Cooperation among heterogeneous prostate cancer cells in the bone metastatic niche.

The growth of disseminated tumor cells into metastatic lesions depends on the establishment of a favorable microenvironment in the stroma of the target organs. Here we show that mice treated with anakinra, an antagonist of the interleukin (IL)-1ß receptor (IL-1R), or harboring a targeted deletion of IL-1R are significantly less prone to develop bone tumors when inoculated in the arterial circulation with human prostate cancer (PCa) cells expressing IL-1ß. Interestingly, human mesenchymal stem cells exposed in vitro to medium conditioned by IL-1ß-expressing cancer cells responded by upregulating S100A4, a marker of cancer-associated fibroblasts (CAFs), and this effect was blocked by anakinra. Analogously, the stroma adjacent to skeletal metastases generated in mice by IL-1ß-expressing cancer cells showed a dramatic increase in S100A4, COX-2 and the alteration of 30 tumor-related genes as measured by Nanostring analysis. These effects were not observed in the stroma associated with the rare and much smaller metastases generated by the same cells in IL-1R knockout animals, confirming that tumor-secreted IL-1ß generates skeletal CAFs and conditions the surrounding bone microenvironment. In skeletal lesions from patients with metastatic PCa, histological and molecular analyses revealed that IL-1ß is highly expressed in cancer cells in which the androgen receptor (AR) is not detected (AR-), whereas this cytokine is uniformly absent in the AR-positive (AR+) metastatic cells. The stroma conditioned by IL-1ß-expressing cancer cells served as a supportive niche also for coexisting IL-1ß-lacking cancer cells, which are otherwise unable to generate tumors after independently seeding the skeleton of mice. This niche is established very early following tumor seeding and hints to a role of IL-1ß in promoting early colonization of PCa at the skeletal level.

Role of the VEGFR3/VEGFD receptor axis in TGFbeta1 activation of primary prostate cell lines.

BACKGROUND: Reports indicate that vascular endothelial growth factor receptor type 3 (VEGFR3) regulates cellular functions such as invasion, proliferation, and chemo-resistance. However, the exact function of the VEGFR3 signaling axis in prostate epithelial cells is poorly characterized. METHODS: The goal of this study was to evaluate whether TGFbeta1 in combination with VEGFD can promote pre-malignant invasive activities of intermediate basal cells (IBC-10a) isolated from human prostate cancer (Gleason score 6). RESULTS: hTERT immortalized IBC-10a cells normally grew as confluent "cobblestoned" monolayers, but treatment with TGFbeta1 (10 ng/ml for 2-6 hr) dissociated the cell-cell junctions and induced VEGFR3 translocation to the cell surface. This event was not inhibited by 10 microM cycloheximide or puromycin, indicating transcription and protein synthesis were not required. We further discovered that TGFbeta1 in combination with VEGFD induced a significant increase in the invasive activity of IBC-10a cells (>26% and 53% after 24 and 48 hr, respectively) in modified Boyden Chamber assays. TGFbetaRII receptor antibodies specifically blocked TGFbeta1 induction of VEGFR3 translocation to the cell surface and blocked VEGFD-induced invasion. Zymograms revealed that TGFbeta1 (and not VEGFR3) stimulated the secretion of MMP-2 and MMP-9, presumably to promote cell invasion. The cell invasion assays confirmed that antibodies specific for TGFbetaII receptor, MMP-2 and MMP-9 and VEGFR3, independently blocked TGFbeta1-induced invasion. CONCLUSIONS: For the first time, we have demonstrated the mechanism by which TGFbeta1 stimulates VEGFD/VEGFR3 receptor axis activation leading to increased cell migration and invasion by primary intermediate basal cell cultures.

Functional characterization of Na(+)/H(+) exchangers in primary cultures of prairie dog gallbladder.

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

Interleukin-10 activation of the interleukin-10E1 pathway and tissue inhibitor of metalloproteinase-1 expression is enhanced by proteasome inhibitors in primary prostate tumor lines.

The interleukin-10 (IL-10) activation of Janus kinase (JAK) family members (JAK1/TYK2) and IL-10E1 is subsequently inactivated by approximately 3-4 h in primary prostate tumor lines. We examined the effect of proteasome inhibition on IL-10 activation of the IL-10E1 pathway following stimulation of HPCA-10a cells. Treatment of HPCA-10a cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-10 receptor and IL-10E1 following stimulation. Further investigation showed that these stable phosphorylation events were the result of prolonged activation of JAK1 and TYK2 plus IL-10E1. IL-10E1 signaling normally induced the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and LLnL treatment of the HPCA-10a and HPCA-10c cells significantly enhanced IL-10 induction of TIMP-1 levels to block tumor cell invasion in modified Boyden chamber invasion assays. These observations were confirmed using pharmacologic inhibitors by Western blot and ELISAs. In the presence of LLnL, stable phosphorylation of IL-10E1 and induction of TIMP-1 was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on IL-10E1 phosphorylation and TIMP-1 could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting that phosphorylated IL-10E1 could be stabilized by phosphatase, but not by proteasome inhibition. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the IL-10E1 pathway and TIMP-1 induction by regulating the deactivation of JAK1/TYK2.

Atypical hemangioma of the breast: a diagnostic pitfall in breast fine-needle aspiration.

We report on the fine-needle aspiration (FNA) cytology of atypical hemangioma of the breast in a 52-yr-old female. The patient presented with a 2-cm palpable left breast mass. An FNA of the mass was performed following a mammogram, corresponding to the palpable breast mass. The FNA demonstrated the presence of numerous atypical single spindle cells scattered throughout a hemorrhagic background. An unequivocal diagnosis of malignancy was not rendered in this case. However, the degree of cytologic atypia suggested a malignant process, and a recommendation for an excisional biopsy was made. Atypical hemangioma should therefore be included in the differential diagnosis of angiosarcoma and other benign and malignant spindle-cell lesions of the breast encountered on cytologic samples.

Clinical experience with open renal cryoablation.

OBJECTIVES: To evaluate the safety and efficacy of open renal cryoablation of small solid renal masses, since the delivery of freezing temperatures has been shown to effectively ablate solid neoplasms of the liver, uterus, and prostate. METHODS: A total of 29 patients were treated with open renal cryoablation since December 1996 and followed up to evaluate the treatment safety and initial radiographic response. RESULTS: The median preoperative lesion size was 2.2 cm, with 22 solid renal masses and 7 complex renal lesions. Five serious adverse events occurred in 5 patients, with only one event directly related to the procedure. One patient experienced a biopsy-proven local recurrence, and 91.3% of patients (median follow-up 16 months) demonstrated a complete radiographic response with only a residual scar or small, nonenhancing cyst. CONCLUSIONS: Open renal cryoablation appears to be a safe technique for the in situ destruction of solid or complex renal masses. However, inadequate freezing of renal cell carcinoma may result in local disease persistence. The expected slow growth rate of small renal cancers necessitates prolonged radiologic follow-up. Continued clinical research is required before renal cryoablation can be considered an acceptable curative treatment for renal cancer.

Increased cellularity of tumor-encased native vessels in prostate carcinoma is a marker for tumor progression.

Changes in the native vasculature of the prostate gland associated with prostate adenocarcinoma have not been well characterized. Eighty-nine whole mounts of entirely submitted radical prostatectomies were reviewed. Thirty prostates with a minimum of five native arteries surrounded by carcinoma with corresponding control arteries were found and included in this study. The number of nuclei in the media of native arteries was recorded per 0.138 mm2 using a 40x objective. The number of nuclei in vessels embedded in carcinoma (n = 204) was increased when compared with controls (26.37 versus 20.58 mean nuclei per 0.138 mm2; P < .001). Pathologic Stage T3 carcinomas contained vessels that were more cellular than stage T2 (P < .001). Vessels embedded in Gleason Grade 4 showed more cellularity than arteries embedded in Gleason Grade 3 (P < .002). Increased media cellularity of native prostate vessels encased in carcinoma is a histologic feature of higher grade/stage prostate carcinoma and provides positive indicator of advanced prostate cancer.

Fine-needle aspiration biopsy in the evaluation of lymphadenopathy associated with cutaneous T-cell lymphoma (mycosis fungoides/Sézary syndrome).

We studied the role of fine-needle aspiration (FNA) in the evaluation of lymphadenopathy associated with cutaneous T-cell lymphoma (CTCL) in 11 patients with lymphadenopathy and compared findings with corresponding histologic material. Molecular genetic analysis for T-cell clonality by polymerase chain reaction (PCR) was performed on all aspirates. Immunophenotyping was successful in 4 of 7 cases in which flow cytometry was attempted from the aspirated material. Cytologic evaluation of FNA samples correlated strongly with histologic rating of involvement based on numbers of atypical cerebriform lymphocytes in the nodal specimen. Of 7 nodal specimens with scattered or small groups of atypical cells in the background of dermatopathic lymphadenopathy (LN1-2), the cytologic diagnosis was interpreted as reactive in all instances. Of 4 specimens with highly suspect (LN3) or definite histologic involvement (LN4), the cytologic diagnosis was likewise suspect or malignant. The correlation between molecular genetic studies on FNA samples and studies on tissue was not significant; in 2 cases, a T-cell clone was detected in the nodal tissue sample but not in the FNA sample, suggesting undersampling. A T-cell clone was detected by PCR in 5 of 7 nodal specimens judged reactive by FNA biopsy or histologic assessment. FNA for cytologic and molecular genetic analysis is a useful method to evaluate lymphadenopathy associated with CTCL and may obviate the need for surgical biopsy.

Prostate-specific antigen expression and lipochrome pigment granules in the differential diagnosis of prostatic adenocarcinoma versus seminal vesicle-ejaculatory duct epithelium.

BACKGROUND: Lipochrome pigment granules (LPGs) and prostate-specific antigen (PSA) localization have been cited as helpful adjuncts in differentiating atypical histologic patterns of seminal vesicle-ejaculatory duct (SVED) from prostatic adenocarcinoma. However, LPGs have been described in both benign and neoplastic prostatic acini, and PSA expression within the intraprostatic SVED has not been fully explored. DESIGN: Fifty radical prostatectomy specimens were studied for LPGs and 9 cases for PSA expression. RESULTS: Two morphologic types of LPGs (type 1 and type 2) were observed. The reproducibility in classifying LPGs was evaluated by kappa statistics, which demonstrated a strong agreement between 4 observers. Type 1 was restricted to SVED in all 50 specimens. Type 2 was subclassified into 2A and 2B. Type 2 LPGs were observed in prostatic acini of different zones, high-grade prostatic intraepithelial neoplasia, prostatic adenocarcinoma, and occasionally with type 1 LPG in SVED. Focal reactivity for PSA in the distal portion of SVED near urethra was noted in 1 of 9 cases. CONCLUSION: Awareness about morphologic differences between the 2 types of LPGs could help to avoid a potential diagnostic pitfall of misinterpreting SVED epithelium for adenocarcinoma. Caution is recommended in interpreting PSA expression, since rare focal PSA reactivity was observed in the distal SVED.

Growth of HPV-18 immortalized human prostatic intraepithelial neoplasia cell lines. Influence of IL-10, follistatin, activin-A, and DHT.

Cultures from high grade prostatic intraepithelial neoplasia (HGPIN) have been established and immortalized by HPV-18 infection. The cultures were identified as PIN by Western blotting with anti-cytokeratin (34betaE12) and prostate specific antigen (PSA) antibodies. We examined the growth capabilities of the cultures in the presence of TGF-beta1, activin-A, follistatin (FS), androgens (DHEA, DHT) and several cytokines (IL-10, IL-2, IL-4). IL-10, FS, and DHT stimulated cell proliferation and colony forming ability, while the other cytokines and growth factors had no discernable effect. In addition, DHT and to a lesser extent IL-10 both stimulated PSA production. Activin-A blocked IL-10, FS, and DHT stimulated growth and PSA production. We interpret the data to mean that IL-10 induction of FS secretion (and FS binding of activin A) restores the normal growth capabilities of HGPIN cultures.

Mucin stain on frozen sections: a rapid 3-minute method.

CONTEXT: The importance of frozen-section diagnoses in the practice of pathology cannot be overemphasized. In some cases, the use of a mucin stain can greatly aid in the diagnosis. Since few methods for mucin staining have been described that can be used in the frozen-section setting, we developed one such staining procedure for mucin. OBJECTIVE: To develop a rapid mucicarmine staining technique to be used on frozen sections that does not significantly delay overall turnaround time. DESIGN: A standard mucicarmine staining technique was modified by using a concentrated mucicarmine stain and a microwave oven, to reduce the total staining time to 3 minutes or less. Frozen tissue from normal colonic mucosa was used as a control, and skin from extramammary Paget disease for evaluation of margins was used as a case. RESULTS: The rapid mucicarmine stain successfully demonstrated the presence of mucin on frozen-tissue sections. Mucin stained deep rose, and the connective tissue stained green. CONCLUSION: This rapid and simple mucin staining technique can be used on frozen sections with no significant effect on the overall turnaround time, thereby aiding rapid diagnosis on frozen sections.

Role of interleukin 10 and transforming growth factor beta1 in the angiogenesis and metastasis of human prostate primary tumor lines from orthotopic implants in severe combined immunodeficiency mice.

Transfection of primary human prostate tumor cells (i.e., HPCA-10a, 10b, 10c, and 10d lines) with the transforming growth factor (TGF)-beta1 gene stimulated anchorage-independent growth and promoted tumor growth, angiogenesis, and metastasis after orthotopic implantation in severe combined immunodeficiency mice. In contrast, interleukin (IL)-10 transfected cells or cells cotransfected with these two genes exhibited reduced growth rates and significantly reduced angiogenesis and metastasis after 8, 12, and 16 weeks. Enzyme-linked immunosandwich assays confirmed that the respective tumors expressed elevated levels of TGF-beta1 and IL-10 in vivo. ELISAs further showed that TGF-beta1 expression induced matrix metalloproteinases-2 (MMP-2) expression, whereas IL-10 down-regulated MMP-2 expression while up regulating TIMP-1 in the transfected cells. Also, tumor factor VIII levels correlated with TGF-beta1 and MMP-2 expression and inversely with IL-10 and TIMP-1 levels. More importantly, mouse survival was zero after 4-6 months in mice bearing TGF-beta1- and MMP-2-expressing tumors and increased significantly in mice implanted with IL-10- and TIMP-1-expressing tumors (i.e., to >80% survival). Analysis of the metastatic lesions showed that they expressed TGF-beta1 and MMP-2 but barely detectable levels of IL-10 or TIMP-1, suggesting that IL-10 and TIMP-1 might normally block tumor growth, angiogenesis, and metastasis.

Malignant phyllodes tumor with chondrosarcomatous differentiation: report of a case with cytological presentation.

Malignant phyllodes tumor is a rare breast tumor with neoplastic epithelial and stromal components. The stromal component may show homologous and heterologous sarcomatous elements, including chondrosarcomatous and osteosarcomatous differentiation. Because these tumors may present with an almost exclusively sarcomatous component, it is important for the pathologist to include this entity in the diagnostic considerations of fine-needle aspirations of breast neoplasms showing sarcomatous differentiation. Following surgical excision, careful examination of the gross specimen and thorough sampling of the specimen is recommended before rendering a definitive histologic diagnosis. We describe the cytologic and histologic findings in a case of malignant phyllodes tumor with sarcomatous overgrowth showing predominantly chondrosarcomatous differentiation.

Low-grade fibromyxoid sarcoma: clinicopathologic case report with review of the literature.

Low-grade fibromyxoid sarcoma is a rare, benign-appearing soft tissue neoplasm with an aggressive clinical course characterized by multiple local recurrences over several years, with ultimate spread to lung and occasionally to bone. Thus far, a total of 24 cases of low-grade fibromyxoid sarcoma have been reported in the literature. The authors present an additional case that grossly and microscopically emphasizes a pronounced lobular pattern of contrasting areas of cellularity showing high proliferative activity, as demonstrated by a proliferation marker, Ki 67 with MIB-1, and hypocellular areas with prominent myxoid component and abundant collagen fibrils. There was predominance of delicate capillary-sized stromal vessels with collagenized walls in both cellular and myxoid areas. The unusual features in this case were osseous metaplasia, prominent intranuclear pseudoinclusions, DNA tetraploidy, and membrane-bound intracytoplasmic fat vacuoles. The immunoprofile and cytologic and ultrastructural features are described. After the excision of the tumor, the patient was treated with radiotherapy without chemotherapy. The patient has been observed for 26 months and is alive without the evidence of disease. The postoperative follow-up with axial computed tomography at 24 months showed no evidence of disease, except postsurgical fibrotic changes.

Breast abnormalities in patients with cystic fibrosis: previously unrecognized changes.

We reviewed breast tissue from 19 female postpubertal and three prepubertal patients with cystic fibrosis to determine the histopathologic changes present. Autopsy material was available from 19 patients who died from complications of cystic fibrosis. Two patients had lumpectomies for breast carcinoma and an additional patient had a breast biopsy and bilateral prophylactic mastectomies for breast carcinoma. The postpubertal patients ranged in age from 18 to 50 years (median age, 32 years). The histologic changes in the breast of female patients with cystic fibrosis have not been fully studied, and the only case reported suggests that these patients suffered from complete lobular agenesis. This study shows that postpubertal breasts had normal development and varying degrees of fibrosis affecting lobular units and ducts, and that proliferative lesions and carcinoma could be encountered.

Specific transcription factors prognostic for prostate cancer progression.

We have previously identified (M. Wang et al., Oncol. Res., in press, 1998) an enhancer element [human tissue inhibitor of metalloproteinase-1 enhancer-1 (HTE)] for the human tissue inhibitor of metalloproteinase-1 promoter that binds a novel zinc finger, cysteine-rich transcription factor (CRTF). In this study, we have used electrophoretic mobility shift assays to examine the relative level of expression of CRTF, jun/fos, and IFN-gamma responsive signal transducer activators of transcription (STATs) that bind specific HTE, activator protein, and IFN-gamma (Fcy and interferon regulatory factor) response motifs in tumor lines and human prostate tissue [i.e., normal (n = 3); benign prostatic hyperplasia (BPH; n = 12); high grade prostate intraepithelial neoplasia (PIN; n = 10); and prostate cancer adenocarcinoma (PCA; n = 61) plus seminal vesicle (n = 10) tissues]. The data showed that CRTF was overexpressed in PCA (Gleason's score, 10>8>6>5>4) compared with BPH, PIN, seminal vesicle, and normal tissues. To a much lesser degree, jun/fos and STAT 1 were also elevated in PCA compared to BPH, PIN, and normal tissues. In addition, blinded studies showed that CRTF and jun/fos were present at low levels in organ-confined specimens but at significantly elevated levels (P < 0.001) in samples exhibiting capsular penetration and localized spread, which indicated that CRTF and perhaps jun/fos were markers for cancer progression.

Laryngeal glycogenic acanthosis presenting as leukoplakia.

We report a case of an incidental finding of glycogenic acanthosis of the larynx on autopsy in a 79-year-old man who died of myocardial infarction. The lesion was grossly recognized as a white plaque (leukoplakia) on the subglottic compartment of the left side of the larynx. Histological sections revealed thickened squamous mucosa positive for abundant glycogen on periodic acid-Schiff staining. No epithelial dysplasia was noted. The patient had a history of smoking. This case represents the first report of glycogenic acanthosis involving the larynx. This benign condition should be added to the vast differential diagnosis of leukoplakia in this anatomical location.

Focal hematopoietic hyperplasia of the rib. Report of a case diagnosed by fine needle aspiration.

BACKGROUND: Focal hematopoietic hyperplasia (FHH) of the rib is a rare, benign, localized proliferation of bone marrow to such a degree that it produces a tumorlike expansion of the rib that can be the source of considerable clinical alarm. In the appropriate clinical setting, this lesion needs to be included in the differential diagnosis of solitary bone lesions, in particular when assessing the adequacy of a specimen at the time of aspiration. CASE: A large, lytic mass on the posterior aspect of the sixth rib was incidentally discovered on a chest roentgenogram from a 46-year-old male during a routine presurgical evaluation for diverticulitis. The radiologic characteristics of the tumor were thought to be consistent with a neoplasm; that prompted a recommendation for fine needle aspiration biopsy (FNAB). The mass was thoroughly sampled under radiologic guidance, performing multiple aspirations of different areas. All smears prepared at the time of the aspiration for the evaluation of specimen adequacy showed abundant marrow tissue without any evidence of malignancy. Although it was initially thought that the tissue was probably obtained from the periphery of the lesion, this notion was discarded after multiple passes from different areas showed only marrow tissue and since there was radiologic evidence that the sample was obtained from within the lesion. CONCLUSION: The diagnosis of FHH of the rib by FNAB or other small-biopsy techniques requires strict radiologic-pathologic correlation. Awareness of this entity will avoid unnecessary repeated biopsy procedures and potentially large, complicated surgical procedures. This case shares several features with the other two reported cases: a solitary lesion on the rib presenting in an asymptomatic patient with no evidence of associated hematologic disease.

Retrospective DNA ploidy analysis by image and flow cytometry in head and neck cancer.

A retrospective analysis of DNA content (DNA ploidy) was conducted in formalin-fixed specimens from patients with advanced, unresectable head and neck cancer who were treated on a single defined protocol with accelerated fractionation radiation therapy and concomitant cisplatin (Platinol) chemotherapy. Specimens from 31 tumor sites were analyzed by image cytometry using the Feulgen staining method. Fifteen specimens were analyzed by flow cytometry after deparaffinization, nuclear disaggregation, and staining with propidium iodide. By image analysis, 10 (32%) of 31 specimens contained only diploid tumor cells, while 21 (68%) specimens exhibited at least one aneuploid tumor component. Seven of the eight tumors with a single G0/G1 peak by image analysis had a single peak by flow analysis. Five of the seven tumors with multiple peaks by image analysis had multiple peaks by flow analysis. Histology was also reevaluated, and tumor grade was determined, reflecting tumor cell differentiation based on keratinization, mitotic activity and the degree of nuclear polymorphism. For this well-defined patient population managed according to a uniform therapeutic approach, DNA ploidy status and histology provided a suggestion of prognostic separation; however, statistical significance was not obtained, most likely due to the small number of patients.

Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry.

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.