Alicia M. Terando

CpG Island Methylator Phenotype Predicts Progression of Malignant Melanoma

Purpose: The CpG island methylator phenotype (CIMP) may be associated with development of malignancy through coordinated inactivation of tumor suppressor and tumor-related genes (TRG) and methylation of multiple noncoding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMP pattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigenetic inactivation of TRGs also has been shown in malignant melanoma, we hypothesized the existence of a clinically significant CIMP in cutaneous melanoma progression. Experimental Design: The methylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology (WIF1, TFPI2, RASSF1A, RARβ2, SOCS1, and GATA4) and a panel of MINT loci (MINT1, MINT2, MINT3, MINT12, MINT17, MINT25, and MINT31) in primary and metastatic tumors of different clinical stages (n = 122) was assessed. Results: Here, we show an increase in hypermethylation of the TRGs WIF1, TFPI2, RASSF1A, and SOCS1 with advancing clinical tumor stage. Furthermore, we find a significant positive association between the methylation status of MINT17, MINT31, and TRGs. The methylation status of MINT31 is associated with disease outcome in stage III melanoma. Conclusions: These findings show the significance of a CIMP pattern that is associated with advancing clinical stage of malignant melanoma. Future prospective large-scale studies may determine if CIMP-positive primary melanomas are at high risk of metastasis or recurrence.

Activation of CCR9/CCL25 in Cutaneous Melanoma Mediates Preferential Metastasis to the Small Intestine

Purpose: Specific chemokines and their respective receptors have been implicated in distant tumor cell metastasis. Cutaneous melanoma has a distinct pattern of metastasis, preferentially targeting the submucosa of the small intestine. However, the underlying pathogenic mechanism remains unknown. Migration of CCR9(+) lymphocytes to the small intestine is known to occur in response to the chemoattractant effects of CCL25 (thymus-expressed chemokine). The integrin heterodimers αβ are also known to be important mediators of cellular adhesion. We hypothesize that the mechanism of small intestinal metastasis by melanoma is via the CCR9-CCL25 axis and specific integrins. Experimental Design: Quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to assess melanoma tumors for CCR9 and CCL25. Integrin expression was assessed using flow cytometry. CCR9 expression by quantitative reverse transcription-PCR was assessed in primary (n = 23) and metastatic (n = 198) melanomas, and melanoma lines derived from small intestinal metastases (n = 23). Results: We showed CCR9 expression in 88 of 102 paraffin-embedded metastatic melanomas from the small intestine, 8 of 8 melanoma lines derived from metastases in the small intestine, and 0 of 96 metastatic melanomas from other sites. In vitro migration and invasion studies done on CCR9(+) melanoma lines showed migration in response to CCL25 that was inhibited by anti-CCR9 antibody or by short interfering RNA CCR9. Flow cytometric analysis confirmed CCR9 expression by melanomas to the small intestine and showed concomitant α4β1 integrin expression. Conclusions: Our findings show that functionally active CCR9 on melanoma cells facilitates metastasis to the small intestine. The CCR9-CCL25 axis may explain the high incidence of melanoma metastasis to this specific location.

Osteomyelitis of the Foot and Toe in Adults Is a Surgical Disease: Conservative Management Worsens Lower Extremity Salvage

Objective:To characterize the national epidemiology of adult osteomyelitis (OM) and, using a single institutions’ experience, test the hypothesis that early surgical therapy as compared with antibiotics alone results in an improved chance of wound healing and limb salvage. Background:Foot and digit OM is a very common problem for which management is variable and for which few guidelines exist. Methods:The Nationwide Inpatient Sample (NIS) and a single institution review from 1993 to 2000 form the basis of this study, using ICD-9CM codes for lower extremity foot and digit OM. Demographics, risk factors, and treatments were analyzed against the outcomes of a healed wound, limb salvage, and death. Results:The NIS included 51,875 patients (incidence = 9/10,000 patients per year) with a mean age of 60 years, and 59% were men. The median length of stay decreased from 9 to 6 days (P < 0.001), but the average admission charge of

Chemokine gene modification of human dendritic cell-based tumor vaccines using a recombinant adenoviral vector

19,000 did not significantly decrease over 7 years. Of these patients, 23% underwent a digit amputation and 8.5% suffered proximal limb loss. Single-institution analysis of 237 consecutive patients with OM confirmed a similar mean age (58 years), gender (67% men), and most presented with a foot or digit ulcer (56%). Wound healing was achieved in 56% and overall limb salvage was 80%. Decreased wound healing was associated with peripheral vascular occlusive disease (odds ratio, 0.4; 95% confidence interval, 0.2–0.8, P = 0.006) and preadmission antibiotic use (odds ratio, 0.2; 95% confidence interval, 0.05–1.1, P=0.07), while surgical debridement (odds ratio, 2.2; 95% confidence interval, 1.2–4.2, P = 0.02) was associated with increased healing. Limb salvage was improved with an arterial bypass (odds ratio, 3.9; 95% confidence interval, 1.1–14, P = 0.04), while preadmission solid organ transplant (odds ratio, 0.37; 95% confidence interval, 0.14–0.96, P = 0.04), peripheral vascular occlusive disease (odds ratio, 0.25; 95% confidence interval, 0.12–0.5, P = 0.001), and preadmission antibiotic use (odds ratio, 0.34; 95% confidence interval, 0.15–0.77, P = 0.009) were associated with greater limb loss. Conclusion:Digit OM is an expensive and morbid disease. Aggressive surgical debridement/digit amputation and selected use of arterial bypass should improve wound healing and limb salvage, respectively. In contrast, antibiotic therapy alone is associated with decreased wound healing and limb salvage.

On combining antineoplastic drugs with tumor vaccines

Previous animal studies conducted in our laboratory have shown that tumor antigen-pulsed dendritic cells (TP-DC) can mediate antitumor effects in vivo. However, durable and complete regression of established tumors has been difficult to achieve through the administration of TP-DC alone. To better augment immune priming to tumors in vivo, we have hypothesized that it is necessary to achieve an increased number of host-derived, naïve T cells at the site of TP-DC vaccine injections. To accomplish this goal, we have embarked on a series of studies that utilize defined chemokines. One of these molecules, secondary lymphoid tissue chemokine (SLC), has been shown to be uniquely chemoattractant for naïve T cells and dendritic cells. We propose that gene modification of DC-based tumor vaccines to produce human SLC will enhance T-cell recruitment and immune priming to tumor-associated antigens, and thereby translate into improved antitumor vaccine efficacy in vivo. Utilizing an E1-, E3-deleted adenoviral vector containing the gene for human SLC, we have been able to transduce human DC to produce biologically active human SLC that chemoattracts human T cells in vitro. SLC production by transduced DC was markedly enhanced upon DC maturation. Additionally, these SLC-secreting DC were found to be viable to a large extent despite the cytopathic effect inherent in adenoviral gene transfer and, most importantly, functional as determined by their ability to prime autologous T cells to a known melanoma-associated antigen, MART-1. Based on these encouraging results, we plan to initiate Phase I clinical studies utilizing DC-SLC to treat patients with advanced solid tumors.

Safety and Feasibility of Minimally Invasive Inguinal Lymph Node Dissection in Patients With Melanoma (SAFE-MILND): Report of a Prospective Multi-institutional Trial

Over the past 2 decades, the classical paradigm of trimodal cancer therapy has been expanded to include immunotherapy, encompassing both passive, adoptive T-cell transfer techniques as well as active vaccination strategies. As the mainstay of anticancer therapy, antineoplastic drugs have long been used for their direct tumoricidal properties, while the immunosuppressive adverse effects have been merely tolerated and supported. With the advent of the increasing use of immunotherapy in the clinical setting, investigators have sought to determine ways in which to combine accepted chemotherapeutic regimens with innovative immunotherapeutic techniques, and have discovered that the lymphodepletion that results from antineoplastic drug administration may be, in some cases, advantageous in eliciting clinically relevant responses to cancer immunotherapy. As well, several of these drugs have been found, paradoxically, to actually augment antitumor immunity. There is a paucity of preclinical and clinical data to date on combining chemotherapy and antitumor vaccines, as this is a strategy in its infancy. However, it may ultimately be found that chemotherapy combined with vaccine therapy offers therapeutic advantages over single-modality treatment. Here we will explore the available data regarding the mechanisms behind enhancement of antitumor efficacy through the combination of antineoplastic drugs with tumor vaccines.

Training High-Volume Melanoma Surgeons to Perform a Novel Minimally Invasive Inguinal Lymphadenectomy: Report of a Prospective Multi-Institutional Trial

Background: Minimally invasive inguinal lymph node dissection (MILND) is a novel approach to inguinal lymphadenectomy. SAFE-MILND (NCT01500304) is a multicenter, phase I/II clinical trial evaluating the safety and feasibility of MILND for patients with melanoma in a group of surgeons newly adopting the procedure. Methods: Twelve melanoma surgeons from 10 institutions without any previous MILND experience, enrolled patients into a prospective study after completing specialized training including didactic lectures, participating in a hands-on cadaveric laboratory, and being provided an instructional DVD of the procedure. Complications and adverse postoperative events were graded using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events Version 4.0. Results: Eighty-seven patients underwent a MILND. Seventy-seven cases (88.5%) were completed via a minimally invasive approach. The median total inguinal lymph nodes pathologically examined (SLN + MILND) was 12.0 (interquartile range 8.0, 14.0). Overall, 71% of patients suffered an adverse event (AE); the majority of these were grades 1 and 2, with 26% of patients experiencing a grade 3 AE. No grade 4 or 5 AEs were observed. Conclusions: After a structured training program, high-volume melanoma surgeons adopted a novel surgical technique with a lymph node retrieval rate that met or exceeded current oncologic guidelines and published benchmarks, and a favorable morbidity profile.

Applications of gene transfer to cellular immunotherapy

BACKGROUND Minimally invasive inguinal lymphadenectomy (MILND) is a novel procedure with the potential to decrease surgical morbidity compared with the traditional open approach. The current study examined the feasibility of a combined didactic and hands-on training program to prepare high-volume melanoma surgeons to perform this procedure safely and proficiently. STUDY DESIGN A select group of melanoma surgeons with no MILND experience were recruited. After completing a structured training program, surgeons enrolled patients with melanoma who required inguinal lymphadenectomy and performed the procedure in the minimally invasive fashion. A proficiency score composed of lymph node yield, operative time, and blood loss (or adverse events) was assigned for each case. After performing six cases, surgeons meeting a threshold score were considered proficient in the procedure. RESULTS Twelve surgeons from 10 institutions enrolled 88 patients. The majority of surgeons were deemed proficient within 6 cases (83%). No differences in operative time or lymph node yield were noted during the course of the study. The rate of conversion was higher during an individual surgeons early experience (9 of 49 [18%]), and only 1 procedure was converted in the 39 cases performed after a surgeon had performed 5 cases (late conversion rate, 3%; p = 0.038); however, this did not remain significant after controlling for surgeon. CONCLUSIONS After a structured training program, experienced melanoma surgeons adopted a novel surgical technique with acceptable operative times, conversions, and lymph node yield. Eighty-four percent of the surgeons who completed at least 6 MILND procedures were considered proficient based on our predetermined definition.

Plasma microRNA levels following resection of metastatic melanoma

Adoptive cellular therapy remains a potentially powerful method of eradicating established tumors. T-cells have been particularly potent effector cells, as demonstrated in animal models and clinical studies, and it is apparent that the stimulation of certain subpopulations of T-cells that are reactive to tumor antigens can lead to more therapeutic T-cells. The use of gene transfer techniques has resulted in more effective and specific methods to generate these tumor-specific T-cells. Another area of tremendous interest is in the adoptive transfer of DCs manipulated to present tumor antigen to resting, naive T-cells. Gene transfer techniques may offer more optimal ways to generate therapeutic DCs. Adoptive immunotherapy may ultimately [figure: see text] have its greatest use in patients undergoing cellular rescue after ablative chemotherapy; the infusion of immunocompetent T-cells, genetically modified stem cells, or programmed DCs may offer the opportunity to direct a patients immune response to eliminate residual microscopic disease.

Primary Squamous Cell Carcinoma Arising from a Breast Implant Capsule.

Melanoma remains the leading cause of skin cancer–related deaths. Surgical resection and adjuvant therapies can result in disease-free intervals for stage III and stage IV disease; however, recurrence is common. Understanding microRNA (miR) dynamics following surgical resection of melanomas is critical to accurately interpret miR changes suggestive of melanoma recurrence. Plasma of 6 patients with stage III (n = 2) and stage IV (n = 4) melanoma was evaluated using the NanoString platform to determine pre- and postsurgical miR expression profiles, enabling analysis of more than 800 miRs simultaneously in 12 samples. Principal component analysis detected underlying patterns of miR expression between pre- vs postsurgical patients. Group A contained 3 of 4 patients with stage IV disease (pre- and postsurgical samples) and 2 patients with stage III disease (postsurgical samples only). The corresponding preoperative samples to both individuals with stage III disease were contained in group B along with 1 individual with stage IV disease (pre- and postsurgical samples). Group A was distinguished from group B by statistically significant analysis of variance changes in miR expression (P < .0001). This analysis revealed that group A vs group B had downregulation of let-7b-5p, miR-520f, miR-720, miR-4454, miR-21-5p, miR-22-3p, miR-151a-3p, miR-378e, and miR-1283 and upregulation of miR-126-3p, miR-223-3p, miR-451a, let-7a-5p, let-7g-5p, miR-15b-5p, miR-16-5p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-26a-5p, miR-106a-5p, miR-17-5p, miR-130a-3p, miR-142-3p, miR-150-5p, miR-191-5p, miR-199a-3p, miR-199b-3p, and miR-1976. Changes in miR expression were not readily evident in individuals with distant metastatic disease (stage IV) as these individuals may have prolonged inflammatory responses. Thus, inflammatory-driven miRs coinciding with tumor-derived miRs can blunt anticipated changes in expression profiles following surgical resection.