse effects usually are not experienced permanently. Total dose and TDF should not be chosen higher than necessary.

JDDG | 2 ̇2006 (Band 4) Die Behandlung von Hauttumoren mit Röntgenstrahlen hat in der Dermatologie eine lange Tradition. Miescher [1] und Schreus [2] haben sich in den 40er Jahren des letzten Jahrhunderts durch die klinische Erprobung weicher Röntgenstrahlen besonders um die dermatologische Radiotherapie verdient gemacht. Nach einem großen Aufschwung in der Nachkriegszeit ist die Strahlentherapie von Hauttumoren seit Ende der 60er Jahre im Rückgang. Die Verbesserung plastisch-chirurgischer Techniken und die Verbreitung neuer Methoden wie Kryochirurgie, photodynamische Therapie und topische Anwendung von Imiquimod haben diese Entwicklung beeinflusst. In Deutschland haben viele dermatologische Kliniken und niedergelassene Hautärzte die Bestrahlung von Hauttumoren aufgegeben. Dennoch ist die Radiotherapie auch heute noch eine wichtige Behandlungsoption bei Hauttumoren am Kopf. Das wird belegt durch Publikationen aus den letzten 10 Jahren [3–8] und zwei Tagungen mit internationaler Beteiligung, die der Strahlentherapie dermatologischer Erkrankungen gewidmet waren: 1995 in München [7] und 2004 in Berlin. Sehr eindrucksvoll werden Ergebnisse der Strahlentherapie von Basalzellkarzinomen in der Arbeit von Olschewski et al. in dieser Ausgabe [9] dargestellt. 85 Patienten mit 104 Basalzellkarzinomen wurden bestrahlt. In der Nachbeobachtungszeit wurde kein Rezidiv und kein spätes (mehr als 90 Tage nach der Behandlung) Ulcus im Bestrahlungsfeld gefunden. Das kosmetische Ergebnis war in 94 % der Bestrahlungsfelder „exzellent“ oder „gut“. Die Autoren führen ihre hervorragenden Resultate auf ihr Bestrahlungsschema zurück. Olschewski et al. [9] zitieren in ihrer Publikation Arbeiten, in denen über eine geringe lokale Rezidivquote (4 %–7 %) und seltene späte Ulcera im Bestrahlungsfeld nach Radiotherapie von Basalzellkarzinomen berichtet wird. Größere [8] und sklerosierende [5] Basalzellkarzinome rezidivieren häufiger. Die meisten Ulcera in Bestrahlungsfeldern heilen unter Therapie mit Salben und feuchten Umschlägen oder spontan ab [8]. 99 % der Patienten von Olschewski und Mitarbeitern waren mit dem kosmetischen Ergebnis sehr zufrieden oder zufrieden. Viele ältere Patienten schätzen trotz der zu erwartenden meist geringfügigen Veränderungen im Strahlenfeld die Radiotherapie, weil ihnen dadurch die Belastung durch eine mittlere oder größere Operation erspart wird. Wenn der Tumor mit einem Eingriff in Lokalanästhesie mit anschließender primärer Naht entfernt werden kann, sollte nicht bestrahlt werden. Der relativ kleine Eingriff belastet nicht wesentlich mehr als die vor der Bestrahlung erforderliche Probebiopsie. Ein Patient von Olschewski und Mitarbeitern war wegen einer lokalisierten Alopezie mit dem Bestrahlungsergebnis unzufrieden. Meist lässt sich das vermeiden, wenn man vor der Radiotherapie in dem angezeichneten Bestrahlungsfeld die Haare abschneidet. Der Patient (oder die Patientin) sollte dann versuchen, durch Überkämmen der benachbarten Haare die kahle Stelle zu verstecken und überlegen, ob er (oder sie) den bleibenden Haarverlust im Bestrahlungsfeld Radiotherapie des Basalzellkarzinoms

In their article on radiotherapy for cutaneous malignant melanoma, Drs. Ballo and Ang discuss four key points, which we will address below.

The aim of the presented study was to evaluate side-effects of soft x-ray therapy for basal and squamous cell carcinomas of the skin. 888 patients suffering from basal and/or squamous cell carcinomas were followed up for five years after treatment with soft x-rays. 47 (4.7%) ulcerations developed in 1005 roentgenoderms. 44 of these ulcerations cured by ointments and moist compresses, 2 required an operation, and one appeared to be recalcitrant. Cosmetic disorders, especially depigmentation (90,6% of 403 irradiated fields), were observed frequently. Lacrimation was noted in 76 of 165 (46%) roentgenoderms around the eye. This symptom decreased with time. After 8 of 97 (8.3%) irradiation treatments of tumours of the lips patients had difficulties closing their mouth completely. Severe side effects of soft x-ray therapy are rare and therefore do not diminish the value of this treatment.