Is the non-sentinel lymph node compartment the next site for melanoma progression from the sentinel lymph node compartment in the regional nodal basin?

Melanoma patients with additional positive lymph nodes in the completion lymph node dissection (CLND) following a positive sentinel lymph node (SLN) biopsy would have a poorer prognosis than patients with no additional positive lymph nodes. We hypothesize that the progression of disease from the SLN to the non-SLN compartment is orderly and is associated with the worsening of the disease status. Thus, the SLN and non-SLN compartments are biologically different in that cancer cells, in general, arrive in the SLN compartment before spreading to the non-SLN compartment. To validate this concept, we used a large cohort of melanoma patients from our prospective SLN database in an academic tertiary medical center. Adult cutaneous melanoma patients (n = 291) undergoing CLND after a positive SLN biopsy from 1994 to 2009 were analyzed. Comparison of 5-year disease-free survival and 5-year overall survival between positive (n = 66) and negative (n = 225) CLND groups was made. The 5-year disease-free survival rates were 55% (95% CI 49-62%) for patients with no additional LN on CLND versus 14% (95% CI 8-26%) in patients with positive LN on CLND (p < 0.0001, log-rank test). The median disease-free survival time was 7.4 years with negative CLND (95% CI 4.4-15+ years) and 1.2 years with positive CLND (95% CI 1.0-1.8 years). The 5-year overall survival rates were 67% (95% CI 61-74%) for negative CLND versus 38% (95% CI 28-52%) for positive CLND (p < 0.0001, log-rank test). The median overall survival time was 12.1 years for negative CLND (95% CI 9.3-15+ years) and 2.5 years for positive CLND (95% CI 2.2-5.7 years). This study shows that CLND status is a significant prognostic factor for patients with positive SLNs undergoing CLND. Also, it suggests an orderly progression of metastasis from the SLN to the non-SLN compartment. Thus, the SLN in the regional nodal basin draining the primary melanoma may serve as an important gateway for metastasis to the non-SLN compartment and beyond to the systemic sites.

The role of BPTF in melanoma progression and in response to BRAF-targeted therapy.

BACKGROUND: Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we explore the oncogenic effects of BPTF in melanoma. METHODS: The consequences of differential expression of BPTF were explored using shRNA-mediated knockdown in several melanoma cell lines. Immunoblotting was used to assess the expression of various proteins regulated by BPTF. The functional role of BPTF in melanoma progression was investigated using assays of colony formation, invasion, cell cycle, sensitivity to selective BRAF inhibitors, and in xenograft models of melanoma progression (n = 12 mice per group). The biomarker role of BPTF in melanoma progression was assessed using fluorescence in situ hybridization and immunohistochemical analyses. All statistical tests were two-sided. RESULTS: shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF copy number (mean ≥ 3) was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival P = .03, and disease-specific survival P = .008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell subclones, indicating the continued presence of drug-responsive subclones within tumors demonstrating overall resistance to anti-BRAF agents. CONCLUSIONS: These studies demonstrate multiple protumorigenic functions for BPTF and identify it as a novel target for anticancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF.

Antitumor activity of miR-1280 in melanoma by regulation of Src.

MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3' untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma.

Prognostic impact of PHIP copy number in melanoma: linkage to ulceration.

Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced distant metastasis-free survival (DMFS; P=0.01) and disease-specific survival (DSS; P=0.009) by Kaplan-Meier analyses. PHIP FISH scores were independently predictive of DMFS (P=0.03) and DSS (P=0.03). Increased PHIP copy number was an independent predictor of ulceration status (P=0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (P<0.0001). Stable suppression of PHIP in human melanoma cells resulted in significantly reduced glycolytic activity in vitro, with lower expression of lactate dehydrogenase 5, hypoxia-inducible factor 1 alpha subunit, and vascular endothelial growth factor, and was accompanied by reduced microvessel density in vivo. These results provide further support for PHIP as a molecular prognostic marker of melanoma, and reveal a significant linkage between PHIP levels and ulceration. Moreover, they suggest that ulceration may be driven by increased glycolysis and angiogenesis.

Is head and neck melanoma different from trunk and extremity melanomas with respect to sentinel lymph node status and clinical outcome?

BACKGROUND: Previous studies showed conflicting and inconsistent results regarding the effect of anatomic location of the melanoma on sentinel lymph node (SLN) positivity and/or survival. This study was conducted to evaluate and compare the effect of the anatomic locations of primary melanoma on long-term clinical outcomes. METHODS: All consecutive cutaneous melanoma patients (n=2,079) who underwent selective SLN dissection (SLND) from 1993 to 2009 in a single academic tertiary-care medical center were included. SLN positive rate, disease-free survival (DFS), and overall survival (OS) were determined. Kaplan-Meier survival, univariate, and multivariate analyses were performed to determine predictive factors for SLN status, DFS, and OS. RESULTS: Head and neck melanoma (HNM) had the lowest SLN-positive rate at 10.8% (16.8% for extremity and 19.3% for trunk; P=0.002) but had the worst 5-year DFS (P<0.0001) and 5-year OS (P<0.0001) compared with other sites. Tumor thickness (P<0.001), ulceration (P<0.001), HNM location (P=0.001), mitotic rate (P<0.001), and decreasing age (P<0.001) were independent predictive factors for SLN-positivity. HNM with T3 or T4 thickness had significantly lower SLN positive rate compared with other locations (P≤0.05). Also, on multivariate analysis, HNM location versus other anatomic sites was independently predictive of decreased DFS and OS (P<0.001). By Kaplan-Meier analysis, HNM was associated significantly with the worst DFS and OS. CONCLUSIONS: Primary melanoma anatomic location is an independent predictor of SLN status and survival. Although HNM has a decreased SLN-positivity rate, it shows a significantly increased risk of recurrence and death as compared with other sites.

Analysis of sentinel lymph node positivity in patients with thin primary melanoma.

BACKGROUND: A minority of patients with T1 melanoma will have a positive sentinel lymph node (SLN) biopsy (SLNB) finding. Identifying who will develop metastatic disease is important in determining prognosis and treatment. OBJECTIVE: We sought to identify clinical and histologic features predictive of a positive SLNB result and determine its prognostic significance in patients with T1 melanoma. METHODS: Clinical and histologic parameters were evaluated in 484 patients with T1 melanoma for their ability to predict a positive SLNB result. The impact of various factors on SLN positivity was evaluated. SLN status was examined as a predictor of overall survival. RESULTS: In all, 34 patients had a positive SLNB finding. Four factors predicted a higher risk of SLN positivity: age 43 years or younger, Breslow depth 0.8 mm or greater, tumors on the lower extremity and trunk, and tumor-infiltrating lymphocyte level. By multivariate analysis, low tumor-infiltrating lymphocytes (P = .0015) and decreasing age (P = .0058) independently predicted SLN positivity. If 0 to 2 of these factors were present, the rate of a positive SLNB result was 3%; this increased to 15% with 3 factors present and to 30% if all 4 factors were present (P < .002). SLN-positive patients had significantly decreased survival (P = .003), and SLN status was the most powerful predictor of survival (P = .009). LIMITATIONS: Our data analysis includes patients from 1994 to 2007 and therefore information on mitotic rate, a recently defined T1b criterion, is not recorded for all patients. CONCLUSIONS: Combining clinical and histologic prognostic factors may help identify subgroups of T1 patients at higher risk of SLN positivity. SLN status has significant prognostic impact in patients with thin melanomas.

Inoperable bulky melanoma responds to neoadjuvant therapy with vemurafenib.

A patient with a bulky inoperable stage IIIC melanoma involving the left axilla and neck from a primary of the left medial elbow received vemurafenib as neo-adjuvant treatment. Based on the molecular analysis, BRAF V600E mutation was present. After 4 months of vemurafinib treatment, the tumours shrank to less than 50% of original clinical size and allowed the surgeons to perform a left modified radical neck dissection and left radical axillary dissection. Pathological analysis of specimen revealed viable metastatic cells only in 1 of 40 nodes resected in the neck and axillary dissection, accounting for over 98% pathological response. Other lymph nodes had a mixture of foamy histiocytic inflammatory reaction fibrosis and islands of necrotic tissues. After recovery from surgery, vemurafenib was resumed and continued for 6 months. He remained disease free 6 months after surgery.

The effect of delay time between primary melanoma biopsy and sentinel lymph node dissection on sentinel node status, recurrence, and survival.

For primary melanoma, there is a delay between the initial skin biopsy and sentinel lymph node dissection, which may cause anxiety for the patient. The consequences of this delay on disease progression are unknown. The goal of this study was to determine whether delay time for sentinel node dissection from the initial cutaneous melanoma biopsy affects patient outcomes. A retrospective analysis of 492 patients with melanoma who underwent a sentinel node dissection between 1993 and 1999 was carried out. The endpoints assessed were sentinel node tumor status, recurrence, and mortality. Time to sentinel node dissection was compared between patients with positive and negative sentinel nodes. Long-term survival and recurrence were evaluated in relation to the time between the cutaneous biopsy and the sentinel node dissection (delay time), comparing less than 40 days with at least 40 days. In total, 15.9% of patients had positive sentinel nodes. The median follow-up was 11.7 years. Positive sentinel node patients had a median delay of 35 days between the primary melanoma biopsy and the sentinel node dissection compared with 41 days for negative sentinel node patients (P=0.5). Kaplan-Meier survival curves showed that a delay time of less than 40 days versus at least 40 days was not related to recurrence of melanoma (log-rank P=0.13) or overall survival (log-rank P=0.14). On multivariate analysis of age, thickness, ulceration, and sentinel node status, there was no difference in disease-free survival (P=0.58) or overall survival (P=0.53) between the less than 40 days and the at least 40 days groups. A modest delay in sentinel node dissection from the initial melanoma biopsy does not adversely affect sentinel node status, recurrence, nor survival.

Pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis.

Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.

Long-term prognosis and significance of the sentinel lymph node in head and neck melanoma.

OBJECTIVE: To report the long-term significance of sentinel lymph node (SLN) biopsy on prognosis, determine false-negative SLN occurrences, and determine risk factors for death and recurrence in a large series of patients with head and neck melanoma. STUDY DESIGN: Case series with tumor registry review. SETTING: Academic tertiary care medical center. SUBJECTS AND METHODS: A database review was performed of all patients who underwent SLN biopsy for head and neck melanoma from 1994 to 2009. End points assessed were SLN status, recurrence, false-negative SLN results, and survival comparing SLN-positive and SLN-negative patients and different locations. Survival curves and multivariate analyses were performed. RESULTS: SLN biopsy was performed in 365 patients. SLNs were identified in 98.6% of patients with a mean of 3.7 nodes removed from 1.6 nodal basins per patient. Median follow-up was 8 years. The SLN was positive in 40 (11%) patients. SLN-positive patients had significantly thicker melanomas, higher recurrence (P < .0001), and a significant decrease in overall survival compared with SLN-negative patients (P < .002). Scalp melanoma patients had significantly thicker melanomas and an elevated risk of SLN positivity, recurrence, and death compared with other sites. Seventeen of 365 SLN-negative patients developed regional nodal disease for a false-omission rate of 5.2% and a negative predictive value of a negative SLN to be 94.8%. Risks for false negative-SLN occurrences included thick melanomas and scalp melanomas. CONCLUSION: SLN biopsy is accurate in head and neck melanoma and provides significant prognostic data. Scalp melanoma patients present with thicker tumors with an increase in SLN positivity and false-negative SLN occurrences.

In-transit intramammary sentinel lymph nodes from malignant melanoma of the trunk.

OBJECTIVE: Our goal was to determine the incidence and outcomes of intramammary in-transit sentinel lymph nodes (IMSLN) from primary malignant melanoma (MM) of the trunk. We hypothesize that regional metastasis to the breast from anterior trunk MM also occurs via the lymphatic system to these intramammary in-transit sentinel lymph nodes. BACKGROUND: MM is the most common solid tumor metastasis to the breast. The mechanism of intramammary (IM) metastasis is generally attributed to hematogenous rather than lymphatic spread. METHODS: We retrospectively reviewed medical records from all patients who underwent selective sentinel lymph node dissection at the UCSF Melanoma Center from 1993 to 2008 after the approval of UCSF Committee on Human Research. Of the 1911 cases, we found 614 patients with primary MM located on the trunk, and queried their medical records for in-transit SLN and SLNs in the breast. Data from preoperative lymphoscintigraphy, intraoperative lymphatic mapping, operative notes, and pathology and clinic notes were gathered. RESULTS: Of the 1911 patients with MM, 169 (8.9%) and 420 (22.0%) had anterior and posterior trunk lesions, respectively, and 25 patients (1.3%) with flank lesions (lateral abdominal wall below the rib cage, above the iliac crest). Of the anterior trunk population, 18 patients had in-transit SLNs. The vast majority of these patients (14 of 18, 77.8%) had in-transit IMSLN. Of patients with posterior trunk melanoma, 27 patients had in-transit nodes with 1 patient having IMSLNs. Of patients with flank melanomas, 3 patients had in-transit nodes with 1 patient having IMSLNs. Interestingly, all patients with IMSLNs had primary lesions located inferior to the breasts. Two of the 16 patients with IMSLNs had micrometastasis to IMSLN; 1 patient died and the other currently is disease free 4 years after initial SLND. Four of the 32 patients with non-IM in-transit nodes had micrometastases to these in-transit nodes. Of all patients with trunk melanomas, 4 patients had micrometastases to axillary SLNs (AxSLNs). Three of the 4 patients with positive AxSLNs also had positive in-transit nodes whereas only half of the patients with positive in-transit SLNs had positive AxSLNs. CONCLUSIONS: IMSLNs exist in the breast. Our results establish an anatomic basis for lymphatic metastasis to the breast from primary cutaneous melanoma mainly from the anterior trunk inferior to the breasts. For anterior trunk melanomas, IMSLNs should not be overlooked during SLND as they may harbor micrometastasis.

Selective sentinel lymph node dissection for melanoma: importance of harvesting nodes with lower radioactive counts without the need for blue dye.

BACKGROUND: Determining how many sentinel lymph nodes (SLNs) should be removed for melanoma is important. The purpose of this study is to determine the frequency at which nodes that are less radioactive than the "hottest" node (which is negative) are positive for melanoma, how low of a radioactivity should warrant harvest, and if isosulfan blue is necessary. METHODS: We reviewed 1,152 melanoma patients who underwent lymphoscintigraphy with technetium, with or without blue dye, and SLN dissection from 1996 to 2008. SLNs with radioactivity ≥10% of the "hottest" SLN, all blue nodes, and all suspicious nodes were removed and analyzed. The miss rate was calculated as the proportion of node positive cases in which the "hottest" SLN was negative. RESULTS: SLNs were identified in 1,520 nodal basins in 1,152 patients. SLN micrometastases were detected in 218 basins (14%) in 204 patients (18%). In 16% of SLN-positive patients (33/204 patients), the positive SLN was found to have a lower radioactive count than the "hottest" SLN, which was negative. In 21 of these cases, the positive SLNs had radioactivity ≤50% of the "hottest" SLN. The 10% rule significantly reduced the miss rate to 2.5% compared with removal of only the "hottest" SLN (miss rate = 16%). Also, blue dye did not significantly decrease the miss rate compared with radiocolloid alone using the 10% rule. CONCLUSIONS: To decrease the miss rate, all SLNs with ≥10% of the ex vivo radioactivity of the "hottest" SLN should be removed and blue dye is not essential.