Spindle cell carcinoma of the larynx: review of 26 cases including DNA content and immunohistochemistry

Spindle cell carcinoma (SpCC) is uncommon, with a predilection for the upper aerodigestive tract. Its histogenesis has not been resolved, although most authors support the sarcomatoid carcinoma concept. Ploidy analysis and proliferation indices have not been reported for laryngeal SpCCs. The authors examined the pathological and clinical features of 26 patients (25 men, 1 woman; mean age, 64 years) with laryngeal SpCC treated at the Mayo Clinic from 1960 to 1990. Twenty-three tumors were examined with digital image analysis for DNA content of the spindle cell population (13 tumors had a sufficient squamous component to be analyzed separately). The glottis was involved most frequently (19 patients); 21 tumors were grossly polypoid. Twenty-three tumors were biphasic, and three were monophasic. Overall, 17 tumors (65%) showed keratin positivity in the spindle cell component. Polyclonal antikeratin (15 positive cases), 34betaE12 (15 positive), and AE1/AE3 (12 positive) were the most sensitive markers. Spindle cells were diploid in 5 tumors (22%) and nondiploid in 18 (78%); conventional squamous cell carcinoma was diploid in 4 cases and nondiploid in 9. DNA ploidy results were concordant between the two populations in 11 of 13 tumors (85%). Mean percent MIB-1 staining was 31% in the sarcomatoid component and 45% in the squamous component. In our primary treatment group of 22 patients (median follow-up, 6.4 years), 4 (18%) had local recurrence, 3 (14%) had distant metastasis, and 4 (18%) died of disease. Presence of a nondiploid spindle cell population in 78% of cases of laryngeal SpCC is interpreted as evidence of a neoplastic rather than reactive process. Keratin positivity in nearly two thirds of tumors supports the theory of epithelial origin of these tumors (sarcomatoid carcinoma).     

Evaluation of loss of heterozygosity and microsatellite instability in human pterygium: clinical correlations

The histogenesis of carcinosarcoma of the breast is controversial. In the current case, the demarcation between the carcinomatous and sarcomatous components was distinct in all microscopic fields. Immunohistochemical analysis was negative for epithelial membrane antigen (EMA) and keratin in the sarcomatous component and was negative for desmin in the carcinomatous component, suggesting that this tumor could be derived from the two different stem cells. To determine the histogenesis of this tumor, both carcinomatous and sarcomatous lesions were microdissected from formalin-fixed tissues and DNAs were prepared by proteinase K digestion. PCR amplification of the human androgen receptor (HUMARA) short tandem repeat (STR), after Hpa II digestion of the genomic DNA, indicated that the patterns of X-chromosome inactivation were identical in both components. Moreover, both components contained the identical TGT --> TTT transversion in codon 275 of the p53 gene. These observations strongly support the hypothesis that this tumor is derived from a single totipotent stem cell.     

DNA aberrations in the epithelial cell component of adamantinoma of long bones

Adamantinoma of long bones is a rare malignant tumor composed of cells with epithelial characteristics in various differentiation patterns surrounded by fibrous cells. Evidence as to whether this neoplasm should be designated as an epithelial bone tumor or a biphasic sarcoma with both epithelial and mesenchymal features is lacking. In this study the nature of the mesenchymal and epithelial components of adamantinoma was investigated by DNA flow cytometry, DNA image cytometry, p53 immunohistochemistry, and polymerase chain reaction-based loss of heterozygosity detection at the p53 locus. Specimens from 6 of 15 patients (40%) analyzed by flow cytometry had an aneuploid DNA index. Image cytometry analysis of Feulgen-stained paraffin sections of 6 aneuploid and 2 diploid tumors revealed that aneuploid nuclei were detected in cells with an epithelial phenotype only, whereas all fibrous cells were diploid. Immunohistochemistry for p53 on specimens from 25 patients revealed moderate or strong immunoreactivity in 12 tumors (48%) restricted to the epithelial cells. Loss of heterozygosity at the p53 locus could be confirmed in the epithelial component of an immunohistochemically p53-positive tumor. Additionally, sections of 7 lung metastases were studied histologically. Only keratin-positive epithelial cells, predominantly in the spindle cell pattern, were present in these metastases, whereas the osteofibrous tissue present in the primary tumors was not detected. These results suggest that either adamantinoma consists of a malignant epithelial part with a reactive osteofibrous stroma or that the malignant epithelial cells develop next to a proliferating benign fibrous component. Additional analysis of common genetic abnormalities in the fibrous and epithelial cells of adamantinoma is therefore indicated.     

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis

Oral squamous cell carcinoma develops through a series of precancerous stages manifested at the microscopic level as epithelial dysplasia. Mutation of the p53 tumour suppressor gene is thought to be an important component of oral carcinogenesis. p53 regulates cell proliferation and DNA repair by inhibiting the cell cycle at G1/S; loss of p53 function may therefore lead to aberrant cell kinetics. To date, no studies have examined the relationship between p53 protein and alterations in cell kinetics in oral epithelial dysplasia from a single anatomical site. Serial sections were studied from 40 routinely processed biopsy specimens of epithelial dysplasia from the floor of the mouth. The expression of p53 protein was determined by immunohistochemistry and cell proliferation was studied by immunostaining for the cell cycle-dependent protein Ki-67. The number of positive cells per millimetre of basement membrane was determined using computer image analysis and compared with site-matched normal controls. The mean p53 labelling index (LI) in normal mucosa was low, 3.48 +/- 0.92 [mean +/- 95 per cent confidence interval (CI)], and increased sharply in the transition from mild (42.49 +/- 21.71) to moderate (104.86 +/- 51.39) epithelial dysplasia. The mean p53 LI for severe dysplasia was 119.09 +/- 56.50. Differences were also observed in the distribution of p53-positive cells between grades of dysplasia, with the development of compact p53-positive foci in severe dysplasia. Mean proliferative indices, as determined by Ki-67 expression, were significantly associated with grade of epithelial dysplasia. Furthermore, there was a significant correlation between p53 LI and Ki-67 score (r2 = 0.37, P = 0.01). It is concluded that altered p53 protein expression is probably an early event in oral carcinogenesis in the floor of the mouth and is associated with dysregulation of cell proliferation at this site.     

Salivary gland malignant myoepithelioma: a clinicopathologic and immunohistochemical study of ten cases

BACKGROUND: Malignant myoepithelioma (MME) of the salivary gland, also known as myoepithelial carcinoma, is rare and its biologic behavior has not been clarified fully. METHODS: Ten cases of MME were analyzed for their clinicopathologic features and immunohistochemical characteristics, focusing on prognostic factors and tumor differentiation. In addition, six cases of benign myoepithelioma (BME) also were examined for comparison. RESULTS: The ten patients with MME (3 men and 7 women) ranged in age from 48-81 years (mean, 61.9 years). Seven cases of MME arose in the parotid salivary gland, two in the submandibular salivary gland, and one in minor salivary glands of the soft palate. In the current series, the incidence of MME was 0.45% among 1945 cases of major salivary gland tumors. Seven cases of MME developed from a benign preexisting tumor (six in pleomorphic adenoma and one in BME). Four of nine patients with MME died of the disease and two patients developed a recurrence. It was shown that MMEs were comprised of one cell type or a combination of two cell populations; these included, in order of incidence, epithelioid, spindle, and plasmacytoid cells. Patients with MME with marked cellular pleomorphism and perineural invasion had a poor prognosis. Immunohistochemically, putative myoepithelial markers such as muscle actins, cytokeratin 14, vimentin, and calponin, and S-100 protein were expressed highly in MME. High and low molecular weight cytokeratins and epithelial membrane antigen also frequently were positive in MME. p53 expression was observed in five MME cases, four of which either recurred or were fatal. Cellular proliferative activity assessed by mitotic count and the Ki-67 labeling index was significantly higher in MME cases than in BME cases. In limited cases, such cellular proliferative activity was shown to have prognostic value. Ultrastructurally, the tumor cells displayed certain myoepithelial characteristics. CONCLUSIONS: MME is a rare salivary gland tumor showing clinicopathologic diversity and presenting with various stages of myoepithelial differentiation. Histologic aggressiveness, marked cellular pleomorphism, p53 expression, and high cell proliferative activity were found to be correlated with a poor clinical outcome.     

Carnosine, a protective, anti-ageing peptide?

Spindle cell squamous esophageal carcinomas are distinctive polypoid "biphasic" tumors in which the sarcoma-like phenotype usually predominates over the epithelial component. To biologically assess both phenotypes, we compared the tumoral proliferative activity and DNA ploidy between the two histological components of 13 polypoid spindle cell squamous carcinomas of the esophagus. We studied the tumoral proliferative index (TPI) using MIB 1 monoclonal antibody (Ki-67) and determined the DNA histogram by image cytometry on Feulgen-stained sections. The DNA histograms were classified into four types (I to IV) according to the degree of dispersion of the DNA. The TPI of the carcinomatous regions ranged from 0.20 to 0.63 (mean, 0.44) and from 0.55 to 0.85 for the sarcoma-like areas (mean, 0.68) P < .0001. In all cases, the sarcoma-like areas were aneuploid, and 37.5% of the carcinomatous regions were diploid. Also, in all instances the carcinomatous areas were of either histogram type I or II, and the sarcoma-like areas showed histograms of type II or III. We conclude that in esophageal spindle cell squamous carcinomas the sarcoma-like phenotype differs biologically in two ways from the carcinomatous: (1) it has a higher TPI and (2) it has higher aneuploidy with a greater dispersion of the DNA content. We postulate that these characteristics could give a "growth" advantage to the sarcoma-like component of these tumors and explain its predominance over the carcinomatous component.     

Down-regulation of laminin-5 in breast carcinoma cells

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.     

Carcinosarcoma arising in eccrine spiradenoma of the breast. Report of a case and review of the literature

Although the vast majority of eccrine spiradenomas behave in a benign fashion, 23 cases of malignant transformation have been reported to date. We describe a unique example of malignant eccrine spiradenoma that arose in the right breast of a 68-year-old woman. The quiescent mass, which was present for approximately 50 years, experienced sudden enlargement with erythematous changes of the overlying skin and nipple discharge. Microscopically, the tumor showed the typical features of an eccrine spiradenoma with areas of adenocarcinoma, squamous cell carcinoma, and sarcoma. The sarcomatous component consisted of rhabdomyosarcoma and osteosarcoma. The immunoperoxidase staining revealed p53 protein expression only in the carcinomatous and sarcomatous components. This suggests that accumulation of p53 protein may be an important event in the malignant transformation of spiradenomas. Because of its location and biphasic nature, this malignant eccrine spiradenoma should be distinguished from metaplastic breast carcinoma. To our knowledge, this represents the first carcinosarcomatous transformation of eccrine spiradenoma in the breast. This case led us to conclude that breast tissue, which often undergoes apocrine metaplasia and gives rise to apocrine neoplasms, is also capable of originating benign and malignant tumors with eccrine sweat duct phenotype.     

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.     

Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro

An organotypic, tridimensional cell culture, also called a raft system, was used to study the influence of fibroblasts on epithelial carcinogenesis in a cell line derived from laryngeal squamous cell carcinoma and harboring a mutated p53. Differences between the effects of normal fibroblasts and those of tumor-derived fibroblasts were compared by means of fibroblasts taken from the normal skin and from the tumor of a cancer patient and cultivated with epithelial carcinoma cells in an organotypic culture. To study cell contact-mediated changes, the fibroblasts were either simply embedded in collagen matrix or additionally brought into direct contact with epithelial cells. Control epithelial cells were cultivated without any fibroblasts in an organotypic model. A protein panel [p53, p21, PCNA, bcl-2, Ki67, total cytokeratin (CK), CK 8, CK 10, CK 17, CK 18, CK 19, vimentin] involved in cell cycling and epithelial differentiation was assessed immunocytochemically in all organotypic cultures with fibroblasts, in tumor cells cultivated as a monolayer, and in the original tumor sample. The most dysplastic phenotype was obtained when tumor-derived fibroblasts were used in direct contact with epithelial cells, whereas the most benign phenotype was seen when skin fibroblasts had no contact with them. The intensive staining seen for p53 can be explained by p53 mutations also reflecting the weak expression of p21 and abundant expression of PCNA. The intensive Ki67 staining seen in all sections paralleled that of PCNA and marked active cellular proliferation. The CK staining pattern seen in cultured epithelia toward embryonic CKs, CK 8 and CK 18, suggested a simple epithelial phenotype. CK 19 was found only in the epithelium where no direct contacts had occurred. Vimentin expression increased when the raft epithelium was shifting toward a more benign phenotype. The results stress the importance of the origin of fibroblasts as well as the role of direct cellular contacts in modifying the epithelial phenotype even when the epithelial cells are malignant.     

Coexpression of hepatocyte growth factor and c-Met proto-oncogene product in synovial sarcoma

Hepatocyte growth factor (HGF) is a heterodimeric polypeptide growth factor that has pleiotropic roles, including those of mitogen, motogen and morphogen. The HGF receptor is characterized as a c-Met proto-oncogene product (c-Met), which is a heterodimeric tyrosine kinase receptor. Hepatocyte growth factor acts as a mediator between the mesenchymal and epithelial tissues because HGF is produced by mesenchymal cells and c-Met is mainly expressed on various epithelial cells. Furthermore, the HGF/c-Met system plays an important role in embryogenesis and the regeneration of various organs. Synovial sarcoma (SS) are unique sarcoma that show epithelial differentiation, but little is known about their histogenesis. The expression of HGF and c-Met was examined by immunohistochemistry in SS specimens from 12 patients (six each of biphasic and monophasic fibrous types). Immunohistochemical coexpression of HGF and c-Met was demonstrated in the epithelial component of five biphasic SS, while only c-Met was expressed in the epithelioid nests of three monophasic fibrous SS. The spindle cell component was negative for HGF and c-Met. In SS, positivity for epithelial markers, such as cytokeratins and epithelial membrane antigen, was diffusely observed in the epithelial component and was focally observed in spindle cells, while vimentin was positive predominantly in the spindle cell component. The areas expressing HGF and c-Met corresponded to distinct epithelial structures; however, HGF and c-Met expression were not found in any other tumor cells expressing epithelial markers in the spindle cell component of SS. Considering the morphogenic effect of HGF, which has been known to be one of its most important roles, the unique immunohistochemical localization of HGF and c-Met in SS suggests that the HGF/c-Met system may be closely related to the formation of epithelial (glandular) structures in biphasic SS.     

Gestational squamous cell carcinoma of the breast: an unusual mammary tumor associated with aggressive clinical course

We report two cases of squamous carcinoma of the breast detected during the gestational period. One woman was post-partum and lactating; one was in the first trimester of pregnancy. The lesions were clinically palpable, multifocal, and measured more than 5 cm in their largest dimension; both had a cystic appearance. They were treated with radical mastectomy. One patient received pre-operatory chemotherapy. Histologically, the tumors were poorly differentiated squamous cell carcinomas. No areas of ordinary duct differentiation were seen. Lymph nodes contained metastatic squamous carcinoma in both cases. Tumor cells were negative for estrogen and progesterone receptors. Also, they expressed a high proliferative index and several markers of tumor progression, including cErb-B2, p53 protein, bcl-2, and epidermal growth factor receptor. One patient died of tumor 5 months following breast surgery and had extensive metastases proven at autopsy. The other patient had evidence of pulmonary metastases: following cisplatin therapy, she underwent clinical remission. This study shows that squamous carcinoma of the breast may occur in pregnant or lactating women: it appears clinically distinguishable from the non-gestational type that is usually associated with a better prognosis and occurs in peri- or postmenopausal women.     

Recommendations for the reporting of resected neoplasms of the kidney. Association of Directors of Anatomical and Surgical Pathology

Expression of c-erbB-2 oncogene and mutant p53 protein were detected immunohistochemically in 14 cases of myoepithelioma and 6 cases of myoepithelial carcinoma of the salivary gland. Five of the 6 myoepithelial carcinomas and nine of the 14 myoepitheliomas were overexpression of c-erbB-2 oncoprotein. The salivary ductal epithelial cells near tumors were overexpression of c-erbB-2 oncoprotein. These results indicate that c-erbB-2 may be an initial oncogene during the histogenesis of myoepithelial tumors. The p53 protein was positive in five cases of the 6 myoepithelial carcinoma, whereas all myoepitheliomas were negative. The results indicate that the mutant p53 gene may play an important role in the carcinogenesis of myoepithelial carcinoma.     

Validation of p53 accumulation as a predictor of distant metastasis at 10 years of follow-up in 1400 node-negative breast cancers

Most studies are in favor of a prognostic relevance of p53 accumulation determined by immunohistochemistry in breast cancer, but negative results are not lacking. On a series of 1400 patients with lymph node-negative cancers treated with local-regional therapy alone until relapse and with a median follow-up of 10 years, we validated the prognostic relevance for overall relapse and death of p53 accumulation observed in a pilot study and analyzed its predictivity on different adverse events. p53 protein accumulation was immunocytochemically detected using PAb1801. The case series had also been previously characterized for hormone receptor content [estrogen receptors (ERs) and progesterone receptors (PgRs)] and for cell proliferation [[3H]thymidine labeling index ([3H]dT LI)]. p53 expression, considered as a dichotomous variable with a cutoff value of 5% positive cells, significantly predicted the occurrence of overall relapse, distant metastasis, and death with an interaction with cell proliferation. p53 accumulation, cell proliferation, and their interaction term, along with tumor size and patient age, retained a predictive role for overall relapse, and together with tumor size and PgR, also for overall survival. When considered as continuous variables, we observed that the hazard of metastasis increased linearly with the increase of [3H]dT LI and decreased linearly with the increase of ER and PgR. Conversely, the hazard increased with the increase of p53-positive cells only for tumors with a [3H]dT LI lower than 7.5%. In multivariate analysis, the same prognostic factors for distant metastasis were identified when the biomarkers were considered as continuous or dichotomous variables.     

Myxoid and sclerosing sarcomatoid transitional cell carcinoma of the urinary bladder: a clinicopathologic and immunohistochemical study of 25 cases

We report 25 sarcomatoid carcinomas of the urinary bladder with a prominent myxoid and/or sclerotic appearance. The average age of the patients was 72 years (range, 50-92 yr); 14 were men, and 11 were women. The cystoscopic appearance varied from a large polypoid mass to an intramural mass with bladder wall thickening, often with necrosis and ulceration. The tumors ranged from 3 to 10 cm and were typically rubbery or gelatinous with a brown, pink, or gray color. Microscopy revealed tapering spindle cells with a variable admixture of cohesive non-spindled cells. Twenty-two cases had an invasive overtly epithelial carcinomatous component, and in situ transitional carcinoma was present in 12 cases. All of the cases had areas with myxoid change, ranging from extensive to focal, separating the spindle cells. Fourteen cases had areas of sclerosis. In all the cases, the spindle cells were atypical, at least focally, with hyperchromatic pleomorphic nuclei, prominent nucleoli, and coarse chromatin. Mitotic activity was prominent in the majority of cases, and abnormal mitotic figures were frequent. In eight cases, the myxoid histologic pattern was very reminiscent of an inflammatory pseudotumor, a diagnosis frequently entertained and erroneously made in one case; many of the spindle cells in three of these cases were mildly atypical, with minimal mitotic activity. The spindle cells were immunoreactive for cytokeratin (12 of 19), vimentin (16 of 17), carcinoembryonic antigen (3 of 15) and muscle-specific actin (4 of 16), and nonreactive for epithelial membrane antigen, desmin, S-100, KP1, CD34, and Leu-M1. The epithelioid carcinomatous areas were highlighted by the cytokeratin immunostain. These features and the conventional light microscopic features indicative of a diagnosis of carcinoma distinguish this tumor from reactive of neoplastic mesenchymal lesions.     

Small giant cell carcinoma of the lung diagnosed preoperatively by transthoracic aspiration cytology. A case report

Giant cell carcinoma (GCC) of the lung is considered an aggressive form of lung cancer. Most patients with GCC have large tumors with widespread metastases on hospital admission. We describe a small GCC in the lower lobe of the right lung. Preoperative aspiration cytology showed numerous bizarre, giant epithelial cells, highly suggestive of GCC. The resected tumor measured 1.2 x 1.0 x 0.7 cm in diameter, with an ill-defined margin. Histologically the tumor was composed of pleomorphic mononucleate or multinucleate giant cells without adenocarcinoma or squamous cell differentiation. The tumor cells were loosely organized and noncohesive or free in the alveolar space. The histology was interpreted as GCC of the lung. The clinicopathologic features of giant cell carcinoma are discussed.     

Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer

Urokinase plasminogen activator (uPA) is a serine proteinase involved in degradation of the extracellular matrix during cancer invasion. uPA is up-regulated in breast cancer, and high levels of uPA in tumor extracts are strongly associated with poor prognosis. Like several other matrix proteinases, uPA is in some types of cancer, including breast cancer, expressed by stromal cells. The present study was undertaken to determine the identity of the uPA-expressing stromal cells in breast cancer tissue. By in situ hybridization, a positive signal for uPA mRNA was in 26 of 28 ductal and four of five lobular carcinomas demonstrated in stromal cells adjacent to nests of cancer cells, whereas only one ductal carcinoma showed a positive reaction in the epithelial component itself. The positive stromal cells were found in both the peripheral and central parts of the tumors. Stromal cells surrounding carcinoma in situ lesions were uPA mRNA positive in a few cases, and no signal was observed in the neighboring nonmalignant tissue. Cell identification was done by immunostaining with Ab to markers for the following cell types: myoepithelial cells, myofibroblasts, smooth muscle cells, macrophages, endothelial cells, and epithelial cells. The only one of these cell types that had a distribution similar to the uPA mRNA-expressing cells was myofibroblasts, recognized as extravascular alpha-smooth muscle actin-positive and cytokeratin-negative cells. On adjacent sections, colocalization was found of cells positive for uPA mRNA and cells positive for alpha-smooth muscle actin and negative for cytokeratin. We concluded that the uPA mRNA-expressing cells are myofibroblasts. The myofibroblasts have previously been found to be abundant in breast cancer tissue. They primarily originate by differentiation of fibroblasts, probably induced by cytokines released from the cancer cells. The present findings suggest that the myofibroblasts, through production of uPA, play an active role in breast cancer invasion.