Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

Mastectomy versus breast-conserving therapy in the treatment of stage I and II carcinoma of the breast: a randomized trial at the National Cancer Institute.

PURPOSE: Mastectomy versus excisional biopsy (lumpectomy) plus radiation for the treatment of stage I and II breast cancer was compared in a prospective randomized study. PATIENTS AND METHODS: From 1979 to 1987, 247 women were randomized and 237 were treated on this study. All patients received a full axillary dissection and all node-positive patients received adjuvant chemotherapy with cyclophosphamide and doxorubicin. Radiation consisted of external-beam therapy to the whole breast with or without supraclavicular nodal irradiation followed by a boost to the tumor bed. RESULTS: The minimum time on the study was 18 months and the median time on the study was 68 months. No differences in overall survival or disease-free survival were observed. Actuarial estimates at 5 years showed that 85% of mastectomy-treated patients were alive compared with 89% of the lumpectomy/radiation patients (P2 = .49; 95% two-sided confidence interval [CI] about this difference, 0% to 9% favoring lumpectomy plus radiation). The probability of failure in the irradiated breast was 12% by 5 years and 20% by 8 years according to actuarial estimates. Of 15 local breast failures, 14 were treated with and 12 were controlled by mastectomy; the ultimate local-regional control was similar in both arms of the trial. CONCLUSION: These data add further weight to the conclusion that breast conservation using lumpectomy and breast irradiation is equivalent to mastectomy in terms of survival and ultimate local control for stage I and II breast cancer patients.

Murine tumor cells transduced with the gene for tumor necrosis factor-alpha. Evidence for paracrine immune effects of tumor necrosis factor against tumors.

Studies of the anti-tumor activity of TNF-alpha in vivo have been hampered by the need to administer systemically toxic doses of the cytokine to obtain a curative response. To facilitate studies of the effect of high local concentrations of TNF-alpha on tumor growth and host immunity, a newly induced murine sarcoma was transduced with the gene for human TNF-alpha and the biologic characteristics of these cells were examined. We identified high and low TNF-producing tumor clones which exhibited stable TNF secretion over time. Significant amounts of membrane associated TNF were found in a high-TNF producing clone as well. No difference in the in vitro growth rates between TNF-producing and nonproducing cell lines was observed. In contrast, in vivo studies demonstrate that although unmodified parental tumor cells grew progressively when implanted s.c. in animals, tumor cells transduced with the TNF gene were found to regress in a significant number of animals after an initial phase of growth. This effect correlated with the amount of TNF produced and could be blocked with a specific anti-TNF antibody. Regressions of TNF-producing cells occurred in the absence of any demonstrable toxicity in the animals bearing these tumors. TNF-producing tumor cells could function in a paracrine fashion by inhibiting the growth of unmodified, parental tumor cells implanted at the same site. The ability of tumor cells to regress was abrogated by in vivo depletion of CD4+ or CD8+ T cell subsets and animals that had experienced regression of TNF-producing tumors rejected subsequent challenges of parental tumor. Our studies thus show that tumor cells elaborating high local concentrations of TNF regress in the absence of toxicity in the host and that this process requires the existence of intact host immunity. Studies of the lymphocytes infiltrating the gene modified tumors and attempts to use TNF gene modified tumor infiltrating lymphocytes to deliver high local concentrations of TNF to the tumor site without inducing systemic toxicity are underway.

Cytologic diagnosis of prostatic carcinoma by fine-needle aspiration biopsy. Community hospital experience.

Transrectal fine-needle aspiration and transrectal or perineal core biopsies were simultaneously performed on 31 patients with suspected prostatic cancer over an eighteen-month period. Of the 29 aspirations that were adequate for cytologic diagnosis, there was histologic correlation in 24 (83%). The sensitivity of aspiration for the diagnosis for prostatic cancer was 92 per cent (11 of 12) compared with 85 per cent (11 of 13) for the core biopsy method. There were no apparent false negative or false positive diagnoses with the aspiration biopsy technique. Insufficient material was obtained by aspiration in 2 cases. A febrile urinary tract infection occurred in 1 patient after transrectal aspiration and core biopsy. Our results suggest that fine-needle aspiration may be utilized by the practicing urologist in conjunction with a pathologist trained in the interpretation of fine-needle aspirates as a safe, relatively inexpensive, and sensitive diagnostic procedure for suspected prostatic cancer.

Antitumor effect of recombinant tumor necrosis factor-alpha against murine sarcomas at visceral sites: tumor size influences the response to therapy.

We examined the antitumor efficacy of rTNF-alpha administration on established tumor at two visceral sites, lungs and liver. Treatment of B6 mice harboring multiple (greater than 100 foci of less than or equal to 0.5 mm diameter) 10-day pulmonary macrometastases from the MCA-106 sarcoma, with dosages of rTNF-alpha (5-10 micrograms, single dose i.v.) that caused hemorrhagic necrosis and regression of a 6 mm MCA-106 s.c. tumor, had no impact on the number (or size) of lung nodules. Similarly, rTNF-alpha failed to show an antitumor effect in B6 mice with advanced day 8 or 10 multiple (greater than 100 foci of less than or equal to 0.5 mm diameter) hepatic metastases at single i.v. doses up to 20 micrograms, as measured by either enumeration of residual liver nodules or survival. B6 mice injected s.c. with MCA-106 sarcoma and treated with rTNF-alpha as a single i.v. dose on day 0, 3, 5, or 7 experienced marked tumor regression only after the day 7 rTNF-alpha injection, when the tumor had achieved a size of 5-6 mm in diameter. Since tumor size appeared important for rTNF-alpha susceptibility in vivo, we next induced a single hepatic tumor of the MCA-106 sarcoma by the direct injection of cells into the left lobe of the liver and treated these mice at day 10 when the nodule had achieved a size of 5-6 mm in diameter. Increasing doses of rTNF-alpha (up to 8 micrograms) given as a single i.v. injection resulted in increasingly greater reductions in hepatic tumor as well as significant survival benefit of the treated mice. Sites of regressing hepatic tumor exhibited central necrosis accompanied by polymorphonuclear leukocytes and lymphocytes. Collectively, these results show that rTNF-alpha administration can mediate a significant antitumor effect on visceral tumor and suggest that tumor size is an important factor in rTNF-alpha susceptibility not only for tumors growing at s.c. sites but also for those established at visceral sites.

Studies on the anti-tumor efficacy of systemically administered recombinant tumor necrosis factor against several murine tumors in vivo.

The anti-tumor activity of recombinant human tumor necrosis factor (rHTNF) was examined against four newly induced murine sarcomas (MCA-101, -102, -105, and -106) and a murine adenocarcinoma (MCA-38) transplanted s.c. into C57BL/6 mice. The serum half-life after a single i.v. injection of rHTNF was determined to be 30 +/- 2 min. Tumor-bearing mice were more susceptible to the toxic side effects of rHTNF than were normal mice. Forty-eight percent (41/86) of tumor bearing animals that received 10 micrograms rHTNF died within 48 hr after treatment compared with no deaths in 28 normal animals receiving this dose. Treatment of mice bearing either the MCA-101, -102, -105, or -106 sarcoma or the MCA-38 adenocarcinoma with rHTNF resulted in a marked necrosis of the central portion of each tumor within 24 hr. Animals bearing the weakly immunogenic tumors MCA-105, -106, and -38 experienced a reduction in average tumor area of 47% +/- 5, 46% +/- 6, and 37% +/- 11, respectively, by 3 to 4 days after treatment with rHTNF compared with pre-treatment values (p less than 0.001); increases of 79% +/- 11, 74% +/- 10, and 41% +/- 6 were seen in excipient-treated control animals over the same period. In contrast, animals bearing the non-immunogenic tumors MCA-101 and -102 experienced little if any decrease in tumor area at the doses of rHTNF used. rHTNF failed to mediate cures in animals bearing MCA-38, -101, or -102. In contrast, 67 and 28% of animals bearing MCA-105 and -106, respectively, which received 6 to 10 micrograms rHTNF were cured. Likewise, animals bearing MCA-105 and -106 sarcomas treated with 6 to 10 micrograms rHTNF had significantly increased survival compared with excipient-treated control animals. In contrast, no significant difference in mean survival was observed between excipient and rHTNF treated animals bearing MCA-38, -101, or -102. Histologically, the necrotic response of immunogenic MCA-106 and non-immunogenic MCA-102 tumors to systemically administered rHTNF was very similar. These two tumors differed morphologically, however, by the greater degree of chronic inflammation that was present at the periphery of the MCA-106 tumor in comparison with the MCA-102. By 72 hr after rHTNF administration, the sites of regressed MCA-106 tumors were replaced by a heterogeneous population of inflammatory cells and tumor cell "ghosts".(ABSTRACT TRUNCATED AT 400 WORDS)

Complete axillary lymph node dissection for stage I-II carcinoma of the breast.

We reviewed the complete axillary dissection specimens of 136 patients with stage I-II breast cancer to clarify the distribution of axillary lymph node metastases in this disease. Our series included 71 patients undergoing axillary dissection as part of a modified radical mastectomy (MRM) and 65 patients undergoing axillary dissection in conjunction with conservative surgery of the breast and definitive postoperative breast radiotherapy (CAD). These two groups of patients were comparable according to age, menopausal status, tumor size, and clinical stage. In all patients the pectoralis minor muscle was excised and all axillary tissue removed. Each specimen contained a median of 23 lymph nodes. The axillary levels (I, II, III) were determined according to the relationship of axillary tissue to the pectoralis minor muscle (lateral, inferior, medial). Thirty-nine percent of the lymph nodes were contained in level I, 41% in level II, and 20% in level III. There were no significant differences noted in the number of lymph nodes or in the distribution of lymph nodes according to axillary level between dissections performed as part of the MRM or those done as a single procedure (CAD). Sixty-five patients (47.8%) had one or more positive lymph nodes in their axillary specimen. The clinical and pathologic stage was determined and compared for all patients. Among patients judged to have a clinically negative axilla, 37.6% had histologically positive lymph nodes (clinical false-negative rate). For patients with a clinically positive axilla, 11.1% had, histologically, no evidence of metastatic disease (clinical false-positive rate). When the distribution of lymph node metastases according to axillary level was studied, it was found that 29.2% of lymph node-positive patients (or 14.0% of all patients) had metastases only to level II and/or III of the axilla, with level I being negative (skip metastases). This incidence of skip metastases was greater among clinically node-negative than among clinically node-positive patients, but was not related to the size or location of the primary tumor in the breast. In addition, it was found that 20.0% of lymph node-positive patients (or 9.6% of all patients) were converted from three or fewer to four or more positive nodes by analysis of lymph nodes contained in levels II and III. This conversion from three or fewer to four or more positive nodes was due primarily to information contained in level II, with level III contributing to a smaller degree.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymph node pathology of HTLV and HTLV-associated neoplasms.

Human T-cell leukemia/lymphoma virus I can transform mature T-lymphocytes in vitro and is associated with the human T-cell cancer, adult T-cell leukemia/lymphoma. Adult T-cell leukemia/lymphoma is a distinct clinicopathological entity associated with leukemia, lymphadenopathy, hepatosplenomegaly, skin lesions, hypercalcemia, and lytic bone lesions. Although morphologically diverse it pursues an aggressive clinical course. Human T-cell leukemia/lymphoma virus III is associated with acquired immunodeficiency syndrome, which in its early stages shows follicular lymphoid hyperplasia; however, lymphoid atrophy is progressive and ultimately results in virtually total lymphoid depletion of lymph nodes. Patients with human T-cell leukemia/lymphoma virus III infections appear to have an increased risk of high-grade B-cell lymphomas and perhaps Hodgkin's disease.

Disseminated bilateral chorioretinitis due to Histoplasma capsulatum in a patient with the acquired immunodeficiency syndrome.

A 31-year-old white male homosexual was healthy until March 1984, when he developed Pneumocystis carinii pneumonia, which resolved with treatment. In April 1984 he developed fever, followed by hepatosplenomegaly, headaches, blurred vision, pancytopenia and pulmonary infiltrates. On June 11, intracytoplasmic yeast were noted within leukocytes on a peripheral blood smear, and amphotericin B was started. The patient developed progressive respiratory and renal insufficiency and died on June 13, 1984. Autopsy histopathology demonstrated disseminated histoplasmosis and Histoplasma capsulatum was cultured from numerous tissues. Ocular histopathologic examination using special fungal stains and electron microscopy revealed numerous budding yeasts characteristic of Histoplasma capsulatum in the choroid, retina and central retinal vein. Their identification as H. capsulatum was confirmed by immunofluorescent staining.

Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2.

Incubation of resting lymphoid cells with recombinant interleukin 2 (IL-2) in vitro leads to the generation of lymphokine activated killer (LAK) cells capable of lysing fresh tumor cell suspensions in short-term chromium-release assays. Our previous studies (7) have demonstrated that the injection of LAK cells plus low doses of recombinant IL-2 were capable of inhibiting the growth of pulmonary metastases. We have now explored the ability of high doses of recombinant IL-2, administered systemically, to generate LAK cells in vivo, and to mediate antitumor effects directly. Administration of increasing doses of recombinant IL-2 intraperitoneally resulted in the generation of LAK cells in the spleens of recipient mice. Doses of 100,000 U recombinant IL-2 administered intraperitoneally approximately every 8 h for 5 d were capable of dramatically inhibiting established 3-d pulmonary metastases from the MCA-105 and MCA-106 syngeneic sarcomas and the syngeneic B16 melanoma in C57BL/6 mice. Grossly visible metastases present at 10 d after tumor injection also underwent regression following IL-2 therapy. Surprisingly, established 10 d pulmonary metastases were more susceptible to the effects of IL-2 than were the smaller 3 d pulmonary metastases. All antitumor effects of the systemic administration of recombinant IL-2 were eliminated if mice received prior treatment with 500 rad total body irradiation. The administration of high doses of recombinant IL-2 was also capable of inhibiting the growth of 3-d established subcutaneous tumors from the MCA-105 sarcoma, and of mediating the inhibition of growth and regression of established palpable subcutaneous MCA-105 sarcomas. Lymphocytes, which appeared morphologically to be activated, were present at the site of regressing tumor, and it appears that the mechanism of the antitumor effect of recombinant IL-2 administered systemically is via the generation of LAK cells in vivo, although this hypothesis remains to be proven. The ready availability of high doses of recombinant human IL-2, and the demonstration of antitumor effects seen in animal models have led us to the initiation of the clinical trials of recombinant IL-2 in humans.

Pachydermodactyly.

Pachydermodactyly is a rare distinct form of fibromatosis characterized by dense fibrosis and fibroblastic proliferation around one or more proximal interphalangeal joints. Pachydermodactyly affects the appositional and dorsal aspects of proximal digits II-V. The lateral aspects of the index fingers, the medial aspect of the fifth digits, and the thumbs are usually unaffected. A typical patient was a young adult man with a long history of idiopathic asymptomatic swelling in the fingers. Skin biopsy specimens obtained from three patients with pachydermodactyly showed marked thickening of the dermis with extension of collagenous fibers into the subcutaneous tissues. There was poor demarcation between the papillary and reticular dermis. The cytology of the fibroblasts appeared benign. Isolation of collagen from the involved sites revealed collagen Types I, III, and V in a pattern typical of fibromatoses; i.e. increased Types III and V. Compared with the collagen profile of normal reticular dermis, increased numbers of fine-diameter collagen fibers were notable in electron micrographs.

An erythrocyte Pr auto-antibody with sialoglycoprotein specificity in a patient with purine nucleoside phosphorylase deficiency.

A warm auto-antibody with specificity in the Pr blood group system was demonstrated in the serum and red cell eluate of a patient with purine nucleoside phosphorylase (NP) deficiency. The antibody reacted with all cells tested except En(a-) red cells which lack glycophorin A, the major erythrocyte sialoglycoprotein. However, anti-Ena was ruled out by absorption of the antibody with En(a-) red cells. The antibody demonstrated similar serologic characteristics to Pra antibodies, except that those previously described were inactive with protease-treated red cells, while in this case, reactivity was destroyed by papain and ficin but maintained in the presence of trypsin. Inhibition analysis with purified glycoprotein fragments localized the predominant reactive antigen on the MN sialoglycoprotein between amino acid residues 40 and 61. Serologic tests demonstrated its presence in decreased amount on at least one other erythrocyte membrane structure. The serum from another patient with NP deficiency contained an autoantibody similar to the one described here. It may be of interest to explore the association of auto-antibodies to erythrocyte sialoglycoprotein antigens in NP and other immune deficiency states.

Curative intravenous adoptive immunotherapy of Meth A murine sarcoma. A histologic and immunohistochemical assessment.

Intravenous administration of 1.5 X 10(8) syngeneic spleen cells from immune animals resulted in the complete eradication of established Meth A soft tissue sarcomas in (C57BL/6 X BALB/c) F1 mice. In mice receiving a single injection of immune spleen cells 4 days after tumor implantation in the abdominal wall, the tumors continued to grow for approximately 1 week before undergoing regression. This delay before adoptive immunity is expressed is thought to represent the time needed for the passively transferred cells to give rise to a host response of sufficient magnitude to destroy the tumor. None of the mice receiving a similar number of control spleen cells were cured of their sarcomas. Successful therapy was dependent upon the transfer of viable, immune T lymphocytes and required prior irradiation of the tumor-bearing host in order to remove suppressor T cells. Utilizing sequential histologic and immunohistochemical techniques, we attempted to characterize the cellular events of tumor regression. The earliest histologic difference between animals treated with immune and nonimmune lymphocytes was in the number of lymphocytes detected at the perimeter of the tumor in specifically immunized mice on day 6. There was also a striking difference between animals treated with immune versus nonimmune lymphocytes in the intensity and timing of the acute inflammatory response beginning on day 8. The "front" of immunologically mediated tumor destruction appeared at the lateral and deep borders of the implanted sarcomas and progressed inward. During the period of active tumor regression T lymphocytes reactive with a biotinylated mouse anti-Thy 1.2 monoclonal antibody were increased in frozen sections of tumors in mice receiving immune cells relative to the controls. During the first 3 weeks following adoptive transfer of lymphocytes, T cells reactive with Lyt-1 biotinylated mouse monoclonal antibody (helper/inducer phenotype) outnumbered their Lyt-2 (suppressor/cytotoxic) counterparts in frozen sections of tumor from both specifically immunized and control mice. By the end of the 4th week of the experiment, the sarcomas were completely eradicated in all mice receiving immune cells. The previous tumor beds were occupied by collections of lipid-laden macrophages, lymphocytes, plasma cells, and fibroblasts. Despite vigorous but delayed acute and chronic inflammatory responses at the tumor perimeters in the control mice, these tumors all progressed.(ABSTRACT TRUNCATED AT 400 WORDS)