A 3-dimensional histology computer model of malignant melanoma and its implications for digital pathology.

BACKGROUND: Historically, cancer diagnoses have been made by pathologists using two-dimensional histological slides. However, with the advent of digital pathology and artificial intelligence, slides are being digitised, providing new opportunities to integrate their information. Since nature is 3-dimensional (3D), it seems intuitive to digitally reassemble the 3D structure for diagnosis. OBJECTIVE: To develop the first human-3D-melanoma-histology-model with full data and code availability. Further, to evaluate the 3D-simulation together with experienced pathologists in the field and discuss the implications of digital 3D-models for the future of digital pathology. METHODS: A malignant melanoma of the skin was digitised via 3 µm cuts by a slide scanner; an open-source software was then leveraged to construct the 3D model. A total of nine pathologists from four different countries with at least 10 years of experience in the histologic diagnosis of melanoma tested the model and discussed their experiences as well as implications for future pathology. RESULTS: We successfully constructed and tested the first 3D-model of human melanoma. Based on testing, 88.9% of pathologists believe that the technology is likely to enter routine pathology within the next 10 years; advantages include a better reflectance of anatomy, 3D assessment of symmetry and the opportunity to simultaneously evaluate different tissue levels at the same time; limitations include the high consumption of tissue and a yet inferior resolution due to computational limitations. CONCLUSIONS: 3D-histology-models are promising for digital pathology of cancer and melanoma specifically, however, there are yet limitations which need to be carefully addressed.

Age as a key factor influencing metastasizing patterns and disease-specific survival after sentinel lymph node biopsy for cutaneous melanoma.

In our study, we investigated the impact of the constitutional factor age on the clinical courses of melanoma patients with sentinel lymph node (SLN) biopsy. Descriptive statistics, Kaplan-Meier estimates, logistic regression analysis and the Cox proportional hazards model were used to study a population of 2,268 consecutive patients from three German melanoma centers. Younger age was significantly related to less advanced primary tumors. Nevertheless, patients younger than 40 years of age had a twofold risk of being SLN-positive (p < 0.000001). Of the young patients with primary melanomas with a thickness of 0.76 mm to 1.0 mm, 19.7% were SLN-positive. Using multivariate analysis, younger age, increasing Breslow thickness, ulceration and male sex were significantly related to a higher probability of SLN-metastasis. During follow-up, older patients displayed a significantly increased risk of in-transit recurrences (p = 0.000002) and lymph node recurrences (p = 0.0004). With respect to melanoma specific overall survival the patient's age was highly significant in the multivariate analysis. The unfavorable effect of being older was significant in the subgroups with positive and negative SLNs. Age remained also significant for the survival after the onset of distant metastases (p = 0.002). In conclusion, the patient's age is a strong and independent predictor of melanoma-specific survival in patients with localized melanomas, in patients with positive SLNs and after the onset of distant metastases. Younger patients have a better prognosis despite their higher probability of SLN metastasis. Older patients are less frequently SLN-positive but have a higher risk of loco-regional recurrence.

EANM-EORTC general recommendations for sentinel node diagnostics in melanoma.

The accurate diagnosis of a sentinel node in melanoma includes a sequence of procedures from different medical specialities (nuclear medicine, surgery, oncology, and pathology). The items covered are presented in 11 sections and a reference list: (1) definition of a sentinel node, (2) clinical indications, (3) radiopharmaceuticals and activity injected, (4) dosimetry, (5) injection technique, (6) image acquisition and interpretation, (7) report and display, (8) use of dye, (9) gamma probe detection, (10) surgical techniques in sentinel node biopsy, and (11) pathological evaluation of melanoma-draining sentinel lymph nodes. If specific recommendations given cannot be based on evidence from original, scientific studies, referral is given to "general consensus" and similar expressions. The recommendations are designed to assist in the practice of referral to, performance, interpretation and reporting of all steps of the sentinel node procedure in the hope of setting state-of-the-art standards for good-quality evaluation of possible spread to the lymphatic system in intermediate-to-high risk melanoma without clinical signs of dissemination.

Pathologic reporting and special diagnostic techniques for melanoma.

Pathologists play a central role in the management of cutaneous melanoma in determining that a tumor is a melanoma, whether or not it is primary or metastatic, and whether or not the margins of excision are tumor free and in evaluating prognostic indicators from examination of the primary tumor and, where appropriate, lymph nodes, including the sentinel nodes.

Which melanoma patient carries a BRAF-mutation? A comparison of predictive models.

BACKGROUND: In patients with advanced melanoma the detection of BRAF mutations is considered mandatory before the initiation of an expensive treatment with BRAF/MEK inhibitors. Sometimes it is difficult to perform such an analysis if archival tumor tissue is not available and fresh tissue has to be collected. RESULTS: 514 of 1170 patients (44%) carried a BRAF mutation. All models revealed age and histological subtype of melanoma as the two major predictive variables. Accuracy ranged from 0.65-0.71, being best in the random forest model. Sensitivity ranged 0.76-0.84, again best in the random forest model. Specificity was low in all models ranging 0.51-0.55. METHODS: We collected the clinical data and mutational status of 1170 patients with advanced melanoma and established three different predictive models (binary logistic regression, classification and regression trees, and random forest) to forecast the BRAF status. CONCLUSIONS: Up to date statistical models are not able to predict BRAF mutations in an acceptable accuracy. The analysis of the mutational status by sequencing or immunohistochemistry must still be considered as standard of care.

Preponderance of the oncogenic V599E and V599K mutations in the B-raf kinase domain is enhanced in melanoma lymph node metastases.

Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, amongst other carcinomas, in a substantial subset of primary melanomas, with a preponderance of mutations within the kinase domain, including the activating V599E and V599K transitions. We investigated a representative series of 54 resection specimens of melanoma lymph node metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) gel electrophoresis. Sequencing of cloned PCR-SSCP amplicons resulted in 24 (44%) samples harbouring somatic mutations, which is not significantly different from the mutation frequency found in recently investigated primary cutaneous melanomas (Deichmann M, Thome M, Benner A, Näher H. B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome. Oncology 2004; 66:411-419). The activating mutation T1796A was present in 20 (83%) of these resection specimens, followed in frequency by the oncogenic g1795A mutation in five (21%) cases. With regard to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 15 (62%) and five (21%) of the 24 positive metastases, respectively. The detection of mutations at this hot spot codon was significantly associated with subsequent visceral metastasis (P=0.03; Fisher's exact test). During the transition from primary melanomas (see reference above) to lymph node metastases, the spectrum of B-raf mutations narrows significantly (P=0.00047). The oncogenic B-raf mutations V599E and V599K, as early events in melanocyte transformation, persist throughout metastasis with important prognostic implications.

Nodal Basin Recurrence After Sentinel Lymph Node Biopsy for Melanoma: A Retrospective Multicenter Study in 2653 Patients.

UNLABELLED: The objective of this study was to analyze different types of nodal basin recurrence after sentinel lymph node biopsy (SLNB) for melanoma. PATIENTS AND METHODS: Kaplan-Meier estimates and the Cox proportional hazards model were used to study 2653 patients from 3 German melanoma centers retrospectively.The estimated 5-year negative predictive value of SLNB was 96.4%. The estimated false-negative (FN) rates after 1, 2, 3, 5, and 10 years were 2.5%, 4.6%, 6.4%, 8.7%, and 12.6%, respectively. Independent factors associated with false negativity were older age, fewer SLNs excised, and head or neck location of the primary tumor. Compared with SLN-positive patients, the FNs had a significantly lower survival. In SLN-positive patients undergoing completion lymphadenectomy (CLND), the 5-year nodal basin recurrence rate was 18.3%. The recurrence rates for axilla, groin, and neck were 17.2%, 15.5%, and 44.1%, respectively. Significant factors predicting local relapse after CLND were older age, head, or neck location of the primary tumor, ulceration, deeper penetration of the metastasis into the SLN, tumor-positive CLND, and >2 lymph node metastases. All kinds of nodal relapse were associated with a higher prevalence of in-transit metastases.The FN rate after SLNB steadily increases over the observation period and should, therefore, be estimated by the Kaplan-Meier method. False-negativity is associated with fewer SLNs excised. The beneficial effect of CLND on nodal basin disease control varies considerably across different risk groups. This should be kept in mind about SLN-positive patients when individual decisions on prophylactic CLND are taken.

Pathology of the sentinel lymph node in melanoma.

The success and further evolution of the sentinel lymph node (SLN) concept decisively depend on histological techniques. Fundamental standards were agreed on by a panel of international experts from various disciplines in 1999 and published as "The Augsburg Consensus" in 2000. Conventional histology (hematoxylin and eosin [H&E]) has to be supplemented by immunohistochemistry (eg, S100 and HMB45) using adequate series of paraffin sections. Melanoma cells in SLNs must be carefully differentiated from capsular and trabecular nevocytes, from immigrated Langerhans cells, from interdigitating dendritic leukocytes, and from nerve sheath cells, which all share S100 positivity in the cytoplasm. The micromorphometric S classification is based on the maximum distance of intranodal melanoma cells from the interior margin of the SLN capsule. It has proven its practicability under routine circumstances, as well as its predictive value regarding further nodal and distant metastases as well as overall survival. This has to be considered in prospective randomized trials dealing with the issues of completion lymphadenectomy and adjuvant therapies of melanoma patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, when performed as a supplement to histology on the basis of additional paraffin sections, can further enhance the diagnostic sensitivity for detecting melanoma cells in SLNs.

Lower prevalence of lymphatic metastasis and poorer survival of the sentinel node-negative patients limit the prognostic value of sentinel node biopsy for head or neck melanomas.

Head or neck location of primary cutaneous melanomas has been described as an adverse prognostic factor, but this has to be reassessed after the introduction of sentinel lymph node (SLN) excision (SLNE). Descriptive statistics, Kaplan-Meier estimates and Cox proportional hazard models were used to study retrospectively a population of 2302 consecutive melanoma patients from three German melanoma centres undergoing SLNE. Approximately 10% of the patients (N=237) had a primary melanoma located at the head or neck (HNM). In both the SLN-positive and SLN-negative subpopulation, patients with HNM were significantly older, more frequently men and had thicker primaries compared with patients with tumours in other locations. The proportion of positive SLNs was lower in HNM compared with other locations of the primary (20 vs. 26%, P=0.048). The false-negative rate was higher in HNM (17.5 vs. 8.4%, P=0.05). In patients with HNM, the SLN status was a significant factor for recurrence-free survival but not for overall survival. SLN-negative HNM patients had a significantly worse overall survival than the SLN negatives with primaries at other sites, whereas the prognosis of the SLN-positive patients was similar in both groups. The prevalence of lymph node metastases after SLNE is lower in patients with HNM compared with other melanoma locations. As a result, the prognostic information provided by the SLN for HNM seems less important. Decision making for SLNE in HNM should be carefully balanced considering the potential morbidity of the procedure.

Effectiveness and tolerability of ipilimumab: experiences from 198 patients included in a named-patient program in various daily-practice settings and multiple institutions.

BACKGROUND: Ipilimumab is an approved anti-cytotoxic T-lymphocyte-associated antigen-4 monoclonal antibody introducing immune responses in melanoma patients. Treatment experiences from named-patient programs support the evaluation of the efficacy and tolerability of new medicines under usual circumstances of health care practice. Here, the largest ever reported cohort treated with ipilimumab 3 mg/kg alone is described. METHODS: This report retrospectively analyzes data of 198 patients who were followed up in 15 hospital centers in Germany between April 2010 and March 2013. Patients had received prior therapy for unresectable stage III or IV melanoma before receiving ipilimumab (4 doses of 3 mg/kg every 21 d). Routine staging and tumor response evaluation procedures were applied. RESULTS: Of the patients, 119 received the planned 4-course therapy schedule; in further 79 patients, the number of doses was reduced mainly because of toxicity or fast progression. In all, 196 patients were eligible for evaluation of the efficacy of ipilimumab under routine care conditions. Median overall survival (OS) was 6.8 months [95% confidence interval, 5.6-10.3] from the start of therapy. OS differed significantly among patients who received 4 doses (n=119) and those receiving <4 doses (n=79) (14.2 vs. 2.0 mo; P<0.0001). The overall response rate (ORR) of 11% was in the same range as reported from previous clinical trials; and stable disease (SD) was observed in 11% resulting in a disease control rate (ORR+SD) of 22%. In 23 of the 79 patients with reduced dosing, dose omission was most probably caused by toxicity, whereas 56 patients had progressive disease before receiving all 4 treatment cycles. Immune-related adverse events (irAE) were reported in 30% of all treated patients, the occurrence of irAE correlated significantly with the probability of response to therapy and prolonged OS. CONCLUSION: In this named-patient program including heavily pretreated patients, the efficacy and tolerability of ipilimumab 3 mg/kg corresponds with findings from the confirmatory clinical trial.

Interobserver reproducibility of histologic parameters of melanoma deposits in sentinel lymph nodes: implications for management of patients with melanoma.

BACKGROUND: : Histologic parameters of melanoma deposits in sentinel lymph nodes (SLNs) have been shown to be predictive of clinical outcome and the presence or absence of tumor in non-SLNs, but assessment of these parameters is prone to interobserver variation. METHODS: : Histologic sections of 44 SLNs containing metastatic melanoma were examined by 7 pathologists. Parameters assessed included cross-sectional area of tumor deposits, cross-sectional area of SLNs, percentage of SLN area involved by tumor calculated from the 2 previous parameters, estimated percentage of SLN area involved by tumor, tumor penetrative depth, location of tumor within the SLN, and presence of extracapsular spread. Levels of interobserver agreement were measured by using intraclass correlation coefficients (ICC). RESULTS: : There was good to excellent interobserver agreement on measurement of quantitative parameters: maximal size of largest tumor deposits, calculated area of 3 largest tumor deposits, percentage of the area of SLN involved by tumor, and tumor penetrative depth (ICC, 0.88, 0.73, 0.68, and 0.83, respectively). There was moderate agreement on the evaluation of subcapsular versus nonsubcapsular location of tumor deposits (ICC = 0.50). Agreement on assessment of extracapsular spread was fair (ICC = 0.39). CONCLUSIONS: : Assessment of some of the quantitative parameters was highly reproducible between pathologists. However, evaluation of the location of tumor deposits within SLNs and assessment of extracapsular spread was less reproducible. Clearer definitions and training can be expected to improve the reproducibility of assessment. These results have important implications for reliability and reproducibility of these parameters in staging, prediction of outcome, and clinical management of melanoma patients. Cancer 2009. (c) 2009 American Cancer Society.

Benefit of sentinel lymphadenectomy for patients with nonulcerated cutaneous melanomas in the Breslow range between 0.76 and 1 mm: a follow-up study of 148 patients.

Sentinel lymphadenectomy (SLNE) is now internationally accepted for the primary treatment of melanomas thicker than 1 mm. But it is still controversial whether also patients with nonulcerated melanomas in the Breslow range between 0.76 and 1 mm should be included. At the authors' department, 87 of such patients (Group A) underwent SLNE in combination with wide local excision (WLE) of their primary melanomas in the years 1995 to 2001. SLN micrometastases were found in 10 of these patients (11.5%). Radical completion lymph node dissections (CLND) were added in 4 of the 10 patients without revealing any further nodal metastases. All the 87 Group A patients remained free from recurrent disease at a median follow-up time of 74 months. The control Group B from the same department encompassed 61 consecutive stage Ia patients with melanomas in the identical Breslow range, who had undergone only WLE of their primaries without SLNE in the years 1987 to 1993 (median follow-up time 115 months). Five of these 61 patients (8.2%) developed melanoma metastases within 12 to 68 (median 19) months of follow-up, 3 of them initially in regional lymph nodes. Four of the 5 individuals died because of the final distant dissemination of the melanoma. Kaplan-Meier comparisons between Groups A and B with log-rank testing showed a significantly worse outcome of Group B with respect to recurrence-free survival (p = 0.01), regional nodal progression (p = 0.041), distant metastasis (p = 0.023) and melanoma-related mortality (p = 0.03). The overall survival was not significantly different, because expiries not related to melanoma predominated in both groups. Our data suggest that SLNE seems to nearly completely eliminate the risk of melanoma recurrences in patients with melanomas between 0.76 and 1 mm thick.

Clinical spectrum of cutaneous Langerhans' cell histiocytosis mimicking various diseases.

Langerhans' cell histiocytosis summarizes a spectrum of diseases on the basis of histogenetic criteria. These are characterized by an accumulation of cells with Langerhans' cell phenotype in one or multiple organs. Up to 50% of patients with either single or multi-organ manifestation of Langerhans' cell histiocytosis initially present with cutaneous symptoms. Nevertheless, cutaneous Langerhans' cell histiocytosis is rare and heterogeneous in its clinical features and therefore prone to misdiagnosis. We report on five patients, two infants and three adults, suffering from cutaneous Langerhans' cell histiocytosis, either singly or as part of multi-organ disease. The different skin features morphologically mimicking other entities are shown and the differential diagnoses are discussed. The correct diagnosis in all presented cases is based on immunohistological examination, showing a histiocytic infiltrate positively staining with anti-S100 antibodies, CD1a and--apart from one case--with CD207 (langerin).