Flow DNA-cytometric findings of paraffin-embedded primary cutaneous melanomas related to prognosis

In a retrospective study flow cytometry was performed in 82 paraffin-embedded clinical stage I cutaneous melanomas. A significant correlation of ploidy to melanoma thickness was found. Using a log rank test ploidy was found significant for prognosis. However, using the Cox's proportional hazard test the influence of ploidy vanished and melanoma thickness or Clark level of invasion together with sex and location were the factors most important for prognosis.     

Flow DNA-cytometric findings of paraffin-embedded primary cutaneous melanomas related to prognosis

In a retrospective study flow cytometry was performed in 82 paraffin-embedded clinical stage I cutaneous melanomas. A significant correlation of ploidy to melanoma thickness was found. Using a log rank test ploidy was found significant for prognosis. However, using the Cox’s proportional hazard test the influence of ploidy vanished and melanoma thickness or Clark level of invasion together with sex and location were the factors most important for prognosis.     

Histopathologic abundance of pigmentation correlates with disease-specific survival in malignant melanoma but is not independent of current AJCC pT stage

The increasing number of melanoma patients makes it necessary to develop best possible strategies for prognosis assessment in order to recommend appropriate therapy and follow-up. The prognostic significance of tumor cell pigmentation has not been fully elucidated. Hematoxylin and eosin (H&E)-stained sections of 775 melanomas diagnosed between 2012 and 2015 were independently assessed for melanin pigment abundance by two investigators, and the impact on melanoma-specific survival was calculated. Unpigmented melanomas (n = 99) had a melanoma-specific survival of 67.7%, melanomas with moderate pigmentation (n = 384) had a melanoma-specific survival of 85.9%, and strongly pigmented melanomas (n = 292) had a melanoma-specific survival of 91.4% (p < .001). In an analysis of melanoma-specific survival adjusted for pT stage and pigmentation, we found a nonsignificant impact of pigmentation abundance with a hazard ratio of 1.277 (p = .74). The study presented here provides evidence in a German cohort that patients with pigmented melanomas have a more favorable prognosis than those diagnosed with nonpigmented melanomas. Moreover, the abundance of pigmentation already seems to provide a first prognostic estimate. However, it does not appear to provide significant additional value for prognostic assessment according to the AJCC 2017 pT classification.     

Genomic analysis reveals low tumor mutation burden which may be associated with GNAQ/11 alteration in a series of primary leptomeningeal melanomas

Primary central nervous system melanoma is rare and characterized by a variable prognosis, and no current treatment guidelines exist. We describe the clinical course of a 70‐year‐old female patient diagnosed with primary leptomeningeal melanoma (LMN) whose case represents the diagnostic and management challenges of this tumor. Targeted genomic sequencing of 315 genes from this tumor revealed GNAQ Q209L mutation and low (4 mutations/Megabase) tumor mutation burden (TMB). Wild‐type NRAS, KIT, and BRAF were also observed. A cohort of 4,787 melanomas was subsequently analyzed to identify additional primary central nervous system melanomas, of which 10 additional tumors met pathologic criteria (0.21% of total melanoma cohort). These tumors were genomically assessed according to the same targeted sequencing panel, and 6 of the tumors were also found to harbor a GNAQ mutation. All 10 tumors had low (less than or equal to 2 mutations/Megabase) TMB indicating a potential trend between G‐protein‐coupled receptor (GPCR) alterations and low TMB in LMNs. GPCR alterations were found to significantly correlate with TMB across the cohort of 4,787 melanomas, supporting this potential finding in the limited LMN subset.     

DNA ploidy-characteristics of human malignant melanoma analysed by flow cytometry and compared with histology and clinical course

Flow cytometric analysis of nuclear DNA content is valuable for indicating ploidy- and proliferation abnormalities in surgically removed human malignant melanomas. In 35 primary cutaneous melanomas, 20 metastases of melanoma in skin and lymph nodes, and 16 nevi the DNA distribution was analyzed by flow cytometry and compared with a variety of histological parameters and the subsequent clinical course. Heteroploid DNA distributions with increased polyploid or aneuploid fractions were found in 26 primary melanomas (74%), 14 metastases (70%), and 4 nevi (25%) indicating tumor clones with an abnormal nuclear DNA content. Three or more cell clones in a single biopsy was found in 10 primary melanomas, 2 metastases, and 1 nevus. The frequency of heteroploidy was significantly higher in primary and secondary melanomas than in nevi (p less than 0.001) and was correlated significantly with a high mitotic activity (p less than 0.002), marked nuclear pleomorphism (p less than 0.01), large nucleoli (p less than 0.01) and a thickness of the primary melanoma of more than 2.25 mm (p less than 0.02). Such histologic findings in malignant melanomas have been shown previously to be correlated with a bad prognosis. No significant correlation was found between heteroploidy and the histologic type of melanoma or the level of invasion. A 2-year clinical follow-up showed that more patients died from melanoma if the DNA distribution in the primary or secondary melanoma was heteroploid (6/26; 23% and 8/13; 62% respectively) than if it was diploid (0/9; 0% and 2/5; 40% respectively). However, the differences were not statistically significant. It is concluded that heteroploidy 1) is not an absolute criterion of malignancy, 2) is significantly correlated with histologic features indicating marked cellular anaplasia, and 3) is apparently correlated with a bad prognosis.     

GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis

The main role of growth arrest and DNA damage-inducible (GADD) genes is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The goal of this study was to examine the expression of GADD genes in primary melanomas with respect to prognosis. GADD34 was found in 73% of the primary melanomas investigated. GADD45 and GADD153 were positive in 60% and 80% of primary melanomas, respectively. Cox regression demonstrated that only GADD153 had any independent prognostic impact. We therefore decided to establish a PCR assay for detection of GADD153 in paraffin-embedded tissue. GADD153 deletion was found in 3/26 melanomas. None of the 3 cases with GADD153 deletion showed any expression of GADD153. Sequencing analysis detected polymorphism T-C at amino acid position 10 in 6/23 melanomas. In 6 cases with GADD153 polymorphism, GADD153 expression was found in 2 melanomas with a maximum GADD153 index of 10%. We postulate that the GADD gene family plays an important role in melanoma progression.     

EARLY DIAGNOSIS OF MALIGNANT MELANOMA OF THE SKIN

About five per cent of all malignant lesions of the skin are malignant melanomas. The poor prognosis associated with this malignant lesion emphasizes the importance of early diagnosis. A large proportion of malignant melanomas arise in preexisting lesions such as junction nevi, precancerous melanoses and, much more rarely, blue nevi. Early malignant changes in these precursor lesions include increasing pigmentation, enlargement, thickening, crusting, bleeding, ulceration, tumor formation, and development of satellite lesions.Many pigmented, and some non-pigmented, lesions of the skin must be differentiated from malignant melanoma. Since even with radical surgical treatment the prognosis of malignant melanoma is poor, junction nevi which are subject to continual trauma or have signs of probable malignant degeneration should be prophylactically excised.     

Surgical considerations in the management of malignant melanoma of the ear

Malignant melanomas of the external ear are rare and are difficult lesions to treat because of the cosmetic importance and the reconstructive difficulty of their location. The literature suggests that these lesions have a worse prognosis than melanomas occurring elsewhere and that radical resection is the "correct" treatment. To clarify this issue, we examined 21 consecutive patients (19 male, 2 female) with malignant melanoma of the ear seen at the Yale-New Haven Hospital over the last 10 years. Nineteen patients had a diagnosis of primary malignant melanoma of the ear, one had a local recurrence, and one had an in-transit melanoma from an unknown primary site. The mean thickness of the lesions was 2.7 mm. Two patients had palpable nodes, which in both cases turned out to be histologically positive for tumor. All patients underwent local excision and reconstruction using chondrocutaneous or fasciocutaneous flaps or skin grafts. There was one local recurrence (0.5 mm original thickness); there were two patients with regional recurrences, both of whom died within a year with disseminated disease. Forty-three percent have been followed for 5 or more years and all are alive and free of disease. This suggests that malignant melanoma of the ear may be safely treated by conservative excision and reconstruction.     

Prognostic trees to aid prognosis in patients with cutaneous malignant melanoma. Scottish Melanoma Group.

OBJECTIVES--To design user friendly guides to prognosis for patients who have had invasive primary cutaneous malignant melanomas surgically excised. DESIGN--Adaptation of the classification tree method was used to derive prognostic trees for four different subgroups of malignant melanoma patients in whom known and possible prognostic variables interacted in different ways. SETTING--Scotland. SUBJECTS--Statistical modelling for prognostic trees was based on 1978 patients whose primary malignant melanoma was first diagnosed in 1979-86 for whom five year follow up and all relevant clinical pathological data were available. The resultant model was validated with 300 patients first diagnosed in 1987 for whom the same information was available. MAIN OUTCOME MEASURES--Actual and predicted rate of survival after diagnosis of primary cutaneous malignant melanoma. RESULTS--The four subgroups of patients were men and women with ulcerated and non-ulcerated cutaneous primary melanomas. Validation of the model showed excellent agreement between actual status of patients in the relevant subgroups and their status as predicted by the model. CONCLUSIONS--The prognostic trees are simple to use and give more accurate prognosis for individual patients than is currently available from tumour thickness alone.     

Melanoma in people aged 65 and over in Scotland, 1979-89.

OBJECTIVE--Detailed analysis of primary cutaneous melanoma first diagnosed in Scotland in patients aged 65 and over. DESIGN--Comparison of changing incidence, sex distribution, site, histogenetic type, tumour thickness, and prognosis of all primary cutaneous melanomas in patients aged 65 and over diagnosed in Scotland in the 11 years 1979-89 with similar data for patients aged under 65. SETTING--Data were obtained from the Scottish Melanoma Group''s database, established in 1979, which aims to record detailed clinical, pathological, and surgical follow up details of all primary cutaneous melanomas registered in Scotland. PATIENTS--1430 patients (954 women, 476 men) aged 65 and over; comprising over a third of the 3903 patients with primary melanoma recorded for all age groups in Scotland during this period. RESULTS--The overall incidence of melanoma in patients aged 65 and over increased from 12.2/100,000 in 1979 to 20.7/100,000 in 1989, with the greatest increase seen in older men, from 7.8/100,000 in 1979 to 18.0/100,000 in 1989. The site most commonly affected was the face in both men and women (33% of all tumours). The most common histogenetic type was superficial spreading melanoma. 526 patients (37%) had melanomas with a tumour thickness of 3.5 mm or greater in the older age group, compared with 453 patients (18%) in those aged under 65. The highest proportion of thick tumours was seen in older men. Five year survival figures for 616 patients diagnosed between 1979 and 1984 were 88%, 66%, and 47% for thin, intermediate, and thick tumours respectively. Overall five year survival for the older age group was 64% compared with 78% for the younger age group. CONCLUSION--The increase in melanoma in the elderly and the high proportion of thick tumours, especially in men, require a specific educational programme for both primary and secondary prevention directed towards the older population.     

A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.     

Copy Number Loss of the Interferon Gene Cluster in Melanomas Is Linked to Reduced T Cell Infiltrate and Poor Patient Prognosis

While immunotherapies are rapidly becoming mainstays of cancer treatment, significant gaps remain in our understanding of how to optimally target them, alone or in combination. Here we describe a novel method to monitor levels of immune cells and pathways in expression data from solid tumors using pre-defined groups or modules of co-regulated immune genes. We show that expression of an interconnected sub-network of type I interferon-stimulated genes (ISGs) in melanomas at the time of diagnosis significantly predicted patient survival, as did, to a lesser extent, sub-networks of T helper/T regulatory and NK/T Cytotoxic cell genes. As a group, poor prognosis tumors with reduced ISG and immune gene levels exhibited significant copy number loss of the interferon gene cluster located at chromosome 9p21.3. Our studies demonstrate a link between type I interferon action and immune cell levels in melanomas, and suggest that therapeutic approaches augmenting both activities may be most beneficial.     

Audit of public education campaign to encourage earlier detection of malignant melanoma.

OBJECTIVES--To evaluate a public campaign to encourage earlier referral and treatment of primary cutaneous malignant melanoma and thus reduce mortality related to melanoma. DESIGN--Production and distribution of educational material aimed at adults. Update information sent to general practitioners before campaign. Analysis of data on melanoma before and after campaign in June 1985. SETTING--West of Scotland, population 2.7 million. MAIN OUTCOME MEASURES--Total numbers of referrals per month to melanoma clinic, numbers of melanomas diagnosed, change in distribution of thickness, and mortality before and after introducing the campaign. RESULTS--Referrals to the pigmented lesion clinic increased by 278%, from five a week in June-July 1984 to 19 a week in June-July 1985. Twice as many women as men were referred to the clinic (49% of referrals were of women aged under 65). The numbers of newly diagnosed primary cutaneous melanoma were 63 (12/month) in January-May 1985 and 146 (21/month) in June-December 1985, an increase of 131%. The percentage of tumours detected that were less than 1.5 mm thick rose significantly by 16% (95% confidence interval 11% to 19%), from 38% (328) in 1979-84 to 54% (592) in 1985-9. Mortality began to fall in women from 1988. CONCLUSIONS--The public education campaign succeeded in reducing the absolute number of thick tumours and melanoma related mortality in women.     

Vitamin D Deficiency at Melanoma Diagnosis Is Associated with Higher Breslow Thickness

BackgroundEpidemiological evidence shows that people with thicker, or higher stage, melanomas have lower vitamin D status compared to those with thinner tumours. Evidence from experimental studies is inconsistent, but some suggest that administration of vitamin D metabolites can decrease tumour aggressiveness.ObjectivesDetermine the relationship between vitamin D status at diagnosis and melanoma thickness (as an indicator of prognosis), in a subtropical setting with high melanoma incidence.MethodsWe recruited 100 melanoma patients in Brisbane, Australia within days of their diagnosis. Data on factors previously associated with melanoma risk or prognosis were collected by questionnaire and physical examination. Serum for 25-hydroxyvitamin D3 [25(OH)D] levels was collected prior to wider excision biopsy; histological indicators of prognosis were obtained from pathology reports. We used multivariable logistic regression models to analyse the association between Breslow thickness (≥0.75 mm compared to <0.75 mm), Clark level (2–5 compared to 1) and presence of mitoses, and vitamin D status.ResultsSerum 25(OH)D <50 nmol/L (versus ≥50 nmol/L) was associated with a nearly four-fold increase in risk of having a thicker tumour (Adjusted OR = 3.82, 95% CI: 1.03, 14.14; p = 0.04, adjusted for age, sex, skin phototype, body mass index and season at diagnosis). There was no significant association with Clark level or presence of mitosis. Serum 25(OH)D levels in the highest quartile (≥69.8 nmol/L) were not associated with a more favourable prognosis.ConclusionsVitamin D deficiency at the time of melanoma diagnosis is associated with thicker tumours that are likely to have a poorer prognosis. Ensuring vitamin D levels of 50 nmol/L or higher in this population could potentially result in 18% of melanomas having Breslow thickness of <0.75 mm rather than ≥0.75 mm.     

The clinicopathological and gene expression patterns associated with ulceration of primary melanoma

Ulceration of primary melanomas is associated with poor prognosis yet is reported to predict benefit from adjuvant interferon. To better understand the biological processes involved, clinicopathological factors associated with ulceration were determined in 1804 patients. From this cohort, 348 primary tumor blocks were sampled to generate gene expression data using a 502‐gene cancer panel and 195 blocks were used for immunohistochemistry to detect macrophage infiltration and vessel density. Gene expression results were validated using a whole genome array in two independent sample sets. Ulceration of primary melanomas was associated with more proliferative tumors, tumor vessel invasion, and increased microvessel density. Infiltration of tumors with greater number of macrophages and gene expression pathways associated with wound healing and up‐regulation of pro‐inflammatory cytokines suggests that ulceration is associated with tumor‐related inflammation. The relative benefit from interferon reported in patients with ulcerated tumors may reflect modification of signaling pathways involved in inflammation.     

Evaluation of nodal patterns for melanoma of the ear

Treatment of malignant melanoma of the external ear presents unique challenges. Because of the significant debate regarding the efficacy and validity of using sentinel lymph node mapping for the treatment of ear melanomas, data for a population of patients with melanomas of the ear who underwent surgical excision and reconstruction were reviewed to determine the efficacy of sentinel node mapping. A retrospective chart review of cases treated by a single surgical oncologist was performed. All patients who were treated for malignant melanomas and required reconstruction of the external ear by the plastic surgical service between 1995 and 2001 were identified. Nineteen patients were selected, of whom nine underwent sentinel node mapping. The average age of the patients was 65.2 years. Evaluation of melanoma depth, medical history, surgical margins, lymph node metastasis, and recurrence was performed. Lymphoscintigraphy with technetium-99-sulfur colloid and 1% Lymphazurin (isosulfan blue; Zenith Parenterals, Rosemont, Ill.) demonstrated widely variable lymphatic drainage patterns. The lower tail of the parotid gland and the upper cervical area were the two most common locations. The average number of sentinel nodes identified and removed was 3.7. The average Breslow thickness for these patients was 2.3 mm. None of these patients demonstrated micrometastatic disease in their sentinel nodes. The most common reconstructive procedure after surgical resection was the use of rotational advancement flaps. Localization of radioactivity, as detected with external technetium-99 scanning, was the most reliable method for detection of the sentinel lymph node basins and the individual nodes. The average value for the primary injection site was 8375 counts per second, and the average value for the nodes removed was 973.5 counts per second. Of the nine patients who underwent sentinel lymph node mapping, only one, with an initial lesion depth of 5 mm, developed a local recurrence. The average follow-up period in this study was 21 months (range, 12 to 79 months). All patients in this study were evaluated at least 1 year after the initial surgical resection. Patients were monitored by the same surgical oncologist every 3 months for the first 2 years. Little can be found in the literature regarding the efficacy of sentinel node biopsies for ear melanomas. Larger studies are indicated; however, it seems that this method is practical for designing therapeutic methods for patients with melanoma of the ear.     

Primary malignant melanoma of the nasal cavity: report of two cases and review of the literature

Primary malignant melanomas of the nasal cavity are rare, as only 400 cases have been reported to date. The present paper describes two cases recently seen in Caucasian women. The authors point out the difficult clinical diagnosis, as the symptoms are rather aspecific. From the histopathological point of view, diagnosis is easy in the melanotic cases while can show interpretating problems in the amelanotic ones, when melanoma is almost indistinguishable from other malignant neoplasms. A correlation between histological grading and prognosis was not detected, as both cases showed local recurrences within one year after surgery although they were, respectively, of epithelioid and undifferentiated type. While surgery appears to be the choice treatment of the primary lesion, the treatment of cervical metastasis is still disputable. On the whole, most authors think that the role played by radio- and mainly chemo-therapy is still limited and that cervical adenopathies should be treated by a simple lympho-adenectomy rather than by a neck dissection.     

Infiltration of primary and metastatic melanomas with macrophages of the 25F9-positive phenotype

Summary In order to gain insight into the role of macrophages in human melanoma, we studied fresh-frozen material from 15 dysplastic nevi, 199 primary melanomas, 107 melanoma metastases, and paraffin sections from 98 primary melanomas with the monoclonal antibody 25F9 which recognizes an 86×10³ dalton protein present on a subset of mature human macrophages. Considerable infiltration of tumors with 25F9-positive macrophages was observed in 2 dysplastic nevi (13%), 87 primary melanomas (44%), and 45 metastases (42%). The degree of intratumoral macrophage infiltration correlated with expression of class II HLA-DR antigens on tumor cells, in primary melanoma with a tumor thickness above 0.75 mm, and with the occurence of metastases within 2 years. In paraffin sections, intratumoral 25F9-positive macrophages also correlated with metastatic spread of primary tumors after longer follow-up. Metastases revealed a higher degree of macrophage infiltration following systemic or local immunotherapy, compared with untreated metastases, or metastases removed during chemotherapy. Of 38 patients who died within an observation period of 1 year, 19 (50%) had considerable infiltration of metastases with 25F9-positive macrophages, whereas this was found in only 4 of 12 patients (33%), who survived for longer than 2 years following metastases removal. A higher degree of 25F9-positive macrophages correlated with a shift towards the T8-positive subsets within the T cell compartment of the infiltrate. Our results suggest that accumulation of 25F9-positive macrophages in melanomas indicates more aggressive tumor properties.