Alessandra Filosa

Long-term experience with low-dose interferon-α and PUVA in the management of early mycosis fungoides

Abstract:  Objectives: Combined high‐dose Interferon‐α and psoralen plus ultraviolet A irradiation (PUVA) have been reported to be effective in the treatment of early mycosis fungoides (MF); however, our study is the first controlled prospective study in the literature exploring the activity and tolerability of the combination with low dosages and evaluating further clinical outcome of early‐MF patients. Methods: We carried out a multicentric prospective Phase II clinical study on 89 patients with early‐stage IA to IIA MF treated for 14 months with low‐dose IFN‐α2b (6–18 MU/wk) and PUVA. Treatment success was analysed in terms of freedom from treatment failure. Results and conclusions: Complete remission (CR) was achieved in 84% and an overall response rate in 98% of cases: six‐month CR was associated with a non‐confluent skin infiltrate at histology (P = 0.044) and 14‐month CR with high epidermal CD1a+ dendritic‐cell density (P = 0.030). The combination protocol was successfully tolerated and the most common reason of ‘failure’ was related to relapse and not to toxicity. Sustained remissions were achieved in 20% of patients. High CD8+ lymphoid T‐cell density was associated with a lower relapse rate (P = 0.002). We think that our combination therapy can be considered an alternative approach compared with other modalities. Good immunological host surveillance in the skin lesions seems to be an optimal basis for the therapeutic success.

Prognostic role of tumor necrosis, microvessel density, vascular endothelial growth factor and hypoxia inducible factor-1alpha in patients with clear cell renal carcinoma after radical nephrectomy in a long term follow-up.

Angiogenesis is a critical step in the growth, invasive progression and metastatic spread of solid tumors. We investigated the importance of tumor necrosis, and microvessel density (MVD), vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) immunohistochemical expression in a large series of clear cell renal carcinomas treated with radical nephrectomy and assessed the prognostic value of their expression in terms of patient survival at long-term follow up. Fifty patients with clear cell RCC were examined. The features considered when evaluating the patients were age, tumor size and grade, intratumoral vascular and renal capsula invasion, histological necrosis, and MVD, vascular and tumoral cell VEGF, and vascular, tumoral cytoplasmic and nuclear HIF-1α expression on the histologic specimens. All considered parameters were correlated with patient specific survival. Mean age was 62.06 ± 6.8 years. Median follow-up was 191.66 months; median survival was 120.86 months. Twenty-one patients developed metastases in the follow-up. Tumor necrosis, microvascular invasion and renal capsula infiltration are more likely to occur in high stage and grade RCC; cytoplasmic HIF-1α is highly expressed in high grade RCC. Survival is dependent upon tumor stage and grade, the presence of intratumoral vascular invasion and capsular infiltration, and tumor necrosis; MVD also resulted as being an important prognostic factor. VEGF and HIF-1α correlate with prognosis in high stage tumors where VEGF is the most important independent prognostic factor for cancer specific death. The histological and immunohistochemical parameters considered in our study can influence disease recurrence and survival in RCC.

Prognostic role of global DNA‐methylation and histone acetylation in pT1a clear cell renal carcinoma in partial nephrectomy specimens

Surgery is the main treatment for renal cell carcinoma (RCC); nephron sparing surgery can be performed as a treatment of choice for small peripheral lesions. Epigenetics configures a new entity that regulates gene expression throughout methylation, acetylation and chromatin remodelling. In addition to silencing as a result of mutations, loss of heterozygosity, or classic genetic events, epigenetic modification symbolizes essential events during carcinogenesis and tumour development. We investigated global methylation and histone acetylation expression in a series of small conventional clear cell renal carcinomas (i.e. less than 5 cm) (pT1a) treated with partial nephrectomy, to assess their possible role as diagnostic biomarkers. A total of 54 patients with conventional single RCC were selected and treated with partial nephrectomy; they were followed up to 186 months. Immunohistochemistry was performed on paraffin‐embedded sections, using anti‐5‐methylcytosine (5mc) and anti‐Acetyl‐Histone H3 (Lys 9). Our results confirm that the mean percentage of global cellular methylation in tumoural tissue was significantly higher compared to healthy peritumoural tissue, whereas the mean percentage of histone cellular acetylation in tumoural tissue was significantly lower. The percentage of methylation was significantly higher in grades 3 and 4 (P= 0.033), whereas the percentage of histone acetylation was significantly lower (P= 0.023), suggesting therefore that these markers could correlate with tumour aggressiveness in pT1a RCC. On univariate analysis of patient survival in relation to the different considered factors, Fuhrman grade was the most important survival factor. These epigenetic markers can give us interesting information about chromatin remodelling in RCCs; the percentage of global methylation increases with increasing Fuhrman grade, whereas histone acetylation decreases with increasing grade in small RCC; our results suggest that global hypermethylation and histone hypoacetylation can be assumed to be an early event in RCC and to correlate with tumour aggressiveness.

Bone marrow histopathological and molecular changes of small B-cell lymphomas after rituximab therapy: comparison with clinical response and patients outcome.

This study correlates bone marrow changes after Rituximab (RTX) treatment with the clinical characteristics and outcome of 26 patients with small B-cell lymphomas. The percentage, phenotypic profile and clonality pattern of bone marrow lymphoid infiltrate were analysed before and after RTX treatment. Clinical, histological and molecular responses to RTX were correlated to the clinical outcome of the patients. Sixteen out of twenty-six patients obtained a complete clinical remission (CR). A favourable histology - follicular lymphoma (FL), hairy cell leukaemia (HCL) and marginal zone lymphoma (MZL) - was associated with a higher frequency of clinical CR and histological remission (HR), in comparison with mantle cell lymphoma (MCL), chronic lymphocytic leukaemia (CLL) and lymphoplasmacytic lymphoma (LPL). Two patterns of bone marrow HR were observed: 1) complete lymphoid cell disappearance (9 patients); or 2) nodular/interstitial T-cell infiltration (10 patients). Three histological persistence (HP) patterns were observed: 1) persistence of CD20+ small lymphoid cells in 1 patient with MCL; 2) loss of CD20 antigen expression in 4 patients with CLL; or 3) persistence only of clusters of monotypic plasma cells in 2 patients with LPL. CR and HR were strongly correlated. The percentage of lymphomatous infiltrate after RTX was higher in patients who subsequently died of the disease. Molecular response showed no correlations with the further clinical course in 12 patients achieving a complete clinical remission. In conclusion, bone marrow morphological and immunohistochemical analysis with a restricted panel of antibodies is useful to avoid 42% false positive and 85% false negative interpretations. Persistence of monoclonality after RTX might have a role in evaluating the molecular pattern of CD20-negative clones that can emerge after RTX as a tumoral escape to therapy.

D2-40 immunoreactivity in penile squamous cell carcinoma: a marker of aggressiveness

D2-40 immunohistochemical expression was investigated in tissue specimens from 39 patients with squamous cell carcinoma of the penis who underwent partial or total penectomy between 1987 and 2008. Patient age, tumor size, and grade; D2-40-positive lymphatic vessel density in intratumoral, peritumoral, and normal tissue; cell positivity for D2-40 in intratumoral and normal tissue; and D2-40 staining intensity and distribution were analyzed and correlated with disease-specific survival. Analysis of D2-40-positive lymphatics disclosed that mean lymphatic vessel density was greater in peritumoral tissue than in intratumoral and normal tissue and lower in patients with lymph node metastasis than in those without lymph node metastasis. The receiver operating characteristic curve showed that an intratumoral lymphatic vessel density greater than 2.0 had 83.3% sensitivity and 78% specificity in predicting lymph node metastasis. Analysis of cell immunoreactivity showed cytoplasmic D2-40 positivity in intratumoral and normal tissue in 89.7% and 65.5% of patients, respectively. A strong correlation emerged between grade of cell differentiation and D2-40 immunoreactivity in intratumoral tissue; in particular, 88.9% of tumors with weak podoplanin expression were G1, whereas strong cellular immunoreactivity was detected in 83.3% of G3 patients (P = .003; χ(2) test). A significant correlation was also noted between pattern of reactivity and tumor grade because the basal layer was positive in patients with undifferentiated tumors (100% of G3) and in 72.2% of G1 tumors (P = .021; χ(2) test). D2-40 seems to be a useful marker for the development of node metastasis in squamous cell carcinoma of the penis, although validation in larger series is required to confirm its predictive value.

Nitric oxide synthase expression in rat anorectal tissue after sacral neuromodulation.

BACKGROUND Sacral neuromodulation is becoming established as a valid treatment option for patients with anorectal disorders. Nevertheless, despite its efficacy, little is known regarding its mechanism of action. The purpose of this study was to evaluate whether chronic sacral neuromodulation is able to influence the expression of nitric oxide synthetase (NOS) in the anorectum of rats. MATERIALS AND METHODS Twenty-six female Sprague-Dawley rats were divided into three groups; normal control rats (n = 6); sham treatment (n =10) and group in whom, electrical sacral neuromodulation was performed (n = 10). Bilateral electrode wires were placed in the S1 and electrical stimulation was performed for 14 d. At the end of the procedures the rats were sacrificed, proctectomy was performed, and anorectal specimens were sent to the laboratory for immunostaining with n-NOS and i-NOS. RESULTS In the anal and rectal specimens, n-NOS and i-NOS expression was significantly increased in epithelial and muscle cells after neuromodulation of the anus and rectum of the animals. CONCLUSION Our results showed that this model can be applied in further experimental studies to better understand the mechanism of action of sacral neuromodulation in anorectal disorders.

Do DNA-methylation and histone acetylation play a role in clear cell renal carcinoma? Analysis of radical nephrectomy specimens in a long-term follow-up.

We investigated global methylation and histone acetylation in 50 conventional clear cell renal carcinomas (RCC), treated with radical nephrectomy, to assess their possible role as diagnostic biomarkers. The features considered in this study were patient age, tumor size and grade, percentage and intensity of 5-methylcytosine (5mc) and Acetyl-Histone (Lys 9) expression in tumor tissue. All considered parameters were correlated with patient specific survival. The mean percentage of global cellular methylation in tumoral tissue was significantly higher compared to normal peritumoral tissue (p<0.0001), while the intensity of cellular methylation was significantly higher in normal tissue than in tumoral tissue (p=0.001). The mean percentage of histone cellular acetylation in tumoral tissue was significantly lower compared to normal peritumoral tissue (p=0.0005), while the intensity of mean acetylation in neoplastic tissue was similar to the normal tissue. The percentage of global DNA methylation was significantly higher in grades 3 and 4 tumors (p=0.033). Global DNA methylation and histone acetylation in tumoral tissue did not correlate with survival. Fuhrman grade was statistically significant for prognosis (p=0.031). In conclusion, global hypermethylation and histone hypoacetylation play an important role in RCC carcinogenesis; Fuhrman grade is still considered the most important factor for patient survival; 5mc can have a role as markers of aggressiveness.

Activity and expression of nitric oxide synthase in rat bladder after sacral neuromodulation.

The aim of our study is to investigate the effects of chronic sacral neuromodulation on Nitric Oxide (NO) metabolism in the rat bladder. 26 female Sprangue-Dawley rats were considered: group I, normal control rats; group II, a sham treatment, in whom catheters for electrical stimulation were placed in the S1 foramen bilaterally and left in place for 21 days, without performing neuromodulation; group III in whom electrical sacral neuromodulation was performed for 21 days. Finally a cystectomy was performed and the bladder biopsy specimens were sent for immunostaining with n-NOS and i-NOS. Morphological and immunohistochemical analysis was carried out, and evaluated in urothelial cells, endothelial cells and muscle fibers of the muscularis propria. Differences between the 3 groups were analyzed by Student Newman-Keuls test. We could observe that urothelial and endothelial i-NOS (37.00±4.69 and 59.00±7.42 respectively) and urothelial n-NOS (36.80±7.85) expression are significantly increased in neuromodulated rats, compared to groups 1 and 2 (p < 0.005). In conclusion, the increase of i-NOS expression on endothelial cells after sacral neuromodulation could be in some way related to angiogenetic responses in the microvascular structures; the increase of n-NOS and i-NOS expression on urothelial cells can suggest that NO is able to influence the plasticity of bladder response, inducing the release of messengers within the urothelium. This study can therefore improve our understanding of the mechanisms of sacral neuromodulation on chronic bladder dysfunction; further studies will need to better demonstrate the role of angiogenesis in the bladder after sacral neuromodulation and to investigate the effects of neuromodulation in rats with chronically induced bladder dysfunction.

Severe diarrhoea during Campath-1H treatment for refractory cutaneous T-cell lymphoma

Dear Editor, Alemtuzumab (Campath-1H; Ilex Pharmaceuticals, San Antonio, TX, USA), the monoclonal antibody directed against CD52 [10] introduced recently as firstand secondline therapy for B chronic lymphocytic leukaemia and peripheral T-cell lymphomas, especially the cutaneous types (CTCL), causes a profound and long-lasting depletion of mature B and T lymphocytes, natural killer cells and monocytes [7], as well as neutropaenia [3, 4]. Frequent complications during treatment are infections, which may be life-threatening; detailed recommendations for screening and prophylaxis of infections among treated patients have been recently provided [11]. In studies on alemtuzumab for CTCL [1–3, 5, 6] bacterial complications accounted for 40% of all infectious episodes, including septicaemias, meningitis and pneumonia, occurring early during treatment with alemtuzumab (<8 weeks) or later after treatment in disseminated mycobacterial infection and tuberculosis. Cytomegalovirus (CMV) reactivation was the most common viral infection, followed by herpes simplex virus (HSV) and varicella-zoster virus (VZV); fungal infections were also frequently observed (18%). Diarrhoea is described as a potential complication during therapy with alemtuzumab especially with intravenous infusions [12] and, less frequently, with subcutaneous administration [9]: it is generally mild and self-limiting, recovering after withdrawal of the drug. We herein report a case of a 70-year-old man with a 2-year history of erythrodermic mycosis fungoides treated by several regimens who developed a severe prolonged diarrhoea during treatment with alemtuzumab and anti-infective prophylaxis therapy (valacyclovir and trimethoprim/sulphamethoxazole). Alemtuzumab was administered by subcutaneous injection. The first dose was 3 mg, which was increased to 10 mg on day 3 and to the target dose of 30 mg on day 5. The 30-mg dose was subsequently administered three times weekly for up to 8 weeks when the diarrhoea appeared. Fever was also present for a few days. Biological indices of flogosis were all increased, and CD4 count was low. Stool analysis and hemoculture were not resolutive to show any infectious agent. Investigations for CMV reactivation were also negative. The patient received piperacillin/tazobactam and fluconazole without any relief of diarrhoea. The treatment with alemtuzumab was discontinued to exclude drug toxicity. With time, the patient started to lose weight progressively and showed increased creatininaemia as evidence of renal functionality impairment. After 3 months of progressive wasting syndrome, the patient was submitted to ileal and colonic endoscopy to obtain tissue for histological examination. All biopsy specimens were formalin-fixed and paraffin-embedded. At lower magnification no specific mucosal changes were consistently observed in the colon (Fig. 1a). The crypts were regularly placed and not especially distorted or rich in mitotic figures. There was no cryptitis, and the lamina propria was empty of inflammatory cells. However, at higher magnification, it was evident that the surface epithelium was abnormal: there were foci of epithelial disarray, degeneration and frank necrosis together with scalloped layers of G. Goteri (*) . D. Morichetti . A. Filosa . A. Mandolesi . I. Bearzi Anatomia Patologica, Università Politecnica delle Marche, Ospedali Riuniti di Ancona, Via Conca 71, 60020 Torrette di Ancona, Ancona, Italy e-mail: g.goteri@univpm.it Tel.: +39-071-5964811 Fax: +39-071-889985

Melanoma Diagnosis: The Importance of Histopathological Report

The demand for histopathological diagnosis of pigmented lesions has increased steadily over the last 20 years, partly due to the incidence of melanoma and partly because of increased awareness and referrals from general practitioners. This makes a unified and systematic approach to melanoma histopathology even more important. The histopathological diagnosis of melanocytic lesions is one of the greatest challenges for pathologists. The recognition of a melanocytic tumor as a melanoma is not based on the search of single, objective, and easily reproducible morphological diagnostic features but, instead, it stems from a constellation of diagnostic criteria whose implementation, meaning, and relative weight may vary considerably from one case to another [1]. In the pathological reporting of a primary malignant melanoma, the desirable features include: characteristics of the melanoma, including subtype; whether the lesion has been completely excised; and evaluation of prognostic indicators. This will allow critical clinical decisions in order to avoid local recurrences and possible sources of metastases [1, 2]. There is no single histological criterion that definitely separates benign from malignant melanocytic lesions. Every histological feature that has been reported in melanoma has also been found in benign nevi. Moreover each type of benign nevus may simulate malignant melanoma and vice versa. The histopathological characteristics must be evaluated in conjunction with clinical and macroscopic data, and the histologic features have to be assessed in the clinical context. This stresses the importance of the clinician providing all relevant data in the pathology request form including age, sex, site, pregnancy, history of previous melanomas or nevi in the site of the current lesion or elsewhere, and recent change in the lesion. Accurate diagnosis may sometimes require a close consultation between the Received: December 20, 2017 Accepted: January 8, 2018 Published online: March 13, 2018