Molecular and genomic characterisation of a panel of human anal cancer cell lines.

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

Rhabdoid carcinoma of anal canal: role of electron microscopy and immunohistochemistry in establishing lineage.

ABSTRACT Rhabdoid carcinoma is a high-grade carcinoma with rhabdoid features and it is different from rhabdoid tumors that are broadly defined as malignant neoplasms with rhabdoid cellular appearance found primarily in the pediatric population, but adult cases have been reported in many anatomic locations. To date, no cases of anal canal rhabdoid carcinoma have been reported in the adult or pediatric population. We are reporting the first case of anal canal rhabdoid carcinoma, found in a 75-year-old male. We utilized ultrastructural as well as immunohistochemical studies to arrive at our diagnosis. Ultrastructural studies demonstrated the intermediate filament congregating to impart a rhabdoid appearance to tumor cells, and cytokeratin intermediate filaments and short microvilli indicating nature of tumor as carcinoma. Immunohistochemical phenotype showed neoplastic cells were positive for vimentin, pan-cytokeratin AE1/3, p63 and D2-40, which supports the genesis of tumor from skin adnexa. Even in the modern era of surgical pathology that routinely utilizes immunohistochemistry and molecular studies, adequate use of electron microscopy to help pinpoint the diagnosis in challenging cases is important.

Fibroepithelial polyps of the anus: a histologic, immunohistochemical, and ultrastructural study, including comparison with the normal anal subepithelial layer.

Fibroepithelial polyps of the anus (FEPA) are relatively common lesions that have attracted little attention. A series of 40 FEPA, together with sections from normal anal mucosa, were studied histologically, histochemically, immunohistochemically, and ultrastructurally. The polyps consisted of myxoid or collagenous stroma covered by squamous epithelium. Stromal cells with two or more nuclei were found in 30 polyps (80%). In five cases (12%) these cells showed atypical nuclear features. All the polyps harboring atypical cells were of large size. Mast cells were frequently present and sometimes intimately related to the stromal cells. Stromal cells stained positive for vimentin and negative for actin. Desmin was expressed in 30% of the cases. Electron microscopic examination confirmed the fibroblastic and myofibroblastic nature of the stromal cells. Examination of the subepithelial connective tissue from the normal anal mucosa showed bizarre multinucleated cells and mast cell infiltration. It is concluded that FEPA are benign lesions characterized by the presence of mononucleated and multinucleated, sometimes atypical stromal cells showing fibroblastic and myofibroblastic differentiation. The morphologic similarity between FEPA and normal anal mucosa supports the hypothesis that FEPA may represent a reactive hyperplasia of the subepithelial connective tissue of the anal mucosa. Mast cells, by means of their fibrogenic, fibrolytic, and angiogenic activities may play an important role in the pathogenesis of FEPA.

Perianal ependymoma presenting in the neonatal period.

Extraspinal ependymomas are a rare type of glioma that may arise in the sacrococcygeal region, presenting as a pelvic mass in an infant or child. Ependymoma presenting in the newborn period has not been described previously. Herein we describe a case of a newborn boy who presented with a perianal ependymoma, which was subsequently found to have presacral extension. The major diagnostic challenge this case presented was to rule out the alternative diagnosis of sacrococcygeal teratoma or a developmental malformation/heterotopia.

Clear-cell carcinoma of the anal canal: a variant of anal transitional zone carcinoma.

Optically clear cytoplasm in tumor cells in the absence of mucin is most often associated with renal cell carcinoma. However it is important to recognize the rare occurrence of "clear-cell" variants among tumors arising in other sites. This report describes a clear-cell carcinoma arising in the transitional zone of the anal canal.

[The epithelial junctional zones of the anal canal and cervix uterus: the ultrastructure of tumors of these zones]. / Epitel'ial'nye stykovye zony anal'nogo kanala i sheiki matki: ul'trastruktura nekotorykh opukholei étikh zon.

Relationships between squamous and columnar epithelia in the anal canal and cervix uteri of postnatal period and fetus were studied. The transitional stratified epithelial lining, which is called junctional epithelium, is interposed between the mentioned epithelia. The junctional epithelium has variable numbers of layers of epidermoid cells, which differ from cells of atypical squamous epithelium by some ultrastructural features of the cytoskeleton and cell surface and by a low content of glycogen. The hypothesis on the physiological significance of this epithelium is proposed. Ultrastructural features of the cytoskeleton and cell surface suggest that anal basaloid carcinomas and some cervical squamous carcinomas may develop from so-called junctional epithelium.

[A rare complication of ulcerative colitis: Bowen's disease in perianal condyloma acuminatum]. / Colitis ulcerosa ritka szövödménye: Bowen-kór, perianalis condyloma acuminatumban.

Discharging, itching perianal pointed condyloma acuminatum in a 35 years old male patient who had been suffering from ulcerative colitis for 20 years. Two years following its development the condyloma was excised. Histological examination revealed in the perianal condyloma in situ cancer, characteristic of Bowen's disease. With electronmicroscopic examination the tumor cells were found to be keratinocytes of medium differentiation. Virus could not be detected in the tumor cells. The author was the first to describe in the literature the development of perianal Bowen's disease on the ground of ulcerative colitis. On the basis of literary data the author emphasizes the importance of the radical removal and regular control.

Apoptosis in Bowen's disease. An ultrastructural study.

We performed ultrastructural studies of apoptosis (previously referred to as "malignant dyskeratosis") in a case of genital Bowen's carcinoma in which human papillomavirus (HPV) type 33 genome was identified and in two cases of cutaneous Bowen's disease with no detectable viral DNA; herein we present the sequential stages in the development of apoptotic bodies. The apoptotic process in the HPV-containing genital Bowen's disease was similar to that in the cutaneous lesions with no detectable HPV. The presence of a large number of apoptotic bodies in Bowen's disease may be responsible for the slow progression and noninvasive growth of this carcinoma in situ.

[Primary anorectal malignant melanoma--report of 7 cases and review of literature].

From 1956 to 1985, 7 patients with primary anorectal malignant melanoma were studied by clinicopathology and observed by light and electron microscopes. The sex ratio of male and female was 2:5. The mean age was 59.4 years. The main presenting symptoms were bloody stool and a palpable mass in the anus. Because these symptoms are non-specific, early diagnosis is very difficult. The disease course ranged from 1.5 to 21.5 months with an average of 6.7 months. At present, the combination treatment is advocated, and radical operation is still the treatment of choice. This tumor has a very poor prognosis. Grossly, the tumor often showed nodular or polypoid shape. Histologically, there were various forms, such as epithelial, spindle and balloon-like cells etc.. Ultrastructurally, melanin corpuscles and vimentin (intermediate filaments) were found in all. In this paper, pathogenesis, morphology and prognosis are discussed.

[A case of perineal (extramammary) Paget's disease].

A case of perineal Paget's disease in a 67-year-old male patient is reported. The perineal polyp, having shown no increase in size for 3 years, wks resected. The microscopic features of the polyp was comprised of clusters of a pale, large tumor cell (Paget cell) only in the epidermis. The skin that was resected around the polyp showed almost the same histological appearance as the polyp, except for an infiltration of tumor cells forming a small gland structure on the minimal region of the dermis. Both the Paget's cells and these tumor cells showed positive for mucin staining. Ultrastructurally, we confirmed two types of Paget's cells, one containing much granules and the other lacking in these granules but rich in other organelles. Further, the Paget's cells formed lumen and intercellular canaliculus, suggesting a differentiation to the eccrine sweat gland. From our findings and a review of the literature, we favor presuming that extramammary Paget's disease without a cancer of the neighbouring organs originates from the epidermis and extends to the dermis.

Mammary and extramammary Paget's disease. An immunocytochemical and ultrastructural study.

Twenty-one cases of Paget's disease have been studied using histochemical, ultrastructural, and immunohistochemical methods. Eight of the tumors involved the nipple, and 13 were extramammary (11 vulvar and two anal). The antibodies used were directed against different classes of cytokeratin proteins, epithelial membrane antigen, carcinoembryonic antigen, gross cystic disease fluid protein-15, and S-100 protein. The findings of this study provide conclusive evidence that Paget's cells, regardless of their location, are adenocarcinoma cells. Intracytoplasmic mucin is scanty in Paget's cells within the nipple, but typically plentiful in the extramammary sites where the cells are frequently signet-ring cells. The common mechanism for the evolution of Paget's disease is extension of cells from an underlying carcinoma, but the possibility that some cases, particularly in the vulva, develop from intraepithelial precursors cannot be excluded.

Spindle cell carcinoma (pseudosarcoma) of the anus: a light, electron microscopic and immunocytochemical study of a case.

We report a case of polypoid spindle cell squamous carcinoma (pseudosarcoma) occurring in the anal canal. Electron microscopic findings and the demonstration of keratin by an immunoperoxidase method, gave clear cut evidence of the epithelial nature of the sarcomatoid cells forming this tumour. To our knowledge, this is the first reported case in the literature.

Basal cell carcinoma of the anus: clinical and pathological distinction from cloacogenic carcinoma.

A 54-year-old man developed basal cell carcinoma of the anal verge. This extremely rare tumor behaves rather innocently, whereas cloacogenic carcinoma of the anus, which resembles it histologically, metastasizes early and often proves fatal. Clinical and histopathologic characteristics of the two anal neoplasms are tabulated to facilitate differential diagnosis, and thereby promote their very different therapies.

Intraepithelial carcinoma of the anus in homosexual men.

Anal warts (condylomata acuminata) from seven homosexual men revealed intraepithelial carcinoma (carcinoma in situ) within the condylomatous tissue or in adjacent anal mucosa. All lesions displayed morphologic evidence of papillomavirus infection and two of the seven revealed histologic changes characteristic of herpes simplex infection. This association of viral infection with malignant transformation indicates that persistent or recurrent anal warts should be excised and thoroughly examined by histologic techniques. Since four of the seven patients had histories suspicious for or diagnostic of the acquired immunodeficiency syndrome (AIDS), we further suggest that homosexual men with persistent or recurrent perianal lesions be evaluated for the presence of the syndrome.

[Multiple papilloma virus particles in perianal condylomata acuminata of a drug-dependent patient]. / Multiple papillomviruspartikel in perianalen Condylomata acuminata eines Drogenpatienten.

A 24-year-old male drug-dependent patient with perianal exophytic condylomata acuminata is reported. An increased production of papillomaviruses could be demonstrated by means of electron microscopy and immunocytochemistry (peroxidase-antiperoxidase technique). This is in contrast to earlier investigations of exophytic condylomata acuminata, which revealed characteristically small amounts of mature papillomavirus particles. The basis for the different form of papillomavirus infection of the anogenital region in this drug-dependent patient is discussed.

An unusual type of cytokeratin filament in cells of a human cloacogenic carcinoma derived from the anorectal transition zone.

Epithelia-derived tumors (carcinomas) can be distinguished from mesenchymally derived tumors by the presence of intermediate-sized filaments of the cytokeratin type, which usually coincides with the absence of other types of intermediate-sized filaments such as vimentin filaments. In the course of diagnostic examinations of human tumors, using immunofluorescence microscopy, we have come across a case of an unusual carcinoma (Primary tumor and lymph node metastasis) positively stained not only with cytokeratin antibodies but also with immunoglobulins present in vimentin antisera. Therefore, this tumor, a cloacogenic carcinoma apparently derived from the rectal-anal transitional region, has been examined in greater detail using both immunofluorescence microscopy and immuno-electron microscopy as well as gel electrophoretic analysis of cytoskeletal polypeptides from total tumor tissue and from microdissected nodules enriched in carcinoma cells. The unusual reaction of the carcinoma cells with immunoglobulins present in seven different (rabbit or guinea pig) antisera raised against vimentin, has been found to be diminished after absorption on purified cytokeratin or total epidermal cytoskeletal material, but not after absorption on purified vimentin. Gel electrophoretic analysis of tumor cytoskeletons showed an unusual complex pattern of cytokeratin polypeptides containing relatively large (Mr 68,000 and Mr 58,000) neutral-to-slightly basic cytokeratins, as are typically found in epidermis and other stratified squamous epithelia, as well as several smaller acidic cytokeratins, including a Mr 40,000 polypeptide found in certain nonstratified epithelial such as colon and small intestine. Total tumor also showed the inclusion of some vimentin which, however, was significantly decreased in analysis of excised carcinoma nodules. Examining antibody binding to polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper, we have found that antisera raised against vimentin contained not only vimentin antibodies but also immunoglobulins which specifically bound to the largest cytokeratin component. We conclude that the unusual reaction of immunoglobulins present in vimentin antisera with cytokeratin filament bundles does not represent specific binding to vimentin in these carcinoma cells, but is due to a component obviously widespread in vimentin antisera which binds specifically to a cytokeratin present in this type of tumor but not in most other carcinomas. It is proposed that use is made in diagnostic examinations of vimentin antisera or affinity-purified vimentin antibodies that have been pre-absorbed on cytokeratin protein, in order to eliminate such disturbing reactions.

Inflammatory cloacogenic polyp. A unique inflammatory lesion of the anal transitional zone.

This report describes the clinical and pathologic features of eight cases of an unusual inflammatory polyp arising from the transitional zone of the anus. This lesion has been designated as the inflammatory cloacogenic polyp. Rectal bleeding is the most common presenting clinical symptom. The polyp is usually located on the anterior wall of the anal canal. Morphologically, it is characterized by a tubulovillous pattern of growth, superficial ulceration, displaced groups of crypts into submucosa, and extension of chronically inflamed fibromuscular stroma into the lamina propria. Clinical and morphologic similarities with the solitary rectal ulcer syndrome suggests that prolapse of transitional zone mucosa may be important in its pathogenesis. Simple surgical resection appears to be the treatment of choice.

Melanotic adenocarcinoma of the anorectum.

Epithelial cells of an otherwise typical anorectal mucinous adenocarcinoma contained melanin pigment. Ultrastructurally, three cell populations were present. The largest population contained membrane-bound compound melanosomes in cells having glandular epithelial characteristics including surface specializations, microvilli, and mucin. Additionally, occasional melanocytes were intermixed with the tumor cells and finally very rare cells having both mucin and isolated melanosomes were present. We believe that this phenomenon is better explained by phagocytosis or transfer of melanin from melanocytes to neoplastic cells, rather than by melanin synthesis in adenocarcinoma.