Steve Shivers

Roles of activated Src and Stat3 signaling in melanoma tumor cell growth.

Activation of protein tyrosine kinases is prevalent in human cancers and previous studies have demonstrated that Stat3 signaling is a point of convergence for many of these tyrosine kinases. Moreover, a critical role for constitutive activation of Stat3 in tumor cell proliferation and survival has been established in diverse cancers. However, the oncogenic signaling pathways in melanoma cells remain to be fully defined. In this study, we demonstrate that Stat3 is constitutively activated in a majority of human melanoma cell lines and tumor specimens examined. Blocking Src tyrosine kinase activity, but not EGF receptor or JAK family kinases, leads to inhibition of Stat3 signaling in melanoma cell lines. Consistent with a role of Src in the pathogenesis of melanoma, we show that c-Src tyrosine kinase is activated in melanoma cell lines. Significantly, melanoma cells undergo apoptosis when either Src kinase activity or Stat3 signaling is inhibited. Blockade of Src or Stat3 is also accompanied by down-regulation of expression of the anti-apoptotic genes, Bcl-xL and Mcl-1. These findings demonstrate that Src-activated Stat3 signaling is important for the growth and survival of melanoma tumor cells.

The staging of malignant melanoma and the florida melanoma trial

Lymphatic mapping and sentinel lymph node (SLN) biopsy have changed the standard of care for patients with malignant melanoma, by providing a less morbid procedure to obtain the nodal staging information that is critical for therapeutic decisions. Detailed examination of the SLN identifies patients who have an increased risk for recurrence and death. Patients whose melanoma is upstaged with very sensitive assays based on reverse transcriptase polymerase chain reaction technology are better targeted for clinical trials or surgical or adjuvant therapies. In the future, melanoma may be “ultrastaged” by examining the SLNs, peripheral blood, and bone marrow. This may improve identification of patients who are surgically cured of their disease and therefore can be spared the side effects of more radical surgery or the toxicities of adjuvant therapy. The lymphatic mapping procedure is the most accurate way to determine the tumor status of the regional lymph nodes.

Sentinel lymph node micrometastasis from melanoma

The most powerful predictor of recurrence rates and survival for most solid tumors, including melanoma, is the status of the regional lymph nodes. If a patient has melanoma and regional lymph node disease (Stage III disease), the 5-year survival is decreased 25–50%, depending on the volume of disease in the regional basin. The article by the Boston group describes the emerging new standard of care for the surgical treatment of melanoma, namely, lymphatic mapping and sentinel lymph node (SLN) biopsy. It is a win-win situation for the patient. The ability to perform lymph node staging with only a lymph node biopsy type of procedure inevitably results in lower morbidity for the patient. No postoperative drains are used. It is rare for patients to get lymphedema or paresthesias if their SLN is negative and only a SLN biopsy is performed, and they experience an earlier return to work and other normal activity. However, the true advantage of lymphatic mapping and SLN biopsy is the ability of the surgeon to give the pathologist the lymph node(s) most likely to contain metastatic disease. One would then expect the pathologist to conduct a more detailed examination of the SLNs, which may include making more sections, performing immunohistochemical (IHC) staining, or perhaps even performing molecular biology assays for occult metastases based on the reverse transcriptase–polymerase chain reaction (RT-PCR) technique. In turn, this more detailed examination of the SLN will result in more accurate lymph node staging for the patient. The standard of care for the world in examining the regional lymph nodes is to make one or two sections of the central part of each lymph node and stain the section with a routine hematoxylin and eosin (H & E) stain. With this standard, 1–5% of the submitted material is examined and the number of patients with micrometastatic disease is underestimated. Surgical pathologists make their diagnosis and clinicians make their treatment decisions based on a relatively superficial examination of the lymph nodes from the regional basins. The routine examination misses low volume micrometastatic disease. Hopefully, pathologists are not offended by this revelation. Yu et al. showed that 12% of the patients with melanoma who are called lymph node negative by the pathologist with routine examination are found 551

Molecular staging for patients with malignant melanoma.

Molecular staging of cancers hold the promise of being more accurate compared with routine histology, particularly with regard to determining regional-nodal status. With newer reverse transcriptase-PCR (RT-PCR)-based assays, sensitivities reported are as high as identifying one cancer cell in a background of a million normal cells. Although this sensitivity is 100-times what the human eye can differentiate under the microscope, the new challenge becomes determining the relevance of this low-volume disease in the regional basin, in particular, the sentinel lymph node (SLN). Patients with melanomas greater than 0.75 mm in tumor thickness participated in a research study that examined their SLNs with routine histology, immunhistochemical staining and a RT-PCR assay based on the tyrosinase probe. A total of 311 patients were involved in the study and patients whose SLN were negative from all three assays for metastatic disease had a good survival, with a 92% disease-free survival (DFS) and a 97% overall survival (OS) regardless of the tumor thickness or the ulceration status of the primary melanoma. Patients upstaged with the RT-PCR assay had a significantly decreased DFS and OS compared with patients who were SLN negative. Patients who had enough tumor burden in the SLN that allowed their metastatic disease to be identified with routine histology had a 48% recurrence rate at 5 years. A recently published meta-analysis confirmed that molecular staging of the SLN in melanoma contains important prognostic information. Micrometastatic disease missed by routine histology in the SLN in melanoma patients is clinically relevant disease. Molecular staging has the potential of providing a more accurate staging in the SLN, for prognostication and directing adjuvant therapies.

Molecular Staging of Melanoma

Two papers published in this issue of the Annals of Surgical Oncology involved the emerging field of molecular staging of cancers. For many years investigators have wondered why some patients with melanoma, despite being node negative, recur and die of their disease. Is this due to hematogenous spread from the primary site or perhaps missed micrometastatic disease when the regional basin is sampled? The manuscript by Gradilone and colleagues1 from the University of Rome would suggest that at least some component of these recurrences and deaths are due to missed micrometastatic disease and inaccurate staging in the regional basin. The paper by Ruka et al.,2 from the Skfodouska-Curie Memorial Cancer Center and Institute of Oncology in Poland examined the afferent lymphatic flow into the regional basin after a therapeutic lymph node dissection in patients with melanoma and discovered that some patients shed tumor cells after this operation and those patients are identified to have a worse prognosis. Both groups used the RT-PCR technology, a technology that has the sensitivity of identifying 1 cancer cell in a background of a million normal cells. This sensitivity cannot be matched by routine histology and the human eye under the microscope, which is on the magnitude of being able to identify 1 cancer cell in a background of 10,000 normal lymphocytes. The field of molecular staging for melanoma got its start in 1991 when Smith et al.3 identified circulating tumor cells with a very sensitive RT-PCR assay for tyrosinase. Tyrosinase is an enzyme that catalyzes the first two steps of the biosynthetic pathway of melanin synthesis. All cells in the body have the gene for tyrosinase but only cells that produce pigment, such as melanocytes and melanoma cells, will express the mRNA for this gene. If this gene product is found in the sentinel lymph node (SLN) or afferent lymphatic flow, that is good evidence that metastatic disease exits. The challenge has been to show clinical correlation. In other words, does finding evidence of metastatic disease with RT-PCR assays portend the patient to have a poorer prognosis. Heller and colleagues from the University of South Florida were the first to recognize that routine histologic examination of the regional basin can miss micrometastatic disease.4 Using a simple lymph node culture technique, this group showed that malignant melanoma cells could be grown from lymph nodes called histologic negative by the pathologist. This missed micrometastatic disease was found to be clinically relevant in that these patients up-staged with the method had a significantly higher recurrence rate and poorer survival.5 The tissue culture method proved cumbersome and inefficient in that results were not available for a number of weeks. A more sensitive RT-PCR assay was developed for metastatic melanoma in the SLN6 and approximately 40% of the so-called histologic negative patients were found to have evidence of metastatic disease in their regional basin. Again this missed micrometastasis disease, now termed “submicroscopic disease”, was found to be clinically important in that patients up-staged with the technique had a significantly worse outcome. In multivariate regression analysis that included known prognostic factors for melanoma, including tumor thickness and ulceration, it was the status of the SLN, either determined by routine histology or the RT-PCR assay that was the most important predictor of recurrence and death.7 This finding has now been supported by 6 different laboratories and one national trial throughout the world,8–16 including the recent publication from the University of Rome group. September 10, 2004; September 22, 2004. From the Cutaneous Oncology Program, Lakeland Regional Cancer Center, Lakeland, Florida. Douglas S. Reintgen, MD, Lakeland Regional Cancer Center, 3525 Lakeland Hills Boulevard, PO Box 91057, Lakeland, FL; Fax: 863904-1802; E-mail: doug.reintgen@lrmc.com.

A Randomized Controlled Trial of the Effects of Mindfulness-Based Stress Reduction (MBSR[BC]) on Levels of Inflammatory Biomarkers Among Recovering Breast Cancer Survivors

Purpose: The purpose of this substudy of a large randomized controlled trial was to evaluate the efficacy of the Mindfulness-Based Stress Reduction (Breast Cancer) (MBSR[BC]) program compared to usual care (UC) in normalizing blood levels of pro-inflammatory cytokines among breast cancer survivors (BCS). Method: A total of 322 BCS were randomized to either a 6-week MBSR(BC) program or a UC. At baseline and 6 and 12 weeks, 10 ml of venous blood and demographic and clinical data were collected and/or updated. Plasma cytokines (interleukin [IL]-1β, IL-6, IL-10, tumor necrosis factor [TNF] α, transforming growth factor [TGF] β1, soluble tumor necrosis factor receptor [sTNFR] 1) were assayed. Linear mixed models were used to assess cytokine levels across three time points (baseline and 6 and 12 weeks) by group (MBSR[BC] vs. UC). Results: Of the six measured cytokines, three were nondetectable at rates greater than 50% (IL-10, IL-1β, TGF-β1) and, because of overall low prevalence, were not analyzed further. For the remaining cytokines (TNFα, IL-6, sTNFR1), results showed that TNFα and IL-6 increased during the follow-up period (between 6 and 12 weeks) rather than during the MBSR(BC) training period (between baseline and 6 weeks), while sTNFR1 levels did not change significantly across the 12-week period. Conclusions: Study results suggest that MBSR(BC) affects cytokine levels in BCS, mainly with increases in TNFα and IL-6. The data further suggest that B-cell modulation may be a part of immune recovery during breast cancer management and that increases in TNFα and IL-6 may be markers for MBSR(BC)-related recovery.

A Large Randomized Trial: Effects of Mindfulness-Based Stress Reduction (MBSR) for Breast Cancer (BC) Survivors on Salivary Cortisol and IL-6

Breast cancer survivors (BCS) often experience psychological and physiological symptoms after cancer treatment. Mindfulness-based stress reduction (MBSR), a complementary and alternative therapy, has reduced subjective measures of stress, anxiety, and fatigue among BCS. Little is known, however, about how MBSR affects objective markers of stress, specifically the stress hormone cortisol and the pro-inflammatory cytokine interleukin-6 (IL-6). In the present study, BCS (N = 322) were randomly assigned to a 6-week MBSR program for BC or usual-care control. Measurements of cortisol, IL-6, symptoms, and quality of life were obtained at orientation and 6 weeks. Cortisol and IL-6 were also measured prior to and after the MBSR(BC) class Weeks 1 and 6. The mean age of participants was 56.6 years and 69.4% were White non-Hispanic. Most had Stage I (33.8%) or II (35.7%) BC, and 35.7% had received chemotherapy and radiation. Cortisol levels were reduced immediately following MBSR(BC) class compared to before the class Weeks 1 and 6 (Wilcoxon-signed rank test; p < .01, d = .52–.56). IL-6 was significantly reduced from pre- to postclass at Week 6 (Wilcoxon-signed rank test; p < .01, d = .21). No differences were observed between the MBSR(BC) and control groups from baseline to Week 6 using linear mixed models. Significant relationships with small effect sizes were observed between IL-6 and both symptoms and quality of life in both groups. Results support the use of MBSR(BC) to reduce salivary cortisol and IL-6 levels in the short term in BCS.