Selective changes in EGF receptor expression and function during the proliferation, differentiation and apoptosis of mammary epithelial cells.

Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.

New approaches to the systemic treatment of melanoma.

Metastatic melanoma is an incurable condition with a median survival of about 6 months. Chemotherapy can result in objective tumour responses but only in a minority of cases and remissions are short-lived, 3-6 months. DTJC is the most active single agent with response rates of 15-20% and although combination chemotherapy can result in higher response rates there is no response duration or survival advantage. Phase II studies have suggested that combining chemotherapy with biological response modifiers may result in higher response rates, in the order of 50% and the results of two large randomized trials investigating this approach are awaited. Adjuvant trials currently focus on interferon and/or vaccine strategies. Further data are required before any adjuvant treatment can be regarded as standard.

Changes in ErbB2 (her-2/neu), ErbB3, and ErbB4 during growth, differentiation, and apoptosis of normal rat mammary epithelial cells.

Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs.

D-14 monoclonal antibody to carcinoembryonic antigen: immunohistochemical analysis of formalin-fixed, paraffin-embedded human colorectal carcinoma, tumors of non-colorectal origin and normal tissues.

The reactivity of D-14 monoclonal antibody (mAb) to a specific epitope of carcinoembryonic antigen (CEA) was evaluated on formalin-fixed, paraffin-embedded tissues. A total of 52 normal tissues, 90 colorectal carcinomas and 127 non-colorectal neoplasms were tested using the peroxidase/antiperoxidase technique. D-14 mAb did not react with normal tissues apart from producing a weak staining of normal colonic glands immediately adjacent to the neoplastic structures. All 61 primary and 29 metastatic colorectal carcinomas expressed the carcinoembryonic antigen. However, there was considerable heterogeneity in cellular antigen expression in both primary and metastatic colorectal carcinomas with 10%-99% of tumor cells staining. Of 22 stomach adenocarcinomas, 14 were also immunoreactive, as were 2 of 5 pancreatic carcinomas. Only 6 of 100 neoplasms of non-gastrointestinal origin expressed weak to moderate immunoreactivity. In 7 cases, colorectal micrometastases not recognized in conventional hematoxylin and eosin slides could be identified with D-14 mAb. The specificity of this antibody could be used in differentiating colorectal carcinomas from other types of tumors, including adenocarcinoma from other sites.

Survival of patients with primary fallopian tube carcinoma.

Vaughan MM, Evans BD, Weitzer MJ. Survival of patients with primary fallopian tube carcinoma. Int J Gynecol Cancer 1998; 8: 16-22. Thirty-seven patients with primary fallopian tube carcinoma (PFTC) presenting between 1952 and 1995 were studied. The mean age was 57 years. Seven patients had stage I disease, 20 stage II, 8 stage III, and 2 stage IV. Actuarial 5-year survivals were 73% for stage I, 33% for stage II and 0% for stage III. Stage was a significant predictor of survival at 5 years (Stage I vs. III, P = 0.0006; stage II vs. III, P = 0.0001), however, the majority of patients, even with early stage disease, died of progressive PFTC within 10 years. Grade appeared highly significant at 5 and 10 years (Grades 1 & 2 vs. 3, P = 0. 0023). Neither age nor lymphocytic infiltrate appeared definitely predictive of survival. Eleven of 22 stage II patients received adjuvant treatment. While their median and 5-year survivals were superior to those not receiving adjuvant treatment (51 vs. 30 months, 47% vs. 22%), the difference was not statistically significant. This retrospective analysis confirms the poor prognosis of patients with PFTC. The majority of patients, even with early stage tumors, eventually succumb to their disease. Larger studies may identify a group of patients potentially curable with surgery alone, and clarify the role of adjuvant therapy.

Idiopathic spontaneous tonsillar haemorrhage.

Since the advent of antibiotics major spontaneous tonsillar haemorrhage is extremely rare. The causes are usually ascribed to vessel wall erosion secondary to bacterial infection or infectious mononucleosis. We report the case of a 20-year-old female, presenting to the ENT department with tonsillar bleeding in the absence of any identifiable infective or traumatic process.

An immunohistochemical study of vitamin D receptor expression in canine cutaneous mast cell tumours.

The active form of vitamin D (1alpha, 25-dihydroxycholecalciferol; calcitriol) has potent anti-neoplastic activity in the management of a number of human malignancies. Despite promising data to suggest that calcitriol is an effective adjunct to current chemotherapy modalities, the role of calcitriol in animal neoplasia is poorly understood. Vitamin D inhibits growth of canine mast cell tumours (MCTs) in vitro, presumably due to ligand-mediated activation of the vitamin D receptor (VDR). The aim of the present study was to examine immunohistochemically the expression of the VDR by reactive and neoplastic canine cutaneous mast cells. Expression was graded according to frequency, intensity and score (frequency x intensity). VDR expression was found in all samples containing reactive mast cells (n=9), and in 67 of 69 (97%) MCTs selected from each of the three Patnaik grades. The frequency and score of VDR labelling was greater in MCTs compared with reactive mast cells (P=0.0005 and 0.001, respectively). There was no difference in VDR frequency between the MCT grades, but the frequency of labelling in grade 3 MCTs was higher than for reactive mast cells (P=0.001). There was no association between tumour mitotic index and any of the three VDR variables (all P>0.16). VDR is widely expressed by reactive and neoplastic canine mast cells in vivo. VDR expression is unlikely to represent an independent prognostic factor, but its presence within biopsy specimens might be used to identify patients that are suited to high-dose vitamin D therapeutic trials.

Histopathologic technique for the morphological examination of fixed or frozen solid tumor cells in soft agar.

Two simple techniques are described for preparing sections from soft agar colony cultures of tumor cells. Tumor cells grown in soft agar can be frozen, sectioned, and stained and/or fixed in formalin, embedded in paraffin, sectioned, mounted on glass slides, and stained. The methods are simple and reproducible. These cells can be stained with various stains and the staining quality is excellent. The paraffin blocks and microscope slides can be stored for permanent record. The use of these techniques should provide better understanding of the histomorphologic characteristics of neoplastic cells which grow in soft agar and should expand and refine prognosis and diagnosis of malignant tumors.

MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchyma. To examine whether the endothelial cells of the newly formed capillaries during neoangiogenesis within malignant human brain tumors express MDR1 Pgp, 35 adult surgical brain tumor specimens (29 gliomas and 6 tumors metastatic to the brain) were obtained from previously untreated patients and studied by a new immunohistochemical sandwich method developed in our laboratory using the JSB-1 monoclonal antibody. JSB-1 is specific for the Pgp product of the human MDR1 (and not MDR3) gene. This sensitive method allows the detection of Pgp in capillary endothelial cells of normal brain in conventional paraffin sections after formalin fixation. The endothelial cells of the newly formed capillaries in 25 of 29 gliomas (86%) and 3 of 6 metastatic tumors, immunostained positive for MDR1 Pgp. The tumor cells in 7 of 35 cases were also positive for Pgp. In the 35 brain tumor cases investigated, the endothelial cells were Pgp positive in the tumor-brain border and in the brain further from the tumor. Capillary endothelial cells of neovasculature in 137 malignant tumors (non-brain) obtained from previously untreated patients showed no MDR1 Pgp expression. These results demonstrated that MDR1 Pgp is expressed not only in the capillaries of normal brain but also in the majority of the newly formed capillaries of brain tumors. Multidrug resistance of brain tumors may result not only from the expression of resistance markers in neoplastic cells but also from the MDR1 Pgp expression in endothelial cells of tumor capillaries. Pgp in this special localization can exclude chemotherapeutic agents from tumor cells that are located around the capillaries. The therapeutic benefit and selectivity of chemotherapeutic agents in combination with a Pgp-reversing agent should be evaluated.

Phase I study of carboplatin, doxorubicin and weekly paclitaxel in patients with advanced ovarian carcinoma.

BACKGROUND: Doxorubicin is an active compound in epithelial ovarian cancer (EOC), but adding it to carboplatin-paclitaxel causes toxicity. Toxicity can be reduced by weekly administration. We examined the tolerability of weekly paclitaxel in combination with carboplatin and doxorubicin. PATIENTS AND METHODS: Chemotherapy naïve patients with EOC were treated with doxorubicin (50 mg/m(2) day 1), carboplatin (AUC 6 day 1) and paclitaxel (days 1, 8, 15, 21), 28-day cycle. Three patients were treated at each paclitaxel dose level, starting at 60, 75 and 90 mg/m(2)/week. If more than two patients in a cohort experienced dose-limiting toxicity (DLT) three more patients were treated at the dose level below. RESULTS: Twelve patients with advanced EOC received a median of six cycles (range 2-6) of the three-drug combination. DLT occurred at dose level 3: prolonged grade 4 febrile neutropenia, 1 patient; grade 3 peripheral neuropathy, 1 patient. All six patients treated at dose level 2 experienced short-lived grade 4 neutropenia, which led to dose modifications resulting in an actual delivered dose of paclitaxel of 64 mg/m(2)/week. Eight out of 12 patients had measurable disease on CT scan: four obtained a partial remission; three had stable disease. CONCLUSIONS: The combination of carboplatin, doxorubicin and paclitaxel in patients with EOC is active and its main toxicity is myelosuppression. Dose intensity of paclitaxel can be maintained in a three-drug combination through weekly administration (65 mg/m(2)).

Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01-10 micrograms/ml) and EGF (1-100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland.

Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies.

We used a strain of differentiated rat-thyroid cells in continuous culture (the FRTL-5 strain) to detect the presence of growth-promoting antibodies in serum samples from patients with autoimmune thyroid disease. We found that IgG preparations from 17 of 20 patients (85 per cent) with active Graves' disease and two of five patients (40 per cent) with Hashimoto's thyroiditis could augment thyroid-cell growth. In parallel with IgG-induced elevations in intracellular cyclic AMP levels in the same cell line, all 20 of the patients with active Graves' disease had thyroid-stimulatory antibodies. Patients' IgG preparations fell into three subclasses: those with both potent cyclic AMP stimulation and potent growth-promoting activity; those with potent cyclic AMP stimulation but low-level growth promotion; and those with potent growth promotion and low-level cyclic AMP action. Growth-promoting antibodies were not detected in patients with Graves' disease in remission (seven patients), nodular goiter (seven), subacute thyroiditis (five), or atrophic thyroiditis (one). Simultaneous assays of growth promotion and cyclic AMP stimulation may be useful in the care of patients with autoimmune thyroid disease.

Altered expression and localization of PKC eta in human breast tumors.

Protein kinase C (PKC) eta is a PKC isoform whose upregulation is associated with differentiation in many epithelial tissues, including the rat mammary gland. The purpose of this study was to examine whether PKC eta is altered, in expression or localization, in human breast cancer. Paraffin sections of 49 in situ breast lesions, 29 invasive breast tumors, and nine normal breast biopsies were examined for PKC eta expression by immunohistochemistry. Adjacent regions of normal epithelium, and in situ lesions that were present adjacent to invasive lesions were also analyzed. In normal epithelium, regardless of the presence of adjacent in situ or invasive lesions, PKC eta was present in the cytoplasm of the luminal epithelium, and increased in areas of normal lobular development, similar to normal rat mammary gland. PKC eta staining intensity was homogeneous in normal lobules, but heterogeneous in in situ and invasive lesions, being focally increased in cells with aberrant nuclear morphology. In situ lesions were similar to adjacent normal epithelium in average staining intensity, regardless of whether invasion was also present. However, the invasive lesions themselves were significantly decreased in staining intensity compared to adjacent in situ lesions. In addition, 75% of invasive breast cancer lesions showed decreased staining relative to adjacent normal epithelium, compared to 37% of in situ lesions. The invasive tumors which possessed high PKC eta staining were associated with positive lymph node status. These results demonstrate that quantitative and qualitative alterations in PKC eta occur in human breast cancers.

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.

Continuous low dose Thalidomide: a phase II study in advanced melanoma, renal cell, ovarian and breast cancer.

To grow and metastasize, solid tumours must develop their own blood supply by neo-angiogenesis. Thalidomide inhibits the processing of mRNA encoding peptide molecules including tumour necrosis factor-alpha (TNF-alpha) and the angiogenic factor vascular endothelial growth factor (VEGF). This study investigated the use of continuous low dose Thalidomide in patients with a variety of advanced malignancies. Sixty-six patients (37 women and 29 men; median age, 48 years; range 33-62 years) with advanced measurable cancer (19 ovarian, 18 renal, 17 melanoma, 12 breast cancer) received Thalidomide 100 mg orally every night until disease progression or unacceptable toxicity was encountered. Three of 18 patients with renal cancer showed partial responses and a further three patients experienced stabilization of their disease for up to 6 months. Although no objective responses were seen in the other tumour types, there were significant improvements in patients' sleeping (P < 0.05) and maintained appetite (P < 0.05). Serum and urine concentrations of basic fibroblast growth factor (bFGF), TNF-alpha and VEGF were measured during treatment and higher levels were associated with progressive disease. Thalidomide was well tolerated: Two patients developed WHO Grade 2 peripheral neuropathy and eight patients developed WHO grade 2 lethargy. No patients developed WHO grade 3 or 4 toxicity. Further studies evaluating the use of Thalidomide at higher doses as a single agent for advanced renal cancer and in combination with biochemotherapy regimens are warranted.

GM-CSF with biochemotherapy (cisplatin, DTIC, tamoxifen, IL-2 and interferon-alpha): a phase I trial in melanoma.

BACKGROUND: Ineffective tumour antigen processing is recognised as an important cause of failure of immunotherapy in melanoma. GM-CSF may augment the cytotoxic lymphocyte response by activating antigen-presenting cells. This study evaluates a schedule combining GM-CSF with biochemotherapy. PATIENTS AND METHODS: Nineteen patients with advanced malignant melanoma received cisplatin (25 mg/m2 days 1-3). dacarbazine (220 mg/m2 days 1-3), interleukin-2 (9 MIU/m2/24 h) and interferon-alpha2b (5 MIU/m2) both days 6-10 and days 17-21, and tamoxifen 40 mg/day continuously. Subcutaneous GM-CSF was given in escalating doses to three cohorts: 1) 450 microg/m2 days 4-5 and 15-16; 2) as 1) plus 225 microg/m2 days 6-10 and 17-21; 3) 450 microg/m2 days 4-10 and 15-21. Each cycle was 28 days. RESULTS: Constitutional side effects were the major non-haematological toxicity and lymphopaenia the main haematological toxicity. Six patients responded (32%, 95% confidence interval: 13%-57%), two patients had complete remission. There was an apparent trend for increasing responses with increasing GM-CSF dose; zero of six responses in cohort 1, two of seven in cohort 2 and three of six in cohort 3 (P = 0.016). Median overall survival was 6.2 months. Increasing GM-CSF doses significantly increased serum concentrations of neopterin and TNF-alpha. CONCLUSIONS: The combination of GM-CSF with biochemotherapy is feasible and there appears to be a dose-response relationship with GM-CSF in terms of host immunological response, and possibly clinical efficacy.

Comparison of an immunoperoxidase "sandwich" staining method and western blot detection of P-glycoprotein in human cell lines and sarcomas.

The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated. The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas. The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox). The sarcoma biopsy specimens were selected on the basis of the results obtained in Western blot (WB) detection of Pgp (22 positive and 24 negative by WB) using C219 and C494 monoclonal antibodies to Pgp. The IpS method employed C219. The least resistant cell line in which Pgp could be detected by IpS was fivefold resistant to doxorubicin, whereas Pgp was detected by WB in cells greater than twofold resistant. Cell lines having greater than fivefold resistance to Dox were positive by both IpS and WB analyses. The less resistant cell lines contained more nonreactive cells whereas the highly resistant cell lines showed more homogeneous strong membrane reactions. Among the six cell lines determined to be Pgp negative by WB analysis, no false positive immunostaining by IpS existed. One of 22 WB positive and 7 of 24 WB-negative sarcoma biopsy specimens were positive by IpS methods. Reaction varied and was always focal (a minimum of 3-5 cells, ranging up to 3-4 high power fields) indicating pronounced heterogeneous distribution of Pgp. Thus, WB can detect low average (overall) levels of Pgp in tumor samples but such low concentrations of PgP at the single cell are not detectable by IpS methods. However, IpS can discern among many Pgp-negative cells small subpopulations of immunoreactive cells, which are not detected by WB analysis due to Pgp dilution by the membrane protein of numerous Pgp negative cells. IpS and WB used together as complementary methods can provide more complete information about Pgp distribution and content, particularly in the case of heterogeneous human tumors. The IpS method is more suitable for less drastically treated (not embedded) cell line specimens than for paraffin-embedded (routine) sections. Some modification of the present IpS protocol seems necessary to increase its sensitivity and reduce the disparity with WB results.