Barry M. Kacinski

Identification of the uvrA gene product

Abstract The proteins synthesized by the plasmid pDR2000, a recombinant plasmid with the Escherichia coli uvrA and ssb (lexC) genes cloned on pBR322, were examined using the maxicell procedure developed by Sancar et al. (1979). The proteins synthesized by this plasmid include those expected from pBR322 plus two others, a small protein (Mr = 18,500) and a large one (Mr = 114,000). The molecular weight and DNA binding properties of the 18,500 Mr protein indicate that it is the product of the ssb gene. Derivatives of pDR2000 were prepared in which the uvrA gene was inactivated by insertion of the γδ transposon (Tn1000). In each of these derivatives the 114,000 Mr protein was not made, indicating that it is the product of the uvrA gene. By extrapolating from the relative amounts of ssb and uvrA proteins synthesized in maxicells, we estimate that a normal cell contains only about 20 uvrA polypeptide chains. The uvrA protein binds to both single and double-stranded DNA. A procedure is described for purifying uvrA protein to radiochemical homogeneity, and subsequent gel filtration of this isolated native uvrA protein shows that it is a monomer. When γδ is inserted into the uvrA gene, truncated uvrA polypeptides resulting from premature termination at nonsense codons in the inserted sequence are observed. From the sizes of these truncated polypeptides and the sites of the insertions determined by restriction mapping, the direction of transcription of the uvrA gene was determined to be counter-clockwise on the E. coli genetic map.

CSF-1 and its receptor in breast carcinomas and neoplasms of the female reproductive tract.

The expression and function of CSF‐1 and its receptor were studied in tumors of the human breast, ovary, and endometrium. CSF‐1 and its receptor, initially implicated as essential to normal monocyte development and trophoblastic implantation, have been more recently shown to be expressed by carcinomas of the breast and other epithelia of the female reproductive tract where activation of the receptor by ligand produced either by the tumor cells or by stromal elements stimulates tumor cell invasion by a urokinase‐dependent mechanism.

Smooth muscle releases an epithelial cell scatter factor which binds to heparin

SummaryWe report that culture bovine calf aorta and human adult iliac artery smooth muscle cells release a soluble factor which causes spreading and separation of cells in normally tight, cohesive epithelial colonies, similar to the morphologic changes induced by the fibroblast-derived scatter factor (SF). Smooth muscle-derived SF was heat sensitive, trypsin labile, and nondialyzable, consistent with a protein (or proteins). Its effects on epithelium were not mimicked by a variety of proteolytic enzymes, growth factors, or hormones, and were not blocked by antiproteases or by antibodies to fibronectin and basic fibroblast growth factor. Epithelial cell proliferation was unaffected or only mildly stimulated by partially purified SF at concentrations that produced cell scattering. Both smooth muscle-and MRC5 human embryo fibroblast-derived SFs could be partially purified with similar elution patterns on a number of different chromatographic columns, including DEAE-agarose, heparin-sepharose, Bio-Rex 70, concanavalin A-sepharose, and MonoQ. SF from both sources bound tightly to heparin-sepharose, requiring 1.3 to 1.4M NaCl for elution. The morphologically obvious cell scattering effect was markedly inhibited by soluble heparin at concentrations down to 5 μg/ml, and this inhibition was prevented by protamine. These data suggest that vascular smooth muscle cells produce an epithelial cell scattering factor with properties similar to the fibroblast-produced factor, including a high affinity for heparin. Such factors are potentially important because they may represent a new class of proteins that primarily regulate cell mobility rather than growth and differentiation.

Microsatellite instability in ovarian neoplasms.

Microsatellite instability has been observed in a variety of sporadic malignancies, but its existence in sporadic ovarian cancer has been the subject of conflicting reports. We have performed a polymerase chain reaction-based microsatellite analysis of DNAs extracted from the neoplastic and non-neoplastic tissues of 41 ovarian cancer patients. Tumour-associated alterations were observed in seven (17%) of these cases. Clinicopathological correlations revealed that: (1) alterations among tumours classified as serous adenocarcinomas occurred with relatively low frequency (2/24 or 8%); (2) most of the tumours with microsatellite alterations (5/7 or 71%) were of less common histopathological types (epithelial subtypes such as endometrioid and mixed serous and mucinous, or non-epithelial types such as malignant mixed Müllerian or germ cell tumours); (3) tumour-associated alterations were observed in 3/4 (75%) of the patients with stage I tumours vs 4/37 (11%) of the patients with stage II, III and IV tumours (P = 0.01); (4) tumour-associated microsatellite instability was found to occur with similar frequencies among patients with and without clinical features suggestive of familial disease, including positive family history, early onset, or multiple primary tumours. In summary, we have observed microsatellite alterations in the neoplastic tissues of ovarian cancer patients with diverse genetic backgrounds and clinicopathological features. The pattern of alterations is consistent with the possibility that multiple mechanisms may be responsible for microsatellite instability in ovarian neoplasms.

Expression of colony-stimulating factor 1 receptor during prostate development and prostate cancer progression

Colony-stimulating factor-1 receptor (CSF-1R) is the major regulator of macrophage development and is associated with epithelial cancers of the breast and ovary. Immunohistochemistry analysis of murine prostate development demonstrated epithelial expression of CSF-1R during the protrusion of prostatic buds from the urogenital sinus, during the prepubertal and androgen-driven proliferative expansion and branching of the gland, with a decline in older animals. Models of murine prostate cancer showed CSF-1R expression in areas of carcinoma- and tumor-associated macrophages. Several human prostate cancer cell lines and primary cultures of human prostate epithelial cells had low but detectable levels of CSF-1R. Human prostatectomy samples showed low or undetectable levels of receptor in normal glands or benign prostatic hypertrophy specimens. Staining was strongest in areas of prostatic intraepithelial neoplasia or carcinoma of Gleason histological grade 3 or 4. The activated form of the receptor reactive with antibodies specific for phosphotyrosine modified peptide sequences was observed in samples of metastatic prostate cancer. Immunohistochemistry showed strong expression of CSF-1R by macrophage lineage cells, including villous macrophages and the syncytiotrophoblast layer of placenta, Kupper cells in the liver, and histiocytes infiltrating near prostate cancers. These observations correlate CSF-1R expression with changes in the growth and development of the normal and neoplastic prostate.

Roles of Fas and Fas ligand during mammary gland remodeling

Mammary involution is associated with degeneration of the alveolar structure and programmed cell death of mammary epithelial cells. In this study, we evaluated the expression of Fas and Fas ligand (FasL) in the mammary gland tissue and their possible role in the induction of apoptosis of mammary cells. FasL-positive cells were observed in normal mammary epithelium from pregnant and lactating mice, but not in nonpregnant/virgin mouse mammary tissue. Fas expression was observed in epithelial and stromal cells in nonpregnant mice but was absent during pregnancy. At day 1 after weaning, high levels of both Fas and FasL proteins and caspase 3 were observed and coincided with the appearance of apoptotic cells in ducts and glands. During the same period, no apoptotic cells were found in the Fas-deficient (MRL/lpr) and FasL-deficient (C3H/gld) mice. Increase in Fas and FasL protein was demonstrated in human (MCF10A) and mouse (HC-11) mammary epithelial cells after incubation in hormone-deprived media, before apoptosis was detected. These results suggest that the Fas-FasL interaction plays an important role in the normal remodeling of mammary tissue. Furthermore, this autocrine induction of apoptosis may prevent accumulation of cells with mutations and subsequent neoplastic development. Failure of the Fas/FasL signal could contribute to tumor development.

Macrophage colony-stimulating factor-1 receptor expression is associated with poor outcome in breast cancer by large cohort tissue microarray analysis.

Purpose: Macrophage colony-stimulating factor-1 receptor (CSF-1R) is a transmembrane tyrosine kinase receptor, which is abnormally expressed in invasive breast cancer. Small cohort studies have demonstrated that increased expression of CSF-1R is associated with ipsilateral breast cancer recurrence. Correlation with survival has not been reported. Our aim was to further evaluate the role of CSF-1R in breast cancer, by studying the expression of CSF-1R in a large cohort of clinical specimens. Experimental Design: Tissue microarrays containing 301 node-negative and 280 node-positive cases were used. Immunohistochemical staining was performed and correlated with overall survival, nodal status, and other clinicopathological data. Results: CSF-1R expression was strongly associated with nodal status. Of the node-negative cases, 114 (38.9%) stained positive for CSF-1R, whereas 189 (67.5%) of the node-positive cases expressed CSF-1R (P < 0.0001). CSF-1R expression is also associated with larger tumor size (P = 0.02). Positive staining was strongly associated with decreased survival (P = 0.0003). Among node-negative patients, CSF-1R expression was associated with decreased overall survival (P = 0.045), whereas among node-positive patients, it was not (P = 0.47). In multivariate analysis, CSF-1R was not independent of nodal status as a predictor of survival. Conclusions: CSF-1R expression is a strong predictor of poor outcome in nonmetastatic breast cancer. It is significantly more frequently expressed in patients with nodal involvement. Among the node-negative patients, it has a stronger association with survival than among the node-positive patients. Our findings support other preclinical findings that CSF-1R may be involved in local invasion and metastasis. Thus, this receptor may be an effective target for therapeutic agents.

Total skin electron beam therapy followed by adjuvant psoralen/ultraviolet-A light in the management of patients with T1 and T2 cutaneous T-cell lymphoma (mycosis fungoides)

PURPOSE Patients with mycosis fungoides [cutaneous T-cell lymphoma (CTCL)] may benefit from adjuvant therapy after completing total skin electron beam therapy (TSEBT). We report the results for T1/T2 CTCL patients treated with adjuvant oral psoralen plus ultraviolet light (PUVA) with respect to overall survival (OS), disease-free survival (DFS), salvage of recurrence, and toxicity. METHODS AND MATERIALS Between 1974 and 1993, TSEBT was administered to a total of 213 patients with CTCL. Records were reviewed retrospectively, and a total of 114 patients were identified as having T1 or T2 disease. Radiotherapy was provided via a 6-MeV linac to a total of 36 Gy, 1 Gy/day, 4 days/week, for 9 weeks. Beginning in 1988, patients were offered adjuvant PUVA within 2 months of completing TSEBT. This was started at 0.5-2 J/m2, 1-2 treatments/week, with a taper over 3-6 months. Therapy then continued once per month. There were 39 T1 and 75 T2 patients. Six T1 (15%) and eight T2 (11%) patients were treated with adjuvant PUVA. A further 49% of the 114 patients received adjuvant systemic therapy, 3% received spot external beam, 4% received adjuvant ECP, 2% received topical nitrogen mustard, 22% received a combination of therapies exclusive of PUVA, and 9% received no adjuvant therapy. Patients were balanced in all subgroups based on pre-TSEBT therapy. The median age of the cohort was 58 (range 20-88), with a median follow-up time of 62 months (range 3-179). RESULTS Within 1 month after completing of TSEBT, 97% of T1, and 87% of T2 patients had achieved a complete remission. Stratified by adjuvant therapy, none of six T1 and one of eight T2 patients who received adjuvant PUVA failed within the first 3 years after completion of TSEBT. A total of 43% of the T1 and T2 patients receiving other or no adjuvant treatment failed within the same time course. The 5-year OS for the entire cohort was 85%. Those who received PUVA had a 5-year OS of 100% versus a 5-year OS for the non-PUVA group of 82% (p < 0.10). The 5-year DFS for the entire cohort was 53%. Those who received PUVA had a 5-year DFS of 85% versus a 5-year DFS for the non-PUVA group of 50% (p < 0.02). By T stage, those with T1 receiving PUVA exhibited no relapses, whereas those with T1 not treated with PUVA had a crude relapse rate of 36%. Median DFS was not reached at 103 months for the T1 adjuvant PUVA patients versus 66 months for the non-PUVA patients (p < 0.01). For those with T2, crude relapse rates were 25% and 55%, respectively, with DFS of 60 (median DFS not reached) and 20 months (p < 0.03). The 5-year DFS for patients salvaged with PUVA was 50%. Toxicity of adjuvant and salvage PUVA therapy was acceptable, with only two patients requiring a reduction in PUVA dosage. CONCLUSION PUVA can maintain remissions in patients with CTCL after TSEBT. There is a significant benefit in DFS but no statistically significant improvement in OS. Prospective, randomized data are needed to confirm these results. PUVA is also effective as a salvage therapy after TSEBT in early-stage patients with recurrence, with acceptable toxicity.

Systemic chemotherapy and extracorporeal photochemotherapy for T3 and T4 cutaneous T-cell lymphoma patients who have achieved a complete response to total skin electron beam therapy

PURPOSE To evaluate the impact of systemic adjuvant therapies on relapse-free (RFS) and overall survival (OS) of cutaneous T-cell lymphoma (CTCL) patients treated with total skin electron beam therapy (TSEBT). METHODS AND MATERIALS Between 1974 and 1990, TSEBT (36 Gy at 1 Gy/day; 9 weeks; 6 MeV electrons) was administered with curative intent to a total of 163 CTCL (mycosis fungoides) patients using six fields supplemented by orthovoltage boosts (120 kvp, 1 Gy x 20) to the perineum, soles of feet, and apical scalp (120 kvp, 2 Gy x 3). In this group, all patients who achieved a clinical complete response or a good partial response were offered one of two competing regimens of either adjuvant doxorubicin/cyclophosphamide or adjuvant extracorporeal photochemotherapy (ECP). RESULTS When the results for the group who achieved a complete response (CR) to TSEBT were analyzed, OS for T1 and T2 patients was excellent (85-90% at 5-10 years) and not improved by either adjuvant regimen. However, T3 and T4 patients who received either adjuvant doxorubicin/cyclophosphamide (75% at 3 years) or adjuvant ECP (100% at 3 years) had better overall survival than those who received neither adjuvant regimen (approximately 50% at 5 years). The difference between the OS curves for those who received ECP vs. those who received no adjuvant therapy approached statistical significance (p < 0.06), while a significant survival benefit from the addition of chemotherapy for TSEBT complete responders was not observed. Neither adjuvant therapy provided benefit with respect to relapse free survival after TSEBT. CONCLUSIONS These results suggest that an adjuvant nontoxic regimen of extracorporeal photochemotherapy may prolong survival in advanced stage CTCL patients who have achieved a complete remission after TSEBT. The combination of doxorubicin/cyclophosphamide had no significant impact on overall survival in those patients who achieved CR to TSEBT, and neither adjuvant therapy had an impact on relapse free survival for all T-stages. Such results are the basis for the current development of a prospective, randomized trial studying the impact of ECP after TSEBT in patients with advanced stage CTCL.

Chemoradiation as primary or adjuvant treatment for locally advanced carcinoma of the vulva

PURPOSE To determine the impact of primary or adjuvant chemotherapy and radiation (CRT) on the survival rates of patients with locally advanced vulvar carcinoma. METHODS AND MATERIALS Between 1973 and 1998, 54 patients with vulvar cancer were treated with radiation therapy, among which 20 received CRT, while 34 patients received radiation therapy (RT) alone. Of the 20 patients, 14 were treated for primary or recurrent disease (pCRT), and 6 after radical vulvectomy for high-risk disease (aCRT). Of the 34 patients, 12 were treated primarily (pRT) and 22 received adjuvant treatment (aRT). Chemotherapy consisted of 2 courses of 5-fluorouracil (5-FU) and mitomycin C administered during RT. Six patients received cisplatin in place of mitomycin C. In CRT groups, radiation was administered to the vulva, pelvic, and inguinal lymph nodes to a median dose of 45 Gy with additional 6-17 Gy to gross disease. In RT groups, the median dose to the microscopic diseases was 45 Gy. Nine patients received external beam boost and 16 patients received supplementary brachytherapy in the forms of (226)Ra or (241)Am plaques to sites of macroscopic disease. RESULTS Overall survival was superior in the patients treated with pCRT versus pRT with statistical significance (p = 0.04). There was also a statistically significant improvement in disease-specific (p = 0.03) and relapse-free survival (p = 0.01) favoring pCRT. No statistically significant trends of improved survival rates favoring aCRT over aRT were observed. CONCLUSION Concurrent radiation therapy and chemotherapy decreases local relapse rate, improves disease-specific and overall survival over RT alone as primary treatment for locally advanced vulvar cancer.