Drazen M. Jukic

Neoadjuvant Treatment of Regional Stage IIIB Melanoma With High-Dose Interferon Alfa-2b Induces Objective Tumor Regression in Association With Modulation of Tumor Infiltrating Host Cellular Immune Responses

PURPOSE Adjuvant high-dose interferon-alfa-2b (HDI) improves disease-free and overall survival in patients with high-risk melanoma. However, its mechanism of action is largely unknown. Therefore, HDI was investigated in the neoadjuvant setting to assess clinical and pathologic responses after 4 weeks of HDI and to perform immunohistochemical evaluation of immune cell subsets and melanoma-associated antigens. PATIENTS AND METHODS Patients with palpable regional lymph node metastases from melanoma (American Joint Committee on Cancer stage IIIB-C) underwent surgical biopsy at study entry and then received standard intravenous HDI (20 million units/m2, 5 days per week) for 4 weeks followed by complete lymphadenectomy and standard maintenance subcutaneous HDI (10 million units/m2 3 times per week) for 48 weeks. Biopsy samples were obtained before and after intravenous HDI and subjected to immunohistochemical analysis as well as routine pathologic study. RESULTS Twenty patients were enrolled, and biopsy samples were informative for 17. Eleven patients (55%) demonstrated objective clinical response, and 3 patients (15%) had complete pathologic response. At a median follow-up of 18.5 months (range, 7 months to 50 months) 10 patients had no evidence of recurrent disease. Clinical responders had significantly greater increases in endotumoral CD11c+ and CD3+ cells and significantly greater decreases in endotumoral CD83+ cells compared with nonresponders. No changes in the expression of melanoma-associated lineage antigens, tumor cell proliferation, angiogenesis, or apoptosis were evident. CONCLUSION Neoadjuvant HDI is highly effective for the treatment of palpable stage IIIB-C melanoma, and the findings of this study implicate an indirect immunomodulatory mechanism rather than a direct antitumor mechanism.

Modulation of Signal Transducers and Activators of Transcription 1 and 3 Signaling in Melanoma by High-Dose IFNα2b

Purpose: The Janus-activated kinase/signal transducers and activators of transcription (STAT) pathway of IFN signaling is important to immunoregulation and tumor progression. STAT1 plays a prominent role in the effector immune response, whereas STAT3 is implicated in tumor progression and down-regulation of the response to type I IFNs. The goal of this study was to understand the effects of high-dose IFNα2b (HDI) in relation to the balance of pSTAT1 and pSTAT3. Experimental Design: We evaluated STAT1 and STAT3 jointly as mediators of IFNα effects in the setting of a prospective neoadjuvant trial of HDI, in which tissue samples were obtained before and after 20 doses of HDI therapy. Double immunohistochemistry for pSTAT1 and pSTAT3 was done on paired fixed (9 patients) or frozen (12 patients) biopsies. Results: HDI was found to up-regulate pSTAT1, whereas it down-regulates pSTAT3 and total STAT3 levels in both tumor cells and lymphocytes. Higher pSTAT1/pSTAT3 ratios in tumor cells pretreatment were associated with longer overall survival (P = 0.032). The pSTAT1/pSTAT3 ratios were augmented by HDI both in melanoma cells (P = 0.005) and in lymphocytes (P = 0.022). Of the immunologic mediators and markers tested, TAP2 was augmented by HDI (but not TAP1 and MHC class I/II). Conclusion: IFNα2b significantly modulates the balance of STAT1/STAT3 in tumor cells and host lymphocytes, leading to up-regulation of TAP2 and augmented host antitumor response. The pSTAT1/pSTAT3 ratio in tumor cells at baseline may serve as a useful predictor of clinical outcome in cutaneous melanoma; the modulation of this ratio may serve as a predictor of therapeutic effect.

Microrna profiling analysis of differences between the melanoma of young adults and older adults

BackgroundThis study represents the first attempt to perform a profiling analysis of the intergenerational differences in the microRNAs (miRNAs) of primary cutaneous melanocytic neoplasms in young adult and older age groups. The data emphasize the importance of these master regulators in the transcriptional machinery of melanocytic neoplasms and suggest that differential levels of expressions of these miRs may contribute to differences in phenotypic and pathologic presentation of melanocytic neoplasms at different ages.MethodsAn exploratory miRNA analysis of 666 miRs by low density microRNA arrays was conducted on formalin fixed and paraffin embedded tissues (FFPE) from 10 older adults and 10 young adults including conventional melanoma and melanocytic neoplasms of uncertain biological significance. Age-matched benign melanocytic nevi were used as controls.ResultsPrimary melanoma in patients greater than 60 years old was characterized by the increased expression of miRs regulating TLR-MyD88-NF-kappaB pathway (hsa-miR-199a), RAS/RAB22A pathway (hsa-miR-204); growth differentiation and migration (hsa-miR337), epithelial mesenchymal transition (EMT) (let-7b, hsa-miR-10b/10b*), invasion and metastasis (hsa-miR-10b/10b*), hsa-miR-30a/e*, hsa-miR-29c*; cellular matrix components (hsa-miR-29c*); invasion-cytokinesis (hsa-miR-99b*) compared to melanoma of younger patients. MiR-211 was dramatically downregulated compared to nevi controls, decreased with increasing age and was among the miRs linked to metastatic processes. Melanoma in young adult patients had increased expression of hsa-miR-449a and decreased expression of hsa-miR-146b, hsa-miR-214*. MiR-30a* in clinical stages I-II adult and pediatric melanoma could predict classification of melanoma tissue in the two extremes of age groups. Although the number of cases is small, positive lymph node status in the two age groups was characterized by the statistically significant expression of hsa-miR-30a* and hsa-miR-204 (F-test, p-value < 0.001).ConclusionsOur findings, although preliminary, support the notion that the differential biology of melanoma at the extremes of age is driven, in part, by deregulation of microRNA expression and by fine tuning of miRs that are already known to regulate cell cycle, inflammation, Epithelial-Mesenchymal Transition (EMT)/stroma and more specifically genes known to be altered in melanoma. Our analysis reveals that miR expression differences create unique patterns of frequently affected biological processes that clearly distinguish old age from young age melanomas. This is a novel characterization of the miRnomes of melanocytic neoplasms at two extremes of age and identifies potential diagnostic and clinico-pathologic biomarkers that may serve as novel miR-based targeted modalities in melanoma diagnosis and treatment.

Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay

Defects in cholesterol synthesis result in a wide variety of symptoms, from neonatal lethality to the relatively mild dysmorphic features and developmental delay found in individuals with Smith-Lemli-Opitz syndrome. We report here the identification of mutations in sterol-C4-methyl oxidase–like gene (SC4MOL) as the cause of an autosomal recessive syndrome in a human patient with psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly, and developmental delay. This gene encodes a sterol-C4-methyl oxidase (SMO), which catalyzes demethylation of C4-methylsterols in the cholesterol synthesis pathway. C4-Methylsterols are meiosis-activating sterols (MASs). They exist at high concentrations in the testis and ovary and play roles in meiosis activation. In this study, we found that an accumulation of MASs in the patient led to cell overproliferation in both skin and blood. SMO deficiency also substantially altered immunocyte phenotype and in vitro function. MASs serve as ligands for liver X receptors α and β(LXRα and LXRβ), which are important in regulating not only lipid transport in the epidermis, but also innate and adaptive immunity. Deficiency of SMO represents a biochemical defect in the cholesterol synthesis pathway, the clinical spectrum of which remains to be defined.

SAGE and antibody array analysis of melanoma-infiltrated lymph nodes: identification of Ubc9 as an important molecule in advanced-stage melanomas.

Although patients diagnosed with melanoma of ⩽1.00 mm thickness have a relatively good cure rate, the prognosis for patients with locally advanced and metastatic melanoma is grave. The discovery of new and effective therapies for this disease depends in large part on molecular studies that will resolve why advanced-stage melanoma is refractory to conventional chemotherapy and radiation therapy. To identify genes that have important functions in advanced-stage melanomas, in particular, in melanoma-infiltrated lymph nodes, which are not well characterized at the molecular level, we generated a LongSAGE library from a melanoma-positive lymph node, and subjected melanoma-infiltrated lymph nodes to protein expression profiling. The data document that the molecular signature of melanoma, which has spread to regional lymph nodes, is very similar to the molecular signature of primary melanomas. Equally important, we provide evidence that the ubiquitin-conjugating enzyme, Ubc9, is expressed at high levels in melanoma-positive lymph nodes, and that it plays a crucial role in preventing advanced-stage melanomas from undergoing chemotherapy-induced apoptosis.

OpenSlide: A vendor-neutral software foundation for digital pathology

Although widely touted as a replacement for glass slides and microscopes in pathology, digital slides present major challenges in data storage, transmission, processing and interoperability. Since no universal data format is in widespread use for these images today, each vendor defines its own proprietary data formats, analysis tools, viewers and software libraries. This creates issues not only for pathologists, but also for interoperability. In this paper, we present the design and implementation of OpenSlide, a vendor-neutral C library for reading and manipulating digital slides of diverse vendor formats. The library is extensible and easily interfaced to various programming languages. An application written to the OpenSlide interface can transparently handle multiple vendor formats. OpenSlide is in use today by many academic and industrial organizations world-wide, including many research sites in the United States that are funded by the National Institutes of Health.

An immunohistochemical panel to differentiate metastatic breast carcinoma to skin from primary sweat gland carcinomas with a review of the literature.

CONTEXT Approximately 25% of patients with breast cancer develop cutaneous metastases. Sweat gland carcinomas (SGCs) account for about 0.05% of all cutaneous neoplasms. Cutaneous metastases of breast carcinoma (CMBCs) (especially the ductal type) can be difficult to distinguish from SGCs. Treatment and prognoses for these 2 types of tumors differ radically, making accurate histologic diagnosis crucial. Although a few studies attempt to differentiate these entities employing immunohistochemical (IHC) studies (some of which we review here), to date, no panel of IHC stains exists, to our knowledge, to distinguish these entities. OBJECTIVE To devise a panel of IHC stains to distinguish CMBC from SGC. DESIGN Twelve cases of ductal CMBCs (11 not otherwise specified type, and 1 basal phenotype), 11 cases of SGCs (5 eccrine carcinomas, 3 porocarcinomas, and 3 microcystic adnexal carcinomas), 2 benign sweat gland neoplasm cases, and 2 primary breast cancer cases were retrieved and analyzed with the following IHC panel: mammaglobin, gross cystic disease fluid protein (GCDFP) 15, p63, basal cytokeratins (CK5, CK14, and CK17), androgen receptor, and PAX5. RESULTS The p63 was only weakly expressed in 1 of 12 CMBC cases (8.3%), whereas it was strongly expressed in 10 of 11 SGC cases (90.9%) (P < .001). Basal cytokeratins demonstrated a similar immunoprofile in the SGC group, with 10 of 11 cases (90.9%) expressing all 3 markers, and a variable immunoprofile in the CMBC group with 0% (CK14) (P < .001) to 16.7% (2 of 12 cases; CK5 and CK17) (P < .001) expression. Mammaglobin was expressed in 8 of 12 cases (66.7%) of CMBC. CONCLUSIONS Together, these 5 IHC stains were combined to make a panel that was 100% sensitive and 91% specific in distinguishing between CMBC and SGC.

Pediatric Melanoma: Analysis of an International Registry

The management of pediatric melanoma (PM) has largely been extrapolated from adult data. However, the behavior of PM appears to differ from its adult counterparts. Therefore, an international PM registry was created and analyzed.

Factors Affecting Feeling-of-knowing in a Medical Intelligent Tutoring System – the role of Immediate Feedback as a Metacognitive Scaffold

Previous studies in our laboratory have shown the benefits of immediate feedback on cognitive performance for pathology residents using an intelligent tutoring system (ITS) in pathology. In this study, we examined the effect of immediate feedback on metacognitive performance, and investigated whether other metacognitive scaffolds will support metacognitive gains when immediate feedback is faded. Twenty-three participants were randomized into intervention and control groups. For both groups, periods working with the ITS under varying conditions were alternated with independent computer-based assessments. On day 1, a within-subjects design was used to evaluate the effect of immediate feedback on cognitive and metacognitive performance. On day 2, a between-subjects design was used to compare the use of other metacognitive scaffolds (intervention group) against no metacognitive scaffolds (control group) on cognitive and metacognitive performance, as immediate feedback was faded. Measurements included learning gains (a measure of cognitive performance), as well as several measures of metacognitive performance, including Goodman–Kruskal gamma correlation (G), bias, and discrimination. For the intervention group, we also computed metacognitive measures during tutoring sessions. Results showed that immediate feedback in an intelligent tutoring system had a statistically significant positive effect on learning gains, G and discrimination. Removal of immediate feedback was associated with decreasing metacognitive performance, and this decline was not prevented when students used a version of the tutoring system that provided other metacognitive scaffolds. Results obtained directly from the ITS suggest that other metacognitive scaffolds do have a positive effect on G and discrimination, as immediate feedback is faded. We conclude that immediate feedback had a positive effect on both metacognitive and cognitive gains in a medical tutoring system. Other metacognitive scaffolds were not sufficient to replace immediate feedback in this study. However, results obtained directly from the tutoring system are not consistent with results obtained from assessments. In order to facilitate transfer to real-world tasks, further research will be needed to determine the optimum methods for supporting metacognition as immediate feedback is faded.

Effects of High-Dose IFNα2b on Regional Lymph Node Metastases of Human Melanoma: Modulation of STAT5, FOXP3, and IL-17

Purpose: Signal transducer and activator of transcription 5 (STAT5) and STAT3 oppose one another in regulation of the reciprocal development of CD4+CD25+FOXP3+ regulatory T cells (Treg) and T helper 17 (Th17). A reduction in STAT3 is associated with up-regulation of Treg, and STAT5 activation promotes Treg differentiation or function while constraining Th17 generation. The effects of IFNα on STAT signaling in relation to tumor tissue Treg and Th17 have not been documented in humans beyond the observations that IFNα2b down-regulates STAT3. Experimental Design: Following diagnostic biopsy and before definitive surgery, 20 doses of high-dose IFNα2b (HDI) were administered to patients with stage IIIB melanoma who gave written informed consent. Lymph node biopsies, in which both total STAT3 and phosphorylated STAT3 were down-regulated by HDI, were probed with STAT5, FOXP3, CD4, and interleukin 17 (IL-17) with immunohistochemistry and/or immunofluorescence techniques. Results: The percentage of FOXP3+ lymphocytes determined by immunohistochemistry was up-regulated from 3.06 ± 0.65% to 9.86 ± 1.27% (n = 13, P = 0.0002), and this observation was confirmed by immunofluorescence evaluation of CD4+FOXP3+ Tregs. HDI induced STAT5 up-regulation (five cases observed) in melanoma cells and lymphocytes but did not induce the generation of IL-17–expressing lymphocytes. Increased STAT5 expression was associated with increased FOXP3 expression among lymphocytes, and STAT5 was constitutively activated among both melanoma cells and lymphocytes. Conclusion: IFNα2b up-regulates STAT5 and down-regulates STAT3, in conjunction with up-regulation of Treg and inhibition of IL-17–expressing lymphocytes in melanoma tissues. These findings suggest that the effects of IFNα may be potentiated through interference with the response of Tregs and/or STAT5.