Isolated melanoma cells in sentinel lymph node in stage IIIA melanoma correlate with a favorable prognosis similar to stage IB.

BACKGROUND: The American Joint Committee on Cancer 8th edition (AJCC v8) defines sentinel lymph nodes (SLN) containing any tumor cells as positive SLN. Consequently, even thin melanomas with isolated tumor cells (ic) in SLN are classified as stage IIIA, making them candidates for adjuvant therapy. OBJECTIVES AND ENDPOINTS: We aimed to evaluate survival outcomes of melanoma stage IIIA (ic) and compare them with stage IIIA with lymph node (LN) metastases > 0.1 mm. Primary endpoints were relapse-free survival (RFS) and distant metastases-free survival (DMFS). Secondary endpoint was melanoma specific survival (MSS). RESULTS: The discovery cohort from the Department of Dermatology, University Hospital Tuebingen, included 237 patients; confirmation cohort included 143 patients from the DeCOG trial. The Tuebingen cohort included 95 patients with stage IIIA (ic) and 142 patients with stage IIIA. The DeCOG trial included 39 patients with stage IIIA (ic) and 104 patients with stage IIIA. In the Tuebingen cohort, 10-year RFS rates for stage IIIA (ic) and IIIA were 84% (95% CI 75-94) and 49% (95% CI 39-59), respectively (p < 0.001). 10-year DMFS rates for stage IIIA (ic) and IIIA were 89% (95% CI 81-97) and 56% (95% CI 45-67), respectively; (p < 0.001). In the DeCOG cohort, 10-year RFS for stage IIIA (ic) and stage IIIA were 88% (95% CI 78-99) and 35% (95% CI 7-62), respectively; (p = 0.009). 10-year DMFS for stage IIIA (ic) and IIIA was 88% (95% CI 77-99) and 60% (95% CI 39-80), respectively (p = 0.061). CONCLUSION: Stage IIIA (ic) melanoma exhibits a prognosis similar to stage IB. Recommendation of adjuvant therapy in Stage IIIA (ic) warrants thorough discussion.

Reporting of melanoma cell densities in the sentinel node refines outcome prediction.

INTRODUCTION: Sentinel node biopsy is a key procedure to predict prognosis in melanoma. In a prospective study we compare reporting on melanoma cell densities in cytospin preparations with semiquantitative histopathology for predicting outcome. PATIENTS AND METHODS: Sentinel nodes from 900 melanoma patients were bisected. One half of each node was disaggregated mechanically. The melanoma cell density (number of HMB45 positive cells per million lymphocytes with at least one cell showing morphological features of a melanoma cell) was recorded after examining two cytospins. For the second half the maximum diameter of metastasis was determined after haematoxylin and eosin (H&E) and immunohistological staining of three slides. RESULTS: Cytospins were positive for melanoma in 218 of 900 patients (24%). Routine pathology was positive in 111 of 900 (12%) patients. A more extensive pathological workup in cytospin-only positive patients led to a revised diagnosis (from negative to positive) in 23 of 101 patients (22.7%). We found a moderate but significant correlation between melanoma cell densities (determined in cytospins) and the maximum diameter of metastasis (determined by pathology) (rho = 0.6284, p < 0.001). At a median follow-up of 37 months (IQR 25-53 months) melanoma cell density (cytospins) (p < 0.001), thickness of melanoma (p = 0.008) and ulceration status (p = 0.026) were significant predictors for melanoma specific survival by multivariable testing and were all confirmed as key predictive factors by the random forest model. Maximum diameter of metastases, age and sex were not significant by multivariable testing (all p > 0.05). CONCLUSION: Recording melanoma cell densities by examining two cytospins accurately predicts melanoma outcome and outperforms semiquantitative histopathology.

Modification of Skin Flaps for Single-Stage Reconstruction of the Nasal Ala Using All-Layer Suture: A Prospective Study.

BACKGROUND AND OBJECTIVES: After local flaps, it may be necessary to reconstruct the contour of the nasal ala. This is possible with a single-stage all-layer shaping suture. In the present study, the functional and aesthetic results after single-stage reconstruction of the nasal ala were prospectively evaluated. PATIENTS AND METHODS: Patients who underwent surgery for skin tumors of the nose between 06/2019 and 06/2020 who required reconstruction of the nasal ala as part of the defect closure and had an all-layer suture used were prospectively included in the study. A standardized evaluation of aesthetic and functional outcome was conducted by the patient and a physician at discharge as well as 4 weeks later. Patients additionally underwent a follow-up survey 6 months later. RESULTS: Thirty-seven patients were included in the study. Four weeks postoperatively, all flaps were found to be fully healed and vital. Aesthetic outcome at 4 weeks was rated as very good or good by physicians in 73% and by patients in 78.4%. Persistent complications due to reduced blood flow were not observed. CONCLUSION: The reshaping of the nasal ala as part of the defect reconstruction with an all-layer suture demonstrates very good aesthetic as well as functional results and can be performed in a single-stage procedure. .

Flow Cytometry for Detection and Quantification of Micrometastases in Sentinel Lymph Nodes from Patients with Primary Melanoma.

BACKGROUND: Detection of micrometastases in the regional lymph nodes is one of the most important prognostic factors for melanoma patients. Our aim was to evaluate the suitability of flow cytometry for rapid quantification of disseminated melanoma cells in sentinel lymph nodes (SLN). METHODS: 132 SLNs from 104 patients diagnosed with melanoma were analyzed by flow cytometry, utilizing the extracellular marker melanoma-associated chondroitin sulfate proteoglycan, in addition to quantitative immunocytology and conventional histopathology, including immunohistochemistry. For quantification, the number of melanoma-positive cells per million lymph node cells (disseminated cancer cell density, DCCD) detected by flow cytometry was compared to the DCCD obtained by immunocytology. RESULTS: Compared to histopathology and immunocytology, flow cytometry exhibited a sensitivity of 50% and a specificity of 85%. DCCDs of immunocytology and flow cytometry of the 37 immunocytologically positive SLNs showed a positive correlation (Spearman's ρ = 0.7, P < 0.0001). In 10 SLNs from 9 patients with high tumor load, the flow cytometric DCCD was 8-fold higher on average than the immunocytologic DCCD. CONCLUSIONS: Although flow cytometry is not yet suitable for early detection of metastatic melanoma, it promises to become a valuable tool for rapidly quantifying tumor load in high-risk patients.

Genetic alterations driving metastatic colony formation are acquired outside of the primary tumour in melanoma.

Mouse models indicate that metastatic dissemination occurs extremely early; however, the timing in human cancers is unknown. We therefore determined the time point of metastatic seeding relative to tumour thickness and genomic alterations in melanoma. Here, we find that lymphatic dissemination occurs shortly after dermal invasion of the primary lesion at a median thickness of ~0.5 mm and that typical driver changes, including BRAF mutation and gained or lost regions comprising genes like MET or CDKNA2, are acquired within the lymph node at the time of colony formation. These changes define a colonisation signature that was linked to xenograft formation in immunodeficient mice and death from melanoma. Thus, melanoma cells leave primary tumours early and evolve at different sites in parallel. We propose a model of metastatic melanoma dormancy, evolution and colonisation that will inform direct monitoring of adjuvant therapy targets.

The sentinel lymph node spread determines quantitatively melanoma seeding to non-sentinel lymph nodes and survival.

INTRODUCTION: Complete lymph node dissection (CLND) after a positive sentinel node (SN) biopsy provides important prognostic information in melanoma patients but has been questioned for therapeutic use recently. We explored whether quantification of the tumour spread to SNs may replace histopathology of non-sentinel nodes (NSNs) for staging purposes. PATIENTS AND METHODS: We quantified melanoma spread in SNs and NSNs in 128 patients undergoing CLND for a positive SN. In addition to routine histopathology, one-half of each of all 1496 SNs and NSNs was disaggregated into a single cell suspension and stained immunocytochemically to determine the number of melanoma cells per 106 lymph node cells, i.e. the disseminated cancer cell density (DCCD). RESULTS: We uncovered melanoma spread to NSNs in the majority of patients; however, the tumour load and the proportion of positive nodes were significantly lower in NSNs than in SNs. The relation between SN and NSN spread could be described by a mathematical function with DCCDNSN = DCCDSNc/101-c (c = 0.69; 95% confidence interval [CI]: 0.62-0.76). At a median follow-up of 67 months, multivariable Cox regression analyses revealed that DCCDSN (p = 0.02; HR 1.34, 95% CI: 1.05-1.71) and the total number of pathologically positive nodes (p = 0.02; HR 1.53, 95% CI: 1.07-2.22) were significant risk factors after controlling for age, gender, thickness of melanoma and ulceration status. A prognostic model based on DCCDSN and melanoma thickness predicted outcome as accurately as a model including pathological information of both SNs and NSNs. CONCLUSION: The assessment of DCCDSN renders CLND for staging purposes unnecessary.

Quantitative measurement of melanoma spread in sentinel lymph nodes and survival.

BACKGROUND: Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival. METHODS AND FINDINGS: We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3-123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61-2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02-2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories. CONCLUSIONS: Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.

Treatment of pyoderma gangrenosum with topical factor XIII.

Pyoderma gangrenosum (PG) is a severe ulcerative skin disease. Despite systemic immunosuppressive therapy, PG ulcers often progress and can develop into life-threatening conditions. In this case series, we treated 6 patients suffering from recalcitrant PG with topical coagulation factor XIII, which has been shown to exert beneficial effects on tissue regeneration and wound healing. All 6 patients showed a positive response to treatment with a marked reduction in wound size that was maintained during a 3-month follow-up period. The treatment was well tolerated with no remarkable adverse effects or complications. Topical factor XIII has potential in combination with standard immunosuppressive therapy for the treatment of PG.

Response evaluation of musculoskeletal involvement in patients with deep morphea treated with methotrexate and prednisolone: a combined MRI and clinical approach.

OBJECTIVE: The purpose of this article is to explore the role of MRI in monitoring musculoskeletal involvement in patients with morphea who are undergoing treatment with methotrexate and prednisolone. SUBJECTS AND METHODS: Twenty-two consecutive patients (six men and 16 women; median age, 52 years) with systemic scleroderma and deep morphea prospectively underwent whole-body MRI twice, before treatment (time 1) and during follow-up after 6-12 months (time 2). Images were evaluated for abnormal signal intensity or thickening of sub-cutaneous fatty tissue septa, muscular fasciae, intramuscular perifascial septa, muscle signal intensity, and articular or tendon sheath synovial abnormalities on STIR and gadolinium-enhanced scans. For clinical assessment, the localized scleroderma (morphea) severity index and a 0-6 pain score were applied. RESULTS: From a clinical point of view, none of our patients had progression of the disease, 12 patients were responders (defined as an improvement of localized scleroderma severity index and pain score ≥ 50%), and 10 patients had stable disease. Among responders, the number of patients with subcutaneous septal thickening (time 1, n = 9; time 2, n = 2), fascial enhancement (time 1, n = 8; time 2, n = 3), and articular synovitis (time 1, n = 5; time 2, n = 1) decreased more than in the stable disease group (subcutaneous septal thickening: time 1, n = 9; time 2, n = 8; fascial enhancement: time 1, n = 5; time 2, n = 5; articular synovitis: time 1, n = 8; time 2, n = 6). Subcutaneous thickening, fascial thickening, and fascial enhancement were scored significantly lower at follow-up MRI in responders. CONCLUSION: MRI findings were sensitive to changes in musculoskeletal manifestations in patients with deep morphea undergoing systemic treatment with methotrexate and prednisolone. Thus, MRI can be recommended as an additional tool for response monitoring.

Magnetic resonance imaging findings in patients with systemic scleroderma and musculoskeletal symptoms.

OBJECTIVE: To explore the role of whole-body magnetic resonance imaging (MRI) in detecting musculoskeletal involvement in patients with systemic scleroderma and musculoskeletal symptoms. METHODS: Eighteen consecutive patients (8 men, 10 women) with systemic scleroderma (median age 46 years) presenting with musculoskeletal complaints underwent whole-body MRI at 1.5 T. Images were evaluated for abnormal signal intensity and/or thickening of subcutaneous fatty tissue septa, muscular fasciae, intramuscular perifascial septa, muscle signal intensity and articular or tendon sheath synovial abnormalities on STIR and post-gadolinium scans. Additionally, C-reactive protein, creatinine kinase and the modified Rodnan skin score were determined. RESULTS: MRI indicated evidence of fasciitis, articular synovial inflammation, and subcutaneous thickening in 16 (89 %) patients. MRI findings were compatible with myopathy or myositis in 14 (78 %) patients, tenosynovitis in 11 (61 %) patients and enthesitis in 10 (56 %) patients. Typically, these manifestations were distributed symmetrically and mostly generalised. We only found few correlations with modified Rodnan skin score, C-reactive protein and creatinine kinase. CONCLUSION: In patients with systemic scleroderma experiencing musculoskeletal symptoms, whole-body MRI is able to detect involvement of muscles, fasciae, joints and entheses more confidently compared with clinical and laboratory parameters.

Localized scleroderma: MR findings and clinical features.

PURPOSE: To describe musculoskeletal manifestations seen at magnetic resonance (MR) imaging in patients with localized scleroderma (LS) and to examine the relationship of MR findings to clinical subtypes and clinically suspected musculoskeletal features. MATERIALS AND METHODS: Institutional review board approval was obtained. Forty-three patients (30 female, 13 male; mean age, 42 years) with LS underwent MR imaging with a 1.5-T MR imager between November 2005 and June 2010. Findings were classified into clinical subtypes according to recently published consensus criteria. Images were evaluated for morphologic changes and signal abnormalities of subcutaneous fat septa, muscle fasciae, intramuscular septa, joint and/or tendon sheath synovia, entheses, and bone marrow. Clinically suspicious features of musculoskeletal manifestations-such as articular or periarticular pain, joint contractures, swelling, and increased warmth of the joints or extremities-were recorded. RESULTS: Musculoskeletal involvement was detected with MR imaging in 32 (74%) of 43 patients. It was detected with MR imaging in 26 (96%) of 27 patients in whom it was clinically suspected and in six (38%) of 16 patients in whom it was not clinically suspected. We found fascial thickening (26 [60%] of 43 patients), increased fascial enhancement (23 [53%] of 43 patients), articular synovitis (17 [40%] of 43 patients), tenosynovitis (nine [21%] of 43 patients), perifascial enhancement (seven [16%] of 43 patients), myositis (six [14%] of 43 patients), enthesitis (three [7%] of 43 patients), bone marrow involvement (two [5%] of 43 patients), and subcutaneous septal thickening (28 [65%] of 43 patients). The highest prevalence of musculoskeletal involvement was seen in patients with pansclerotic morphea. CONCLUSION: MR imaging reveals musculoskeletal involvement in patients with localized scleroderma.

Circulating melanoma cells in peripheral blood of patients with uveal melanoma before and after different therapies and association with prognostic parameters: a pilot study.

PURPOSE: To evaluate whether tumour therapy for malignant uveal melanoma leads to a shedding of melanoma cells into the systemic circulation. METHODS: Ninety-four peripheral blood samples from 81 patients with malignant uveal melanoma were collected before and after different tumour therapies and the number of circulating melanoma cells (CMCs) was investigated (seven patients with enucleation, 49 patients with stereotactic radiotherapy, 19 patients with endoresection of the tumour, 15 patients with ruthenium-brachytherapy and four patients with transpupillary thermotherapy). A cellular approach was used to detect CMCs through an immunocytological assay with tumour cell enrichment by immunomagnetic cell sorting. The number of CMCs was analysed further according to specific patient characteristics, tumour parameters and the development of metastasis. RESULTS: There was no significant difference between the number of CMCs before and after the different therapies (p = 0.78). There was also no significant association between established prognostic parameters of primary uveal melanoma and the detection of CMCs (all p >0.05). The number of CMCs was not related to the development of metastasis in a short median follow-up time of 16 months (p > 0.05). CONCLUSION: No changes in CMC values were observed before and after different tumour therapies. In the majority of cases therapy does not lead to a shedding of detectable melanoma cells into the systemic circulation.

Quantification of tumor cell burden by analysis of single cell lymph node disaggregates in metastatic prostate cancer.

BACKGROUND: The size of lymph node (LN) metastases in prostate cancer patients represents an important prognosticator, but histological work-up may not reflect the true extent of tumor invasion. We present a novel technique (1) to detect early tumor cell dissemination and (2) to quantify the true tumor burden. METHODS: Prospectively 232 LN of 20 consecutive patients with prostate cancer after lymph node dissection were longitudinally bisected, one half was subjected to single cell immunocytochemistry for pancytokeratine (CK), the other half underwent routine histopathological work-up and step section analysis. In immunocytochemistry, tumor cell density (TCD) was quantified by calculating the number of CK-positive cells/million leucocytes and compared to routine histopathology and step section analysis. RESULTS: Eight of 20 patients were positive in histopathology and step sectioning, but 14 of 20 patients were positive in single cell analysis. Twenty-five of 232 LN were positive in routine histopathology, whereas 52 of 232 LN were positive in single cell analysis. Median TCD in histopathologically positive LN was 3060.0 x 10(-6) and 9.9 x 10(-6) in histopathologically negative LN (P < 0.0001). Mean TCD of histopathologically negative LN of pN1 patients was significantly higher than the mean TCD of pN0 patients (P < 0.003). Mean TCD per patient correlated with serum-PSA (r(2) = 0.48, P < 0.006). CONCLUSIONS: Single cell analysis has an increased detection rate compared to routine histopathology and even to serial step section analysis. The method can detect early tumor dissemination and enables quantification of the tumor burden. The subgroup of histopathologically negative LN with CK-positive cells represents tumor cell dissemination not depicted histologically.

Visualization of circulating melanoma cells in peripheral blood of patients with primary uveal melanoma.

PURPOSE: In patients with uveal melanoma, tumor cell dissemination and subsequent formation of metastases are confined mainly to the hematogenous route. Here, we sought to isolate circulating melanoma cells in peripheral blood of patients with primary uveal melanoma and clinically localized disease. EXPERIMENTAL DESIGN: Blood samples from 52 patients with clinically localized uveal melanoma and from 20 control individuals were prospectively collected before therapy of the primary tumor. Tumor cells expressing the melanoma-associated chondroitin sulfate proteoglycan were enriched by immunomagnetic cell sorting and visualized by immunocytologic staining. Results were compared with clinical data at presentation. RESULTS: In 10 of 52 patients [19%; 95% confidence interval (95% CI), 10-33%], between 1 and 5 circulating melanoma cells were detected in 50 mL peripheral blood. No melanoma-associated chondroitin sulfate proteoglycan-positive cells were detected in any of the 20 controls examined. The presence of tumor cells in peripheral blood was associated with ciliary body invasion [odds ratio (OR), 20.0; 95% CI, 3.0-131.7], advanced local tumor stage (OR, 6.7; 95% CI, 1.8-25.4), and anterior tumor localization (OR, 4.0; 95% CI, 1.2-12.7), all established factors for uveal melanoma progression. CONCLUSIONS: Immunomagnetic enrichment enables detection of intact melanoma cells in peripheral blood of patients with clinically localized ocular disease. Visualization and capturing of these cells provide a unique tool for characterizing potentially metastasizing tumor cells from a primary melanoma at an early stage of the disease.

Suppression of melanoma-associated neoangiogenesis by bevacizumab.

BACKGROUND: Bevacizumab, a potent antibody against the vascular endothelial growth factor (VEGF), has been shown to be effective for treatment of colorectal cancer. Recently, high effectiveness of bevacizumab in combination with paclitaxel has been reported in a single metastatic melanoma case. To our knowledge, we demonstrate for the first time the antiangiogenetic effect of bevacizumab in a patient with a vitreous melanoma metastasis. OBSERVATIONS: A 68-year-old man with a vitreous melanoma metastasis of the left eye was treated with a revitrectomy combined with intravitreal bevacizumab application because of iris neovascularization and progressive epiretinal tumor plaques. Four days after the treatment, the melanoma-associated neovascularization completely disappeared, but it recurred after 6 weeks. Although repetitive administration of local bevacizumab produced the same antiangiogenetic effect, progression of the epiretinal tumor plaques could not be stopped with the local bevacizumab treatment. CONCLUSIONS: Intraocular administration of the anti-VEGF drug bevacizumab causes immediate and complete regression of melanoma-associated angiogenesis. The rationale for the therapeutic strategy in our patient was an elevated level of VEGF in the vitreous cavity. Because we could not demonstrate a direct antiproliferative effect of bevacizumab on melanoma metastasis, bevacizumab seems most promising if evaluated in combination with antiproliferative agents.