Multiplex Gene Profiling of Cell-Free DNA in Patients With Metastatic Melanoma for Monitoring Disease.

PURPOSE: Hotspot blood cell-free DNA (cfDNA) biomarker assays have limited utility in profiling tumor heterogeneity and burden and in capturing regional metastasis with low disease burden in patients with melanoma. We investigated the utility of a sensitive 54-cancer gene digital next-generation sequencing approach targeting blood cfDNA single nucleotide variants (SNVs) and copy number amplification for monitoring disease in patients with melanoma with regional or distant organ metastasis (DOM). PATIENTS AND METHODS: A total of 142 blood samples were evaluated by digital next-generation sequencing across two patient cohorts. Cohort 1 contained 44 patients with stage II, III, or IV disease with matched tumor DNA at the time of surgery or DOM. Cohort 2 consisted of 12 overlapping patients who were longitudinally monitored after complete lymph node dissection to DOM. RESULTS: In cohort 1, cfDNA SNVs were detected in 75% of patients. Tumor-cfDNA somatic SNV concordance was 85% at a variant allele fraction of ≥ 0.5%. An SNV load (number of unique SNVs detected) of greater than two SNVs and an SNV burden (total cumulative SNV VAF) of > 0.5% were significantly associated with worse overall survival (P < .05) in stage IV patients. In cohort 2, 98 longitudinal blood samples along with matched regional and distant metastases from 12 stage III patients were analyzed before complete lymph node dissection and throughout disease progression. cfDNA SNV levels correlated with tumor burden (P = .019), enabled earlier detection of recurrence compared with radiologic imaging (P < .01), captured tumor heterogeneity, and identified increasing SNVs levels before recurrence. CONCLUSION: This study demonstrates significant utility for cfDNA profiling in patients with melanoma with regional and/or distant metastasis for earlier detection of recurrence and progression and in capturing tumor evolution and heterogeneity, thus impacting how patients with melanoma are monitored.

Impact of Laparoscopic Adrenalectomy on Overall Survival in Patients with Nonmetastatic Adrenocortical Carcinoma.

BACKGROUND: Appropriate use of laparoscopic adrenalectomy (LA) for adrenocortical carcinoma (ACC) remains controversial because complete resection with negative margins is the best chance for potential cure. This study compared the oncologic outcomes and overall survival (OS) of LA and open adrenalectomy (OA) for ACC. STUDY DESIGN: A retrospective analysis of the National Cancer Data Base (NCDB) between 2010 and 2014 identified 423 European Network for the Study of Adrenal Tumors (ENSAT) stage I to III ACC patients who had LA (n = 137) or OA (n = 286). Outcomes and OS were compared between the 2 groups. RESULTS: Patients who underwent OA had more advanced stage disease (p = 0.0001), larger (≥5 cm) tumors (p < 0.0001), and were younger (age less than 55 years, p = 0.05). Nodal assessment was rare in LA (n = 4) compared with OA (n = 88) (p < 0.0001). Margin positivity was affected only by surgical approach in patients with T3 tumors (LA 54.6% vs OA 21.7%; p = 0.0009). Neither surgical procedure nor any socio-demographic factor(s) affected OS for the entire cohort. Only positive margins (p = 0.007), positive nodes (p = 0.02), tumor extension (p = 0.01), and more advanced ENSAT stage (p = 0.004) increased mortality. When stratified by disease stage, LA decreased OS for patients with stage II disease (p = 0.04), and remained an independent risk factor for death on multivariate analysis (hazard ratio [HR] 1.86, 95% CI 1.02 to 3.38; p = 0.04). Only positive margins decreased OS in the entire cohort (HR 2.17, 95% CI 1.32 to 3.57; p = 0.002). CONCLUSIONS: Use of LA may decrease OS in select patients with ACC. Because margin status remains the strongest predictor of mortality, caution should be used in selecting LA for patients with ACC.

Impact of Completion Lymph Node Dissection on Patients with Positive Sentinel Lymph Node Biopsy in Melanoma.

BACKGROUND: Whether patients with positive SLNB should undergo complete lymph node dissection (CLND) is an important unanswered clinical question. STUDY DESIGN: Patients diagnosed with positive SLNB at a melanoma referral center from 1991 to 2013 were studied. Outcomes of patients who underwent CLND were compared with those who did not undergo immediate CLND (observation [OBS] group). RESULTS: There were 471 patients who had positive SLNB; 375 (79.6%) in the CLND group and 96 (20.4%) in the OBS group. The groups were similar except that the CLND group was younger and had more sentinel nodes removed. Five-year nodal recurrence-free survival was significantly better in the CLND group compared with the OBS group (93.1% vs 84.4%; p = 0.005). However, 5-year (66.4% vs 55.2%) and 10-year (59.5% vs 45.0%) distant metastasis-free survival rates were not significantly different (p = 0.061). The CLND group's melanoma-specific survival (MSS) rate was superior to that of the OBS group; 5-year MSS rates were 73.7% vs 65.5% and 10-year MSS rates were 66.8% vs 48.3% (p = 0.015). On multivariate analysis, CLND was associated with improved MSS (hazard ratio = 0.60; 95% CI, 0.40-0.89; p = 0.011) and lower nodal recurrence (hazard ratio = 0.46; 95% CI, 0.24-0.86; p = 0.016). Increased Breslow thickness, older age, ulceration, and trunk melanoma were all associated with worse outcomes. On subgroup analysis, the following factors were associated with better outcomes from CLND: male sex, nonulcerated primary, intermediate thickness, Clark level IV or lower extremity tumors. CONCLUSIONS: Treatment of positive SLNB with CLND was associated with improved MSS and nodal recurrence rates. Follow-up beyond 5 years was needed to see a significant difference in MSS rates.

Predictors and Survival Impact of False-Negative Sentinel Nodes in Melanoma.

BACKGROUND: The status of the sentinel lymph node in melanoma is an important prognostic factor. The clinical predictors and implications of false-negative (FN) biopsy remain debatable. METHODS: We compared patients with positive sentinel lymph node biopsy (SNB) [true positive (TP)] and negative SNB with and without regional recurrence [FN, true negative (TN)] from our prospective institutional database. RESULTS: Among 2986 patients (84 FN, 494 TP, and 2408 TN; median follow-up 93 months), the incidence of FN-SNB was 2.8%. While calculated FN rate was 14.5% [84 FN/(494 TP + 84 FN) × 100], when we accounted for local/in-transit recurrence (LITR) this rate was 8.5% [46 FN/(494 TP + 46 FN) × 100 %]. On multivariate analysis, male gender (OR 2.0, 95% CI 1.1-3.6, p = 0.018), head/neck primaries (OR 2.5, 95% CI 1.3-4.8, p < 0.006), and LITR (OR 3.5, 95% CI 2.1-5.8, p < 0.001) were associated with FN-SNB. Melanoma-specific survival (MSS) for the FN group was similar to the TP group at 5 years (68 vs. 73%, p = 0.539). However, MSS declined more for the FN group with a longer follow up and was significantly worse at 10 years (44 vs. 64%, p < 0.001). On multivariate analysis, FN-SNB was a significant predictor of worse MSS in melanomas <4 mm in Breslow thickness (HR 1.6; 95% CI 1.1-2.5, p = 0.021). CONCLUSIONS: Male gender, LITR, and head and neck tumors were associated with FN-SNB. FN-SNB was an independent predictor of worse MSS in melanomas <4 mm in thickness, but this survival difference did not become apparent until after 5 years of follow-up.

Adrenalectomy for Metastatic Melanoma: Current Role in the Age of Nonsurgical Treatments.

Surgical resection of metastases to the adrenal gland can improve overall survival of patients with stage IV melanoma, but its relative value with respect to current nonsurgical therapies is unknown. We hypothesized that surgery remains an optimal first-line treatment approach for resectable adrenal metastases. A search of our institution's prospectively collected melanoma database identified stage IV patients treated for adrenal metastases between January 1, 2000, and August 11, 2014. The 91 study patients had a mean age of 60.3 years at diagnosis of adrenal metastasis and 24 had undergone adrenalectomy. Improved survival was associated with an unknown primary lesion, surgical resection, and nonsurgical therapies. Median overall survival from diagnosis of adrenal metastases was 29.2 months with adrenalectomy versus 9.4 months with nonoperative treatment. Adrenalectomy, either as complete metastasectomy or targeted to lesions resistant to systemic therapy, is associated with improved long-term survival in metastatic melanoma.

Attitudes and Perceptions of Surgical Oncology Fellows on ACGME Accreditation and the Complex General Surgical Oncology Certification.

BACKGROUND: With the first qualifying examination administered September 15, 2014, complex general surgical oncology (CGSO) is now a board-certified specialty. We aimed to assess the attitudes and perceptions of current and future surgical oncology fellows regarding the recently instituted Accreditation Council for Graduate Medical Education (ACGME) accreditation. METHODS: A 29-question anonymous survey was distributed to fellows in surgical oncology fellowship programs and applicants interviewing at our fellowship program. RESULTS: There were 110 responses (79 fellows and 31 candidates). The response rate for the first- and second-year fellows was 66 %. Ninety-percent of the respondents were aware that completing an ACGME-accredited fellowship leads to board eligibility in CGSO. However, the majority (80 %) of the respondents stated that their decision to specialize in surgical oncology was not influenced by the ACGME accreditation. The fellows in training were concerned about the cost of the exam (90 %) and expressed anxiety in preparing for another board exam (83 %). However, the majority of the respondents believed that CGSO board certification will be helpful (79 %) in obtaining their future career goals. Interestingly, candidate fellows appeared more focused on a career in general complex surgical oncology (p = 0.004), highlighting the impact that fellowship training may have on organ-specific subspecialization. CONCLUSIONS: The majority of the surveyed surgical oncology fellows and candidates believe that obtaining board certification in CGSO is important and will help them pursue their career goals. However, the decision to specialize in surgical oncology does not appear to be motivated by ACGME accreditation or the new board certification.

Sentinel lymph node biopsy and nodal ultrastaging in colorectal cancer.

The tumor status of the regional lymph nodes is the most important prognostic indicator in colorectal cancer (CRC), as it is in other solid tumors. Sentinel lymph node biopsy (SLNB), which has profoundly impacted the treatment of melanoma and breast cancer, has been applied in CRC in an attempt to improve nodal staging accuracy. The challenge lies in identifying patients who have tumor-negative nodes but are at high risk of regional or distant failure and therefore may benefit from adjuvant chemotherapy. Because standard pathological analysis of lymph nodes may incorrectly stage colon cancer, multiple studies have investigated nodal ultrastaging based on identification and immunohistochemical and/or molecular assessment of the sentinel node. This review focuses on the technique of SNLB, its feasibility and validity, and the controversies that remain regarding the clinical significance of nodal ultrastaging in CRC.

Brain metastasis is predetermined in early stages of cutaneous melanoma by CD44v6 expression through epigenetic regulation of the spliceosome.

Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6-positive MBM migration is reduced by blocking with a CD44v6-specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM-specific factor with theranostic utility for stratifying patients.

Epigenetic biomarkers in skin cancer.

Epigenetic aberrations have been associated with cutaneous melanoma tumorigenesis and progression including dysregulated DNA gene promoter region methylation, histone modification, and microRNA. Several of these major epigenetic aberrations have been developed into biomarkers. Epigenetic biomarkers can be detected in tissue and in blood as circulating DNA in melanoma patients. There is strong evidence that biomarkers in cutaneous melanoma will have an important role as companions to therapeutics and overall patient management. Important progress has been made in epigenetic melanoma biomarker development and verification of clinical utility, and this review discusses some of the key current developments and existing challenges.

Management of papillary lesions of the breast: can larger core needle biopsy samples identify patients who may avoid surgical excision?

BACKGROUND: The ability to distinguish benign from atypical/malignant papillary lesions on core needle biopsy is limited by the representative nature of the biopsy method, thus follow-up excision is usually recommended. We aimed to determine if larger samples of tissue obtained by core needle biopsy can more reliably predict the true benign nature of a papilloma. METHODS: We reviewed the pathology slides and medical records of 51 patients who were diagnosed with benign papillomas on core needle biopsy from 2000 to 2010, who subsequently underwent surgical excision. The characteristics of the core needle biopsy that were associated with retention of benign histology on excision were determined and analyzed. RESULTS: Atypical ductal hyperplasia and carcinoma were identified in 5.8 % (3/51) and 5.8 % (3/51) of papillary lesions, respectively, when excised. Patients whose lesions were diagnosed as benign on excision were significantly distinguished by the area (mm(2)) of tissue sampled by core needle biopsy (mean ± standard deviation (SD): 101.5 ± 106.5) compared with those with atypia or carcinoma on excision (mean ± SD: 41.7 ± 24.0, P = 0.003). All biopsies performed with 12-gauge or larger needles retained benign features on excision. Core needle biopsy tissue samples consisting of ≥7 cores, or measuring >96 mm(2) in aggregate, had a negative predictive value for atypia/malignancy of 100 %. CONCLUSIONS: Larger tissue samples significantly improved the predictive value of benign histology on core needle biopsy. A papilloma sampled by a 12-gauge or larger needle, ≥7 cores, or >96 mm(2) retained its benign features upon excision.

Epigenetics of regional lymph node metastasis in solid tumors.

Regional nodal status remains one of the most important prognostic factors in several solid tumors including melanoma, breast cancer, and gastrointestinal malignancies. However, despite the accuracy of lymph node (LN) staging, patients who are LN negative are still at risk for development of recurrence and distant metastasis. As such, numerous molecular studies have focused on genetic and transcriptome changes in primary and metastatic tumors to discover molecular determinants that can predict aggressive metastatic disease and/or correlated to clinical outcomes. More recently, epigenetic aberrations have been investigated in solid cancers and are associated with tumorigenesis and disease progression. These epigenetic alterations have demonstrated potential utility as diagnostic and prognostic biomarkers and are being developed into novel targeted treatment strategies, as epigenetic changes can be reversed by appropriate drugs. If patients who are at increased risk of developing metastases or recurrence can be accurately identified, this will help stratify patients into more appropriate treatment and follow-up. This review discusses some of the recent studies on regional LN metastases in melanoma, breast cancer, and colorectal cancer, focusing on the potential clinicopathological utility of epigenetic aberrations in the management of cancer patients.

Epigenetics of estrogen receptor-negative primary breast cancer.

Increasingly, breast cancer is being recognized as a heterogeneous disease comprised of molecularly and phenotypically distinct intrinsic tumor subtypes with different clinical outcomes. This biological heterogeneity has significant implications, particularly as it relates to expression profiling of estrogen receptor (ER) status, as classifying breast cancers based on hormone receptor expression impacts not only prognosis but also treatment options and long-term outcomes. Epigenetics has emerged as a promising field for the assessment of hormone receptor status. Epigenetic aberrations have been shown to regulate ER and offer reversible targets for development of new therapies. This review covers ER-negative breast tumor epigenetic aberrations and summarizes the major epigenetic mechanisms governing ER expression and how it impacts treatment of ER-negative breast cancer.

Epigenetic regulation of cancer stem cell genes in triple-negative breast cancer.

Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer progression. We hypothesized that promoter methylation regulates specific BCSC-related genes [CD44, CD133, CD24, MSH1 (alias, Musashi-1), and ALDH1] and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1. These sites were screened by treatment with 5-aza-2'-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44, CD133, and Musashi-1, but not for CD24. Methylation of CD44, CD133, and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44, CD133, and Musashi-1, and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.

Stanniocalcin-1: a novel molecular blood and bone marrow marker for human breast cancer.

PURPOSE: Very few tumor molecular markers have been identified that are highly specific for breast cancer cells when applied to blood and bone marrow (BM). Stanniocalcin (STC)-1 is a recently discovered human gene that has been implicated in cellular calcium homeostasis and resistance to hypoxia and is located on chromosome 8p in a region associated with amplification in breast cancer. We investigated STC-1 mRNA as a potential molecular marker for detection of breast cancer metastasis in the blood and BM. EXPERIMENTAL DESIGN: Using the reverse transcriptase-PCR and electrochemiluminescence detection assay to assess for STC-1 mRNA expression, we evaluated 7 breast cancer cell lines, 34 primary breast cancer tumors, and the BM of 71 patients and the blood of 58 patients with American Joint Committee on Cancer stage 0-IV breast cancer. RESULTS: In this cohort of primarily early-stage breast cancer patients, the detection of STC-1 mRNA in the BM and blood significantly correlated with multiple histopathological prognostic factors, including primary tumor size, number of positive lymph nodes, T stage, M stage, N stage, and overall American Joint Committee on Cancer stage. STC-1 mRNA was not detected in the blood or BM of volunteers without cancer. In situ hybridization studies with a STC-1 antisense RNA probe also confirmed STC-1 mRNA expression in breast cancer cell lines and primary breast tumors. CONCLUSIONS: STC-1 is proposed as a novel, specific, and clinically useful molecular marker for detecting occult breast cancer cells in the BM and blood.