Assessing the clinical utility of measuring Insulin-like Growth Factor Binding Proteins in tissues and sera of melanoma patients

<p>Abstract</p> <p>Background</p> <p>Different Insulin-like Growth Factor Binding Proteins (IGFBPs) have been investigated as potential biomarkers in several types of tumors. In this study, we examined both IGFBP-3 and -4 levels in tissues and sera of melanoma patients representing different stages of melanoma progression.</p> <p>Methods</p> <p>The study cohort consisted of 132 melanoma patients (primary, n = 72; metastatic, n = 60; 64 Male, 68 Female; Median Age = 56) prospectively enrolled in the New York University School of Medicine Interdisciplinary Melanoma Cooperative Group (NYU IMCG) between August 2002 and December 2006. We assessed tumor-expression and circulating sera levels of IGFBP-3 and -4 using immunohistochemistry and ELISA assays. Correlations with clinicopathologic parameters were examined using Wilcoxon rank-sum tests and Spearman-rank correlation coefficients.</p> <p>Results</p> <p>Median IGFBP-4 tumor expression was significantly greater in primary versus metastatic patients (70% versus 10%, p = 0.01) A trend for greater median IGFBP-3 sera concentration was observed in metastatic versus primary patients (4.9 μg/ml vs. 3.4 μg/ml, respectively, p = 0.09). However, sera levels fell within a normal range for IGFBP-3. Neither IGFBP-3 nor -4 correlated with survival in this subset of patients.</p> <p>Conclusion</p> <p>Decreased IGFBP-4 tumor expression might be a step in the progression from primary to metastatic melanoma. Our data lend support to a recently-described novel tumor suppressor role of secreting IGFBPs in melanoma. However, data do not support the clinical utility of measuring levels of IGFBP-3 and -4 in sera of melanoma patients.</p

Intra- and Inter-Tumor Heterogeneity of BRAFV600EMutations in Primary and Metastatic Melanoma

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful

Atypia on Breast Core Needle Biopsies: Reproducibility and Significance

Abstract. This study analyzes the interobserver variability in interpreting atypia on breast core needle biopsies and in each category of atypia calculates the upgrade risk of carcinoma in the subsequent surgical excision. We identified 51 cases of atypia on breast core needle biopsies performed at our institution from January 2003 to August 2006. The atypia was classified into 4 categories: atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH), flat epithelial atypia (FEA), and atypia of undetermined significance (AUS). After a tutorial session, these cases were independently reviewed by four pathologists, whose overall multi-rater kappa value for agreement on different categories of atypia was 0.79 (95% CI, 0.69-0.89), which is within the substantial agreement range. The upgrade risk in each category of atypia was as follows: ADH 20% (p = 0.04); ALH 10% (p = 0.6); FEA 16.6% (p = 0.23), and AUS 100% (p = 0.96). Based on our findings, we conclude that follow-up excision should be performed after a diagnosis of ADH. The upgrade risk did not reach statistical significance in ALH or FEA. Although follow-up excision cannot be strongly recommended in ALH and FEA, it should be considered since the upgrade risk is not negligible. Strict adherence to the diagnostic criteria and tutorial sessions can help pathologists to achieve substantial agreement in interpreting atypia on breast core needle biopsies

The novel gamma secretase inhibitor RO4929097 reduces the tumor initiating potential of melanoma.

Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma