Volume 120, Issue 4, Pages 988-994 (March 2001) Human papillomavirus type 16–associated primary squamous cell carcinoma of the rectum  Karl Sotlar, Gerhard Köveker, Christian Aepinus, Hans–Christoph Selinka, Reinhard Kandolf, Burkhard Bültmann  Gastroenterology  Volume 120, Issue 4, Pages 988-994 (March 2001) DOI: 10.1053/gast.2001.22523 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Squamous cell dysplasia/SCC of the rectum. (A) SCC infiltrating the submucosa (right; H&E; original magnification 60×). (B) Tumor cells with morphologic features reminiscent of koilocytotic differentiation (arrows; H&E; original magnification 115×). (C) Dysplastic squamous epithelium replacing normal columnar epithelium by growing into a mucosal crypt (arrows; H&E; original magnification 230×). (D) Rectal mucosa (right)/squamous cell dysplasia (left) sequence (H&E; original magnification 115×). Gastroenterology 2001 120, 988-994DOI: (10.1053/gast.2001.22523) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 PCR amplification of HPV-16 DNA and cDNA by conventional (left) and nested (right) PCR. Conventional PCR with primer pair S3/S4 yielded specific amplification bands of 525-bp length from DNA obtained from biopsy and surgically resected material from the primary rectal SCC, and a lymph node metastasis (lanes 1–3). HPV-16 DNA could not be amplified in an adenocarcinoma of the rectum (lane 4). Amplification of cDNA from the tumor revealed transcriptional activity of HPV-16 E6/E7 oncogenes by the presence of a 343-bp band corresponding to the E6*I splice product (lane 5). No DNA is present in lane 6. HPV-16 DNA and cDNA extracted from CaSki cells served as positive controls (lanes 7 and 8). Nested PCR amplification with primer pair S1/S2 of cDNA extracted from scrapes and biopsy specimens taken at follow-up 8 months after resection of the tumor revealed transcriptional activity of HPV-16 E6/E7 oncogenes by amplification of a 213-bp band corresponding to the E6*I splice product: penile scrape (lane 9) and biopsy specimens from sigmoid (lane 10) and rectum (lane 11). There was no amplification of HPV-16 cDNA from an anal biopsy specimen taken at the same time (lane 12). No cDNA is present in lane 13. CaSki cDNA served as a positive control (lane 14). M, length marker (HaeIII-digested ΦX DNA). Gastroenterology 2001 120, 988-994DOI: (10.1053/gast.2001.22523) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 HPV-16 nonisotopic DNA in situ hybridization. Strong, mostly dotted, nuclear signals are confined to the tumor cells of the SCC (arrows). Adjacent normal mucosa shows no HPV-16 hybridization signals (lower right; original magnification 115×). Gastroenterology 2001 120, 988-994DOI: (10.1053/gast.2001.22523) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Occult tumor metastasis. Immunostaining for cytokeratin reveals a metastatic single tumor cell (arrow) in a pararectal lymph node that was shown to be HPV positive by nested PCR (antibody KL1; Coulter Immunotech, Hamburg, Germany; original magnification 230×). Gastroenterology 2001 120, 988-994DOI: (10.1053/gast.2001.22523) Copyright © 2001 American Gastroenterological Association Terms and Conditions