Dark homogeneous streak dermoscopic pattern correlating with specific KIT mutations in melanoma

Mutations driving melanoma growth have diagnostic, prognostic, and therapeutic implications. Traditional classification systems do not correlate optimally with underlying melanoma growth-promoting mutations. Our objective was to determine whether unique dermoscopic growth patterns directly correlate with driving mutations. OBSERVATIONS: We evaluated common driving mutations in 4 different dermoscopic patterns (rhomboidal, negative pigmented network, polygonal, and dark homogeneous streaks) of primary cutaneous melanomas; 3 melanomas per pattern were tested. Three of the 4 patterns lacked common mutations in BRAF, NRAS, KIT, GNAQ, and HRAS. One pattern, the dark homogeneous streaks pattern, had unique KIT mutations in the second catalytic domain of KIT in exon 17 for all 3 samples tested. Two tumors with the dark homogeneous streaks pattern turned out to be different primary melanomas from the same patient and had different sequence mutations but had an impact on the same KIT domain. CONCLUSIONS AND RELEVANCE: While future study is required, these results have multiple implications. (1) The underlying melanoma-driving mutations may give rise to specific dermoscopic growth patterns, (2) BRAF/NRAS mutations in early melanomas may not be as common as previously thought, and (3) patients may be predisposed to developing specific driving mutations giving rise to melanomas or nevi of similar growth patterns

In Vitro Dedifferentiation of Melanocytes from Adult Epidermis

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation

Postresectional lung injury in thoracic surgery pre and intraoperative risk factors: a retrospective clinical study of a hundred forty-three cases

<p>Abstract</p> <p>Introduction</p> <p>Acute respiratory dysfunction syndrome (ARDS), defined as acute hypoxemia accompanied by radiographic pulmonary infiltrates without a clearly identifiable cause, is a major cause of morbidity and mortality after pulmonary resection. The aim of the study was to determine the pre and intraoperative factors associated with ARDS after pulmonary resection retrospectively.</p> <p>Methods</p> <p>Patients undergoing elective pulmonary resection at Adnan Menderes University Medical Faculty Thoracic Surgery Department from January 2005 to February 2010 were included in this retrospective study. The authors collected data on demographics, relevant co-morbidities, the American Society of Anesthesiologists (ASA) Physical Status classification score, pulmonary function tests, type of operation, duration of surgery and intraoperative fluid administration (fluid therapy and blood products). The primary outcome measure was postoperative ARDS, defined as the need for continuation of mechanical ventilation for greater than 48-hours postoperatively or the need for reinstitution of mechanical ventilation after extubation. Statistical analysis was performed with Fisher exact test for categorical variables and logistic regression analysis for continuous variables.</p> <p>Results</p> <p>Of one hundred forty-three pulmonary resection patients, 11 (7.5%) developed postoperative ARDS. Alcohol abuse (p = 0.01, OR = 39.6), ASA score (p = 0.001, OR: 1257.3), resection type (p = 0.032, OR = 28.6) and fresh frozen plasma (FFP)(p = 0.027, OR = 1.4) were the factors found to be statistically significant.</p> <p>Conclusion</p> <p>In the light of the current study, lung injury after lung resection has a high mortality. Preoperative and postoperative risk factor were significant predictors of postoperative lung injury.</p

Inhibition of Hedgehog Signaling Antagonizes Serous Ovarian Cancer Growth in a Primary Xenograft Model

Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, basal cell, small cell lung, pancreatic, prostate and ovarian. This investigation was designed to determine if inhibition of this pathway could inhibit serous ovarian cancer growth.We utilized an in vivo pre-clinical model of serous ovarian cancer to characterize the anti-tumor activity of Hh pathway inhibitors cyclopamine and a clinically applicable derivative, IPI-926. Primary human serous ovarian tumor tissue was used to generate tumor xenografts in mice that were subsequently treated with cyclopamine or IPI-926.Both compounds demonstrated significant anti-tumor activity as single agents. When IPI-926 was used in combination with paclitaxel and carboplatinum (T/C), no synergistic effect was observed, though sustained treatment with IPI-926 after cessation of T/C continued to suppress tumor growth. Hh pathway activity was analyzed by RT-PCR to assess changes in Gli1 transcript levels. A single dose of IPI-926 inhibited mouse stromal Gli1 transcript levels at 24 hours with unchanged human intra-tumor Gli1 levels. Chronic IPI-926 therapy for 21 days, however, inhibited Hh signaling in both mouse stromal and human tumor cells. Expression data from the micro-dissected stroma in human serous ovarian tumors confirmed the presence of Gli1 transcript and a significant association between elevated Gli1 transcript levels and worsened survival.IPI-926 treatment inhibits serous tumor growth suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may hold promise as a novel therapeutic target, especially in the maintenance setting

Expression of SCF splice variants in human melanocytes and melanoma cell lines: potential prognostic implications

Stem cell factor (SCF), the ligand for c-Kit, is known to regulate developmental and functional processes of haematopoietic stem cells, mast cells and melanocytes. Two different splice variants form predominantly soluble (sSCF or SCF-1) and in addition some membrane-bound SCF (mSCF or SCF-2). In order to explore the prognostic significance of these molecules in melanoma, total SCF, SCF splice variants and c-Kit expression were studied in normal skin melanocytes and in 11 different melanoma cell lines, using reverse transcription polymerase chain reaction, immunocytochemistry and enzyme-linked immunosorbent assay. Nine of the 11 melanoma cell lines expressed SCF-1 mRNA, only two of them SCF-2, and these two also SCF-1. Coexpression of both SCF-1 and c-Kit was noted in five cell lines, and only one cell line as well as normal melanocytes expressed both SCF-1 and SCF-2 as well as c-Kit. Corresponding results were obtained on immunocytochemical staining. Of three exemplary melanoma cell lines studied, two expressing SCF mRNA also released SCF spontaneously and on stimulation, whereas the line lacking SCF and c-kit mRNA (SK-Mel-23) failed to do so. These data demonstrate thus that melanoma cell lines, particularly those known to metastasize in vivo, lose the ability to express SCF-2 mRNA, suggesting that this molecule may serve, next to c-Kit, as a prognostic marker for malignant melanoma. © 2000 Cancer Research Campaig

Multi-Modality Therapeutics with Potent Anti-Tumor Effects: Photochemical Internalization Enhances Delivery of the Fusion Toxin scFvMEL/rGel

BACKGROUND: There is a need for drug delivery systems (DDS) that can enhance cytosolic delivery of anti-cancer drugs trapped in the endo-lysosomal compartments. Exposure of cells to specific photosensitizers followed by light exposure (photochemical internalization, PCI) results in transfer of agents from the endocytic compartment into the cytosol. METHODOLOGY AND PRINCIPAL FINDINGS: The recombinant single-chain fusion construct scFvMEL/rGel is composed of an antibody targeting the progenitor marker HMW-MAA/NG2/MGP/gp240 and the highly effective toxin gelonin (rGel). Here we demonstrate enhanced tumor cell selectivity, cytosolic delivery and anti-tumor activity by applying PCI of scFvMEL/rGel. PCI performed by light activation of cells co-incubated with scFvMEL/rGel and the endo-lysosomal targeting photosensitizers AlPcS(2a) or TPPS(2a) resulted in enhanced cytotoxic effects against antigen-positive cell lines, while no differences in cytotoxicity between the scFvMEL/rGel and rGel were observed in antigen-negative cells. Mice bearing well-developed melanoma (A-375) xenografts (50-100 mm(3)) were treated with PCI of scFvMEL/rGel. By 30 days after injection, approximately 100% of mice in the control groups had tumors>800 mm(3). In contrast, by day 40, 50% of mice in the PCI of scFvMEL/rGel combination group had tumors<800 mm(3) with no increase in tumor size up to 110 days. PCI of scFvMEL/rGel resulted in a synergistic effect (p<0.05) and complete regression (CR) in 33% of tumor-bearing mice (n = 12). CONCLUSIONS/SIGNIFICANCE: This is a unique demonstration that a non-invasive multi-modality approach combining a recombinant, targeted therapeutic such as scFvMEL/rGel and PCI act in concert to provide potent in vivo efficacy without sacrificing selectivity or enhancing toxicity. The present DDS warrants further evaluation of its clinical potential

Rapid Selection and Proliferation of CD133(+) Cells from Cancer Cell Lines: Chemotherapeutic Implications

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates

Skeletal Muscle Phenotypically Converts and Selectively Inhibits Metastatic Cells in Mice

Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals