Expression profile of epidermal differentiation complex genes in normal and anal cancer cells.

Anal cancer originates from a peculiar histological region and provides a useful model for investigating alterations in proliferation and/or differentiation of neoplastic keratinocytes. Epidermal differentiation complex (EDC) genes, which form one of the major gene clusters in the human genome, are involved in the terminal differentiation of epithelial cells and in many instances have been implicated in epithelial tumours. We constructed a DNA macroarray capable of characterising the expression profiles of the entire EDC gene complex in normal mucosa and anal cancer biopsies of seven unrelated patients. Brain tissue and cultured keratinocytes were used as controls. All anal cancer samples showed expression profiles in which none of the EDC genes was silent, as evaluated by phosphor-imager analysis. Variance analysis showed significantly lower expression of SPRR2 with respect to SPRR1 or SPRR3, and significantly higher expression of S100A8 than of other S100A subfamily members. At hierarchical clustering analysis, the four basaloid anal cancer cases conglomerated in the top five positions. The macroarray method used by us provides the first demonstration of the expression profile of the EDC gene family in anal cancer, and is capable of producing significant information on the subgrouping of epithelial tumours such as anal cancer.

Biodistribution of charged 17.1A photoimmunoconjugates in a murine model of hepatic metastasis of colorectal cancer.

Optimizing photodynamic therapy involves attempting to increase both the absolute tumour content of photosensitizer and the selectivity between tumour and surrounding normal tissue. One reason why photodynamic therapy has not been considered suitable for treatment of metastatic tumours in the liver, is the poor selectivity of conventional photosensitizers for tumour compared to normal liver. This report details an alternative approach to increasing this selectivity by the use of antibody-targeted photosensitizers (or photoimmunoconjugates) to target intrahepatic tumours caused by human colorectal cancer cells in the nude mouse, and explores the role of molecular charge on the tumour-targeting efficiency of macromolecules. The murine monoclonal antibody 17.1A (which recognizes an antigen expressed on HT 29 cells) was used to prepare site-specific photoimmunoconjugates with the photosensitizer chlorine6. The conjugates had either a predominant cationic or anionic charge and were injected i.v. into tumour-bearing mice. Biodistribution 3 or 24 h later was measured by extraction of tissue samples and quantitation of chlorine6 content by fluorescence spectroscopy. The photoimmunoconjugates were compared to the polylysine conjugates in an attempt to define the effect of molecular charge as well as antibody targeting. The anionic 17.1A conjugate delivered more than twice as much photosensitizer to the tumour at 3 h than other species (5 times more than the cationic 17. 1A conjugate) and had a tumour:normal liver ratio of 2.5. Tumour-to-liver ratios were greater than one for most compounds at 3 h but declined at 24 h. Tumour-to-skin ratios were high (> 38) for all conjugates but not for free chlorine6. Cationic species had a high uptake in the lungs compared to anionic species. The photoimmunoconjugates show an advantage over literature reports of other photosensitizers, which can result in tumour:normal liver ratios of less than 1.

Experimental photoimmunotherapy of hepatic metastases of colorectal cancer with a 17.1A chlorin(e6) immunoconjugate.

Photoimmunotherapy (using a monoclonal antibody-targeted photosensitizer and red light) may be a strategy to overcome the limitations inherent in photodynamic therapy of liver tumors. The aims of this study were (a) to test the efficacy of selective treatment of hepatic metastases of colorectal cancer in an orthotopic murine xenograft using the murine monoclonal antibody 17.1A conjugated to the photosensitizer chlorin(e6), and (b) to compare the tumor response after the same light dose was delivered at two different fluence rates. Based on previous biodistribution studies that had shown that the photoimmunoconjugate with a polyanionic charge had both a higher absolute tumor chlorin(e6) content and a greater tumor:normal liver ratio than those obtained with a photoimmunoconjugate bearing a polycationic charge, mice were treated 3 h after i.v. injection of the polyanionic 17.1A chlorin(e6) conjugate or unconjugated photosensitizer. Red light was delivered into the liver tumor by an interstitial fiber, and tumor response end points were total tumor weight in the short term and survival in the long term. There was a highly significant reduction (<20% of controls; P = 0.0035) in the weight of the tumors in the mice treated with photoimmunotherapy, and the median survival increased from 62.5 to 102 days (P = 0.015). Photodynamic therapy with free chlorin(e6) produced a smaller decrease in tumor weight and a smaller extension of survival, neither of which were statistically significant. A comparison of photoimmunotherapy with 10 J of light delivered at 30 or 300 mW showed that the higher fluence rate prolonged survival significantly more than the lower fluence rate. This may have been because the high fluence rate gave a contribution of laser-induced hyperthermia to the photodamage. Correlation studies showed that the amount of normal liver remaining at necropsy correlated best with survival. Photoimmunotherapy shows efficacy in destroying liver tumors, and future studies should maximize selectivity to minimize the destruction of normal liver.

Expression analysis and mutational screening of the epithelium-specific ets gene-1 (ESE-1) in patients with squamous anal cancer.

To investigate whether ESE-1 gene abnormalities are involved in alterations of epithelial cell differentiation in squamous anal cancer ESE-1 expression and structure were screened in six patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and automated sequence analysis. The complete cDNA of isoform ESE-1b was always expressed and correctly spliced, with single nucleotide polymorphism being observed in two cases. Presence of ESE-1b point mutations was excluded. Expression of SPRR2A and ENDOA/CK8, two epithelium-specific ESE-1 target genes, were revealed by RT-PCR in all cases. This first report of expression of ESE-1, and of SPRR2A and ENDOA/CK8 (both related to terminal differentiation in different types of epithelia lining) in anal cancer excludes the hypothesis that these genes influenced carcinogenesis in our patients. Despite selecting of patients without clinical evidence of HPV infection, PCR consistently revealed HPV-16 DNA, highlighting the importance of HPV infection in anal cancer.

Targeted photodestruction of human colon cancer cells using charged 17.1A chlorin e6 immunoconjugates.

The goal of this study was to develop a strategy for the selective destruction of colorectal cancer cells. Towards this end, photoimmunoconjugates were prepared between the anti-colon cancer monoclonal antibody 17.1A and the photosensitizer (PS) chlorin(e6) (c(e6)). Polylysine linkers bearing several c(e6) molecules were covalently attached in a site-specific manner to partially reduced IgG molecules, which allowed photoimmunoconjugates to bear either cationic or anionic charges. The conjugates retained immunoreactivity as shown by enzyme-linked immunosorbent assays and by competition studies with native antibody. The overall charge on the photoimmunoconjugate was an important determinant of PS delivery. The cationic photoimmunoconjugate delivered 4 times more c(e6) to the cells than the anionic photoimmunoconjugate, and both 17.1A conjugates showed, in comparison to non-specific rabbit IgG conjugates, selectivity for antigen-positive target cells. Illumination with only 3 J cm(-2) of 666 nm light reduced the number of colony forming cells by more than 90% for the cationic 17.1A conjugate and by 73% for the anionic 17.1A conjugate after incubation with 1 microM c(e6) equivalent of the respective conjugates. By contrast, 1 microM free c(e6) gave only a 35% reduction in colonies. These data suggest photoimmunoconjugates may have applications in photoimmunotherapy where destruction of colorectal cancer cells is required.

Giant retroperitoneal angiomyolipoma. A case report.

We discuss a case of a giant renal angiomyolipoma. A 28 year-old woman presented with a history of a painless and swelling abdominal mass. A computerized tomography (CT) scan of the abdomen demonstrated a 27 x 13 x 11 cm tumor in the right retroperitoneum. At laparotomy, a wide excision of the mass was performed and the histopathological examination of the resected specimen revealed the presence of an angiomyolipoma. The clinical, radiological and pathological findings of this case are reported with a review of the literature.

[Transanal ultrasonography in the study of fistulas of perianal abscess]. / L'ecografia transanale nello studio delle fistole degli ascessi perianali.

The fistulous tracks and abscesses may be related anatomically to the anal sphincters and levator ani muscles. Defining the anatomical relationship between the fistulous lesion and the anal sphincters and/or adjacent organs is essential for correct management. Clinical examination can suggest possible sepsis or the direction of the fistulous track. We report the results of anal endosonography in the evaluation of 54 patients affected by perianal sepsis and fistula in ano. Anal endosonography has correctly identified 22 of 22 internal openings seen at the surgery, 14 of 16 intersphincteric tracts while only 7 of the 9 trans-sphincteric tracks described by anal endosonography were present at the surgery. All the abscesses were correctly identified. Anal endosonography provides a high-resolution image of the anatomy of anal canal and defines the anatomy of tracks and abscesses in relation to the sphincters, determines if there has been trans-sphincteric extension of the sepsis and assesses the state of the sphincters. We have observed a good correlation between the preoperative AES findings and those at surgery. We conclude that anal endosonography is a useful diagnostic procedure in patients with perianal sepsis.