Gennaro Ilardi

Accuracy in melanoma detection: A 10-year multicenter survey

BACKGROUND Early excision is the only strategy to reduce melanoma mortality, but unnecessary excision of benign lesions increases morbidity and healthcare costs. OBJECTIVE To assess accuracy in melanoma detection based on number-needed-to-excise (NNE) values over a 10-year period. METHODS Information was retrieved on all histopathologically confirmed cutaneous melanomas or melanocytic nevi that were excised between 1998 and 2007 at participating clinics. NNE values were calculated by dividing the total number of excised lesions by the number of melanomas. Analyses included changes in NNE over time, differences in NNE between specialized clinical settings (SCS) versus non-specialized clinical settings (NSCS), and patient factors influencing NNE. RESULTS The participating clinics contributed a total of 300,215 cases, including 17,172 melanomas and 283,043 melanocytic nevi. The overall NNE values achieved in SCS and NSCS in the 10-year period were 8.7 and 29.4, respectively. The NNE improved over time in SCS (from 12.8 to 6.8), but appeared unchanged in NSCS. Most of the effect on NNE in SCS was due to a greater number of excised melanomas. Higher NNE values were observed in patients younger than 40 years and for lesions located on the trunk. LIMITATIONS No data concerning the use of dermatoscopy and digital monitoring procedures were collected from the participating centers. CONCLUSION Over the 10-year study period, accuracy in melanoma detection improved only in specialized clinics maybe because of a larger use of new diagnostic techniques such as dermatoscopy.

Epigenetic Disregulation in Oral Cancer

Squamous cell carcinoma of the oral region (OSCC) is one of the most common and highly aggressive malignancies worldwide, despite the fact that significant results have been achieved during the last decades in its detection, prevention and treatment. Although many efforts have been made to define the molecular signatures that identify the clinical outcome of oral cancers, OSCC still lacks reliable prognostic molecular markers. Scientific evidence indicates that transition from normal epithelium to pre-malignancy, and finally to oral carcinoma, depends on the accumulation of genetic and epigenetic alterations in a multistep process. Unlike genetic alterations, epigenetic changes are heritable and potentially reversible. The most common examples of such changes are DNA methylation, histone modification, and small non-coding RNAs. Although several epigenetic changes have been currently linked to OSCC initiation and progression, they have been only partially characterized. Over the last decade, it has been demonstrated that especially aberrant DNA methylation plays a critical role in oral cancer. The major goal of the present paper is to review the recent literature about the epigenetic modifications contribution in early and later phases of OSCC malignant transformation; in particular we point out the current evidence of epigenetic marks as novel markers for early diagnosis and prognosis as well as potential therapeutic targets in oral cancer.

Resveratrol-containing gel for the treatment of acne vulgaris: a single-blind, vehicle-controlled, pilot study.

AbstractBackground: Acne vulgaris is a complex, chronic, and common skin disorder of pilosebaceous units. The major pathogenic factors involved are ductal hyperkeratinization, obstruction of sebaceous follicles resulting from abnormal keratinization of the infundibular epithelium, stimulation of sebaceous gland secretion by androgens, and microbial colonization of pilosebaceous units by Propionibacterium acnes, which promotes perifollicular inflammation. Aim: The aim of the study was to investigate the therapeutic effects of resveratrol, a natural phytoalexin produced by some spermatophytes, such as grapes and other plants, on acneic skin. Methods: Resveratrol was incorporated in a carboxymethylcellulose-based gel. The chemical stability of resveratrol after storage at 4°C for 30 days was investigated by high-performance liquid chromatography (HPLC). The resveratrol-containing hydrogel was administered to 20 patients affected by acne vulgaris enrolled in this single-blind study. The resveratrol-containing formulation was applied daily as a solo treatment on the right side of the face for 60 days, while the hydrogel vehicle was applied to the left side of the face as a control. To objectively evaluate the results, a digital photographic database was used to collect images. The number and type of lesions were recorded for each patient, to compare the Global Acne Grading System (GAGS) score before treatment with that obtained at the end of the study. Moreover, with the innovative technique of follicular biopsy, areas of acneic skin were prepared for histopathology. The average area occupied by microcomedones at baseline was compared with that at the end of treatment. Results: HPLC analysis demonstrated that resveratrol, upon incorporation into the gel, did not convert to its cis-isomer when stored at 4°C for 30 days. All patients were satisfied with the active treatment and none experienced adverse effects. Clinical evaluation showed a 53.75% mean reduction in the GAGS score on the resveratrol-treated sides of the face compared with 6.10% on the vehicle-treated sides of the face. These data were supported by histologic analysis, which showed a 66.7% mean reduction in the average area of microcomedones on the resveratrol-treated sides of the face. The comparison with the vehicle-treated side of the face (9.7% reduction) showed a clinically relevant and statistically significant decrease of lesions in areas treated with resveratrol-containing hydrogel. Conclusion: This pilot study showed positive results for resveratrol gel in acne, and should be considered a valid starting point for further testing of the effectiveness of this molecule in different concentrations and formulations and in a larger group of patients.

FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program.

FK506-binding protein 51 is a possible novel tumoral marker

Dear Editor, FK506-binding protein (FKBP) 51 is a cochaperone, which belongs to the immunophilin family, a group of proteins with peptidyl-prolyl isomerase activity. FKBPs regulate several biological processes through protein–protein interaction.1 In particular, FKBP51 is a component of the steroid receptor complex, with a role in steroid resistance;2 moreover, it is involved in NF-κB activation because of its isomerase activity, which is essential for the function of subunit-α in the IκB kinase complex.3 According to Baughman et al.,4 FKBP51 is abundantly expressed in lymphocytes and in several other tissues, but it is expressed at low levels in the pancreas, spleen, and stomach. There is increasing evidence of an association of FKBP51 hyperexpression with cancer6, 7, 8 and a relevant role of this protein in sustaining cell growth,5 malignancy, and resistance to therapy.6, 7, 8 An immunohistochemistry study of expression of FKBP51 in 50 tumoral samples acquired from our pathology section, including breast, lung, pancreas, ovary, and prostate (10 samples for each tumor), and a comparable number of normal tissue samples showed an intense signal in 38 out of 50 tumors analyzed, whereas normal tissues of the same histotypes showed a weak/absent immunohistochemical signal. The 12 tumor samples with low/negative immunohistochemistry were the 10 breast cancer samples and 2 out of 10 pancreatic tumors. Interestingly, these two pancreatic tumors belonged to the well-differentiated histotype (G1). Figure 1a (upper panel) shows representative immunohistochemical findings. Measurement of FKBP51 mRNA levels in deparaffinized tissues using real-time PCR confirmed the immunohistochemistry results (representative results in Figure 1a, lower panel). Taken together, these findings support the hypothesis that FKBP51 is a promising novel tumoral marker. The association of FKBP51 overexpression with cancer is in line with the rapidly emerging concept that NF-κB drives tumorigenesis in the most common genetic alterations associated with cancer.9, 10 Figure 1 (a) (Upper panel) FKBP51 immunochemical staining of normal and neoplastic tissues. Serial sections of 4 μm from routinely formalin-fixed, paraffin-embedded blocks were cut and mounted on poly--lysine-coated glass slides. (Lower panel) ... Recently, it has been found, in tumor cell lines, that FKBP51 acted as a scaffold to facilitate the interaction between Akt and PH domain leucine-rich repeat protein phosphatase, which mediates dephosphorylation of pAkt (S473).11 This raised the question whether FKBP51 may work out as a tumor suppressor by deactivating Akt. As we previously found that intratumoral injection of FKBP51 siRNA followed by irradiation produced extensive apoptosis in melanoma xenografts,8 we investigated the effect of FKBP51 downmodulation on pAkt (S473) levels in our mouse model of melanoma, in both xenografts and locoregional lymph nodes. Histological examination showed that the mouse lymphatic tissue was not metastatic. Figure 1b shows a representative outcome. On the basis of these results, pAkt (S473) levels were not enhanced with downmodulation of FKBP51 in normal or cancerous tissue. This observation suggests that pathways upstream from Akt, which are often deregulated in cancer, control activated-Akt levels. This hypothesis is supported by the findings that leukemic lymphocytes often display higher levels of pAkt (S473) in comparison with normal lymphocytes (Figure 1c) even in the presence of similar or higher FKBP51 levels. To assess whether an inverse correlation subsisted between FKBP51 and pAkt (S473) levels, we used western blot to measure these levels in samples of primary lymphatic leukemia and normal peripheral blood lymphocytes. Expression levels were quantified by densitometry. Spearmans ρ correlation did not indicate any relationship between the two variables (Figure 1d; P=0.788, left; P=0.199, right). Taken together, these findings do not support an essential role of FKBP51 as a factor that controls the phosphorylation status of Akt inside the cell, thereby weakening the hypothesis that the protein may work out as tumor suppressor. In conclusion, although FKBP51 function is not yet fully elucidated, however, there are clear data suggesting that this immunophilin is often hyperexpressed in tumors and has an active role in pre-neoplastic disorders5 and cancer.6, 7, 8

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis.

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies.

Overexpression of chromatin assembly factor-1 (CAF-1) p60 is predictive of adverse behaviour of prostatic cancer

Aims:  Prostatic cancer may remain organ‐confined indefinitely; in a number of patients, however it gives rise to clinical symptoms and death. The biological behaviour of this tumour mostly remains difficult to predict. A promising tool for diagnosis and prognosis of some human tumours is the chromatin assembly factor‐1 (CAF‐1), involved in the control of higher order chromatin organization. The aim was to explore the role of CAF‐1/p60 protein as a new prognostic marker for prostatic cancer.

BAG3 protein delocalisation in prostate carcinoma

Despite the progressive increase of early diagnosis, a subset of prostate cancers show a metastasizing and lethal course, not always predictable upon the traditional prognostic parameters. The object of this study was to investigate the role of the survival co-chaperone protein BAG3 as a new prognostic marker for prostate cancer. BAG3 was detected by immunohistochemistry in 55 specimens of surgically removed prostate carcinomas and in 15 surgical specimens of non-neoplastic prostate tissues. Results were compared with clinic-pathological data and outcome of patients and statistically evaluated. BAG3 resulted expressed in all the cases: Non-neoplastic prostate tissue showed a cytoplasmatic staining with apical reinforcement, a finding which appears consistent with the reported connection of the protein with the membrane focal cell–adhesion complexes. In prostate carcinomas, BAG3 showed a progressive decrease of the expression level from well- to low-differentiated carcinoma, coupled with the loss of polarisation of the signal in metastasizing cases. These results indicate that BAG3 intra-cytoplasmic delocalisation is a specific feature of cancer versus non-neoplastic prostate and a candidate new marker for prediction of prostate cancer invasiveness and behaviour.

New therapeutic perspectives in CCDC6 deficient lung cancer cells

Non‐small cell lung cancer (NSCLC) is the main cause of cancer‐related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled‐coil‐domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.

Histopathological Determinants of Tumor Resistance: A Special Look to the Immunohistochemical Expression of Carbonic Anhydrase IX in Human Cancers

Intrinsic and acquired drug resistance of tumor cells still causes the failure of treatment regimens in advanced human cancers. It may be driven by intrinsic tumor cells features, or may also arise from micro environmental influences. Hypoxia is a microenvironment feature associated with the aggressiveness and metastasizing ability of human solid cancers. Hypoxic cancer cells overexpress Carbonic Anhydrase IX (CA IX). CA IX ensures a favorable tumor intracellular pH, while contributing to stromal acidosis, which facilitates tumor invasion and metastasis. The overexpression of CA IX is considered an epiphenomenon of the presence of hypoxic, aggressive tumor cells. Recently, a relationship between CA IX overexpression and the cancer stem cells (CSCs) population has been hypothesized. CSCs are strictly regulated by tumor hypoxia and drive a major non-mutational mechanism of cancer drug-resistance. We reviewed the current data concerning the role of CA IX overexpression in human malignancies, extending such information to the expression of the stem cells markers CD44 and nestin in solid cancers, to explore their relationship with the biological behavior of tumors. CA IX is heavily expressed in advanced tumors. A positive trend of correlation between CA IX overexpression, tumor stage/grade and poor outcome emerged. Moreover, stromal CA IX expression was associated with adverse events occurrence, maybe signaling the direct action of CA IX in directing the mesenchymal changes that favor tumor invasion; in addition, membranous/cytoplasmic co-overexpression of CA IX and stem cells markers were found in several aggressive tumors. This suggests that CA IX targeting could indirectly deplete CSCs and counteract resistance of solid cancers in the clinical setting.