Carcinosarcoma of the colon: report of a case with morphological, ultrastructural and molecular analysis

Representative sections of the tumour and all lymph nodes isolated from the surrounding adipose tissue were fixed in 10% buffered neutral formalin, embedded in paraffin and routinely processed. From each block, 5 μm-thick sections were cut and stained with haematoxylin and eosin (H&E).

For immunohistochemical studies, the avidin-biotin peroxidase complex method was used with the following antibodies: cytokeratin 20 (CK20), desmin, vimentin, S-100, c-kit, CD34, sarcomeric actin, smooth muscle actin, osteonectin. The antibodies used are detailed in Table 1.

For ultrastructural examination, small samples were retrieved from paraffin-embedded material, deparaffinised in xylene, rehydrated in ethanol, post-fixed in osmium tetroxyde (OsO₄), dehydrated and embedded in epoxy resin. Ultrathin sections were counterstained in uranile acetate and lead citrate and observed in a Jeol JEM 1010 (Tokyo, Japan) electron microscope operating at 80 kV.

For the molecular detection of somatic genetic alterations, the p53 gene (exons 4 through 9), the c-kit gene (exons 9, 11, 13 and 17) and the EGFR gene (kinase domain, exons 18 through 21) were evaluated by denaturing high-performance liquid chromatography (DHPLC) technique (The WAVE™ Nucleic Acid Fragment Analysis System, Transgenomic Inc., Nebraska, USA), followed by direct sequencing when required. K-Ras gene mutation status was assessed by direct sequencing. Distinct formalin-fixed paraffin-embedded material from both carcinomatous and sarcomatous components of the tumour was isolated by macrodissection. Genomic DNA was extracted by the phenol-chloroform method. Fifty ng of DNA were used for the specific amplification of all the exons listed above, as previously described [5‐7]. After the polymerization reaction, all PCR (Polymerase Chain Reaction) products were visualised on a 2% agarose gel; 10 μl of the PCR product were denatured at 95°C and slowly cooled to room temperature to ensure equimolar homo- and heteroduplexes formation prior being loaded for DHPLC analysis. The running methods used are listed in Table 2. All samples that showed an elution profile different from a wild type control underwent direct sequencing by BigDye^® Terminator v1.1 Cycle Sequencing Kit chemistry, purified by DyeEx 2.0 Spin kit (Qiagen, Hilden, Germany) and analysed by capillary electrophoresis on Abi Prism 310 (Applied Biosystem, Foster City, California, USA). Sequences obtained were aligned with normal sequences and examined for detection of mutations. For allelic status testing, 10 microsatellite markers on chromosomal arms 9p, 11p, 13q, 17q and 18q were investigated (Rb, D9S171, D11S1336, D13S258, D13S317, D13S631, D17S855, D18S35, D18S51 and D18S585). Specific PCRs with fluorescent primers were conducted followed by loss of heterozygosis (LOH) analysis with GeneScan software (Applied Biosystem, Foster City, California, USA).

The surgical specimen included a 4 cm long ileal segment, a 13.5 cm long colonic segment, and the caecal appendix. At the ileocaecal valve, on the colonic side, an exophytic, centrally ulcerated mass, measuring 7 cm in its greatest dimension, was documented. Twenty-two lymph nodes were isolated from the adipose tissue surrounding the bowel wall.

Microscopically, about 70% of the tumour displayed features of moderately differentiated adenocarcinoma, invading the subserosa (pT3). The carcinoma was composed of neoplastic epithelial cells, arranged in nests forming glandular structures with areas of central necrosis. Neoplastic epithelial cells exhibited rare mitotic figures, an increased nuclear/cytoplasmic ratio and prominent nucleoli. Approximately 30% of the tumour presented mesenchymal features. The sarcomatous component was completely separated from its carcinomatous counterpart by fibrous septae (Fig. 1). In the sarcomatous component, fascicular spindle cells admixed with bizarre large cells and focal osteoid-like matrix deposition were found. Fascicular spindle cells showed a moderately eosinophilic cytoplasm, with slightly enlarged nuclei. Large cells were markedly pleomorphic and exhibited enormously swollen nuclei with irregularly dispersed chromatin, focally surrounding areas of osteoid material deposition in the extracellular matrix. Metastasis of adenocarcinoma (N1) was found in 1 out of 22 lymph nodes detected. According to the TNM classification [8], the pathological stage was pT3N1.

The epithelial component was immunoreactive for CK20 in approximately 30% of neoplastic cells, whereas it was negative for vimentin and osteonectin. In the mesenchymal component, spindle cells were diffusely immunoreactive for smooth muscle actin (Fig. 2) and partially positive for vimentin, while large cells were focally immunoreactive for osteonectin (Fig. 3). Both the epithelial component and the mesenchymal component were negative for S100, desmin, CD34, c-kit and sarcomeric actin.

Ultrastructural examination was performed on both epithelial and mesenchymal areas of differentiation. Epithelial cells were organised in glandular structures encircled by basal membrane and showed typical surface microvilla and basal hemidesmosomes (Fig. 4A). Spindle cells exhibited myofibroblastic differentiation with peripheral cytoplasmic thin filaments and interspersed focal densities (Fig. 4B). There was no admixture of the two components.

The molecular screening performed to detect mutations revealed in both tumour components the same mutation R282W in exon 8 of the p53 gene (Figs. 5A, 5B). No mutation was found in the k-ras gene. Although no mutations could be detected, identical polymorphisms were identified in the carcinomatous as well as in the sarcomatous components in p53 exon 4, in EGFR exons 20 and 21, and in c-kit exon 17. The microsatellite analysis evidenced shared allelic retentions and loss among the epithelial and the mesenchymal neoplastic counteparts. Of the 10 STRs (Short Tandem Repeats) investigated, 7 were considered informative and four of them displayed an identical pattern comparing normal and the two tumoral components. An almost complete loss of the second allele was appreciated for D18S585, D18S35 and D18S51 markers (18q21) in both tumor' parts (Fig. 6).