Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma.

BACKGROUND: The clinical outcome is generally positive for patients with node-negative breast carcinoma (i.e., those who do not have detectable metastases in the lymph nodes) who have been treated with surgery or surgery plus radiation therapy. In about 30% of the patients, however, the disease recurs, and they are at risk of death. Determination of valid new prognostic indicators would improve the ability to identify patients at high risk of recurrence. Breast cancer can entail substantial development of new blood vessels within the tumor tissue, and it is known that the growth and metastasis of solid tumors are dependent on such angiogenesis. The conversion of tumor cells to an angiogenic phenotype may be preceded by a change in the balance of angiogenic growth factors and angiogenesis inhibitors. PURPOSE: This study was conducted to determine if the levels of vascular endothelial growth factor (VEGF) protein, a potent endothelial growth factor and mediator of vascular permeability and angiogenesis, measured in the primary tumors of women with node-negative breast cancer are associated with known prognostic factors and patient survival. METHODS: By use of a selective enzymatic immunoassay, levels of VEGF protein were measured in cytosolic extracts of primary tumor tissue surgically obtained from 260 women with node-negative breast carcinoma who had been treated with surgery with or without radiation therapy but not with adjuvant therapy and who had been followed for a median time of 66 months. The relationships between VEGF concentrations and other prognostic dichotomous variables or clinical outcome were tested by the use of the Kolmogorov-Smirnov test and univariate and multivariate Cox analyses, respectively. The relationship between VEGF and hormone receptors (i.e., those for estrogen and progesterone) was examined by the use of Spearman's correlation analyses. All P values resulted from the use of two-sided statistical tests. RESULTS: Tumors from 247 (95%) of the 260 patients had detectable VEGF, ranging in concentration from 5.0 to 6523 pg/mg protein (median, 126.25 pg/mg protein). No statistically significant associations were found between VEGF and the other prognostic factors (e.g., age, menopausal status, histologic tumor type, tumor size, and hormone receptors) examined. Levels of VEGF were found to be prognostic for both relapse-free and overall survival in univariate and multivariate analyses (likelihood ratio tests; all four P values < .001). In the multivariate analysis, the first-order interaction term of VEGF and estrogen receptor was also prognostic for overall survival (likelihood ratio test; P = .05). CONCLUSIONS: The results show that cytosolic levels of VEGF in tumor tissue samples are indicative of prognosis for patients with node-negative breast carcinoma.

Surgical second look in ovarian cancer: a randomized study in patients with laparoscopic complete remission--a Northeastern Oncology Cooperative Group-Ovarian Cancer Cooperative Group Study.

PURPOSE: The usefulness of extensive and repetitive surgery for patients with ovarian cancer still remains unproven (at least for some conditions). We planned an accurate prospective test of the hypothesis that patients with advanced-stage disease, after they had reached a clinical complete remission (CR), may benefit from surgical second look (SSL). PATIENTS AND METHODS: One hundred two patients in CR (as assessed by clinical findings, markers, and visualization by computed tomographic [CT] scan and laparoscopy), after initial debulking and first-line chemotherapy, were randomized to two arms, which were well balanced for predictive criteria such as age, stage at presentation, histology, grading, date of randomization, and residua after first surgery. Forty-eight patients were randomly assigned to receive follow-up evaluation only, while 54 were assigned to receive second surgery (eight of them refused). Of 46 surgical patients, 35 had negative and 11 positive surgical findings (24% clinically false-negative). RESULTS: Despite the microscopic residua found at open surgery, and the fact that the patients were then treated with second-line chemotherapy, SSL did not increase the probability of survival in this setting. In an analysis of the results according to the intention-to-treat criteria, after a 60-month follow-up period, the overall survival rates in the two groups of patients (SSL v no SSL) were 65% and 78%, respectively (P = .14). Multivariate analysis according to predictive criteria confirmed there was no significant difference between the two groups (P = .39). CONCLUSION: Our study shows the following: (1) our second-line treatment is scarcely effective; (2) SSL accurately defines complete responders to first-line chemotherapy; (3) SSL per se does not prolong survival; and (4) if confirmed, a less invasive procedure could replace SSL as a valuable method in new first-line regimens in ovarian cancer patients with clinical CR confirmed by laparoscopy.

A new nested primer pair improves the specificity of CK-19 mRNA detection by RT-PCR in occult breast cancer cells.

Reverse transcription polymerase chain reaction (RT-PCR) of cytokeratin-19 (CK-19) has been widely used to detect small numbers of circulating malignant epithelial cells in the bone marrow or the peripheral blood of patients with breast cancer. However, a high percentage of false positive results has been recorded and conflicting reports question the clinical relevance of this technical approach. We demonstrate that the use of a new nested primer pair for CK-19 RT-PCR avoids false positive results without affecting the sensitivity of the assay. Our experiments were carried out using MCF-7 cells alone or mixed with peripheral blood mononuclear cells (PBMNC) of healthy donors. The results were also validated in a large series of healthy donors and in a preliminary study on a limited number of patients with breast cancer, thus suggesting that our assay is feasible for application in the clinical evaluation of occult malignant epithelial cells.

Autologous bone marrow transplantation for adult advanced stage lymphoblastic lymphoma in first CR. A study of the NHLCSG.

Fourty successive adult patients with lymphoblastic lymphoma entered a study of sequential chemotherapy consisting of an intensive LSA-L2-type protocol to induce first complete remission. Twenty-one patients in first CR (median age 24 years, range 15-43), after receiving a conditioning regimen consisting of cyclophosphamide and total body irradiation, underwent autologous bone marrow transplantation. At this time fourteen patients are alive and well 5-72 months post-transplant (median follow-up 58 months) with an actuarial disease free survival of 66%. These early results suggest that high-dose chemoradiotherapy followed by autologous bone marrow transplantation may improve long-term disease free survival in advanced stage adult lymphoblastic lymphoma.

Tissue polypeptide antigen in breast cancer cytosol: a new effective prognostic indicator.

Since 1982 we have been evaluating oestrogen and progesterone receptors (PgR), cathepsin D and the cytosolic levels of the tumour marker, tissue polypeptide antigen (TPA), in 257 patients radically resected for breast cancer (follow-up 24-81 months). TPA was measured by an immunoradiometric assay previously validated for cytosol. No significant associations were found between cytosolic TPA and age, tumour size, lymph-node status, receptor status and cathepsin D. TPA+ cases showed a significantly longer disease-free survival (DFS) and overall survival (OS) than TPA-patients (log-rank P < 0.0001). The prognostic value of cytosolic TPA was also demonstrated after stratification by nodal status, PgR and cathepsin D. The prognostic value of TPA was independent of the other prognostic indicators, being the most powerful among the evaluated indices (Cox multivariate analysis: chi 2 15.5 for DFS, 11.4 for OS). We conclude that cytosolic TPA is a powerful additional prognostic factor in primary breast cancer. Its prognostic role should therefore be extensively evaluated.

The impact of a psychological intervention on quality of life in non-metastatic breast cancer.

The aim of this study was to determine whether psychological intervention had a beneficial effect on the quality of life and behaviour of women diagnosed with breast cancer. 36 consecutive patients with non-metastatic breast cancer assigned to surgery and systemic chemotherapy were randomised to receive either psychological intervention (weekly cognitive individual psychotherapy and bimonthly family counselling) or standard follow-up. Personality (16-PF and IIQ), quality of life (FLIC), and depression (BDI) scores were the endpoints for this study, and the questionnaires were completed by the patients at diagnosis, and up to 9 months after diagnosis. Cognitive psychotherapy and family counselling improved both depression and quality of life indexes compared with the control group. Better emotional coping behaviours were also revealed by some changes in personality traits in the intervention group.

Randomized study of chlorambucil (CB) compared to interferon (alfa-2b) combined with CB in low-grade non-Hodgkin's lymphoma: an interim report of a randomized study. Non-Hodgkin's Lymphoma Cooperative Study Group.

Alpha interferon has shown initial promise in the treatment of low-grade non-Hodgkin's lymphoma (NHL), especially with the nodular form of the disease. The present study enrolled 70 NHL patients who received either chlorambucil (CB; 10 mg/day) or CB plus interferon alfa-2b (5 million units (MU)/m2 subcutaneously three times a week). Among 63 evaluable patients, similar response rates (62.1% and 64.7% respectively) were recorded for the treatment arms. In patients receiving no maintenance therapy, those who received interferon alfa-2b during the induction phase showed a favourable trend in terms of incidence of relapse compared to those who had received chlorambucil alone. During maintenance therapy with interferon alfa-2b, no significant differences in the occurrence of relapse have yet been seen compared to patients on no maintenance therapy. A longer observation period is needed to make a definitive conclusion about the usefulness of interferon maintenance therapy and to evaluate further the effects of the combined schedule of chlorambucil and interferon induction on the duration of remission.

Phase I-II intraperitoneal mitoxantrone in advanced pretreated ovarian cancer.

36 previously treated patients (25 with anthracyclines) with advanced epithelial ovarian cancer have been treated with intraperitoneal (i.p.) mitoxantrone (M) at increasing doses. The response was evaluated through repeated laparoscopy with multiple biopsies and serial measurement of Ovarian Cancer Antigen 125 (CA 125); 11/36 patients had a complete (6 patients) or partial (5 patients) response. Toxicity (both local and general) was observed starting from 25 mg/m2 of M per cycle. The amount of drug reaching systemic circulation was monitored by measuring M plasma value after i.p. treatment. This study showed wide variations in serum levels obtained after i.p. doses ranging from 23 to 36 mg/m2. The area under the curve (AUC) of mitoxantrone plasma samples, did not correlate with the i.p. administered dose. Conversely, a correlation seems to exist between the plasma AUC and the responder status. Patients who showed clinical responses to i.p. treatment with mitoxantrone had AUCs and plasma peak levels of the drug that were significantly higher than those in non-responders (P = 0.03, Fisher's exact test).

Differentiation, proliferation and apoptosis levels in human leiomyoma and leiomyosarcoma.

A comparative analysis of the differentiation pattern, the proliferative behaviour, and the level of apoptosis between human benign and malignant neoplasms of smooth-muscle (SM) tissue is lacking. The clinical, histopathological, immunochemical, and immunocytochemical features of leiomyomas (LM) and leiomyosarcomas (LMS) were investigated by a panel of monoclonal antibodies specific for some differentiation markers of SM tissue (SM myosin and alpha-actin, desmin, and SM22) and for markers of non-muscle tissue (vimentin and non-muscle myosin). Proliferating normal and neoplastic cells were identified by proliferating-cell nuclear antigen (PCNA)/Ki67 immunostainings and the apoptotic cells were revealed by means of the terminal-deoxynucleotidyltransferase-mediated dUTP nick-end labelling technique. Gel electrophoresis and Western blotting, performed with anti-(SM1/SM2 myosin isoform) antibody, indicated quantitative differences between LMS and LM, which mirrored higher positive to negative nuclear ratios for PCNA, Ki67 and apoptosis in malignant as opposed to benign neoplasms. With LM, however, a similar SM1 to SM2 ratio could be associated with different proliferation levels. Uterine, gastric and intestinal LMS displayed specific patterns of SM1/SM2 and/or non-muscle myosin expression that were not paralleled by different levels of proliferation/apoptosis. While the level of PCNA/Ki67 correlated with the level of apoptosis in normal SM tissues and LM, that of LMS did not. In vivo at the cellular level, LM and uterine LMS displayed a near-uniform SM tissue differentiation, whereas the other LMS displayed a lesser or a heterogeneous immunoreactivity. In vitro, cultured LMS cells showed a limited and peculiar expression of SM myosin. In conclusion, there is no reciprocal relationship between degree of differentiation and the level of proliferation, as exemplified by the finding that the less differentiated intestinal LMS displays the lowest proliferative behaviour and that the relatively more differentiated gastric LMS/metastasis is more proliferative.

Quantitative chemiluminescent immunoassay of p53: prognostic significance in 220 node-negative breast cancer tissue.

The prognostic and predictive role of p53 overexpression in breast cancer samples is usually investigated by using molecular biology or immunohistochemical methods. However, the results are to date controversial, and this is in part due to the methodological pitfalls of both the methods. To study the possibility of overcoming, at least in part, these problems we evaluated a commercially available chemiluminescent immunoassay with which the p53 concentrations of 220 specimens from node negative breast cancer were determined. The assay showed good analytical performance and found detectable levels in 84.7% of cases (median 0.22 ng/mg of proteins, range 0-50 ng/mg of proteins). p53 has been found inversely correlated with estrogen receptors and directly correlated with cathepsin D. The prognostic role of p53 was evaluated in two different ways: a) two previous studies (Borg et al 1995, DeWitte et al. 1996) using the same method found almost 30% of samples had significantly shorter DFS and OS. We subdivided our cases in order to identify the same positivity rate and to verify if the previous cathegorizations were effective also in our patient series. We confirmed the independent association with DFS (p = 0.006) and OS (p = 0.0005); b) considering that any categorization of quantitative parameters could cause a loss of clinical information, we also evaluated p53 as a continuous variable. Multivariate analysis showed a significant quantitative relationship between p53 and both disease free (p = 0.026) and overall survival (p = 0.02).

A Randomized Study of Epithelial Ovarian Cancer: Is Chemotherapy Useful after Complete Remission?

Objective. The aim of this study is to verify whether consolidation chemotherapy with Cisplatin improves disease-free survival and/or overall survival in patients affected by epithelial ovarian cancer.Methods. A multicenter study examined 122 randomized patients in complete remission as judged by laparoscopy or laparotomy following first-line chemotherapy consisting of ACy (Adriamycin + Cyclophosphamide), PCy (Cisplatin + Cyclophosphamide), or Mitoxantrone + Carboplatin. Sixty-one of these patients were treated with 3 cycles of 5-Fluorouracil (FU) 500 mg/m2 for 5 days followed by Cisplatin at 100 mg/m2 on the 6th or 7th day every 28 days; the other 61 received no further treatment (nihil group).Results. Sixty patients in the Cisplatin arm were evaluable. There were 36 relapses in the FU+Cisplatin arm and 30 in the nihil arm. Peritoneal relapses were 25% for Cisplatin treatment vs. 16.4 % for nihil. There were 29 deaths in the Cisplatin arm vs. 27 for nihil. Median overall survival time (95 months with Cisplatin vs. 96 months in the nihil group) and median disease-free survival (66 months with Cisplatin vs. 73 in the nihil group) were similar in both arms (p=0.66 and p=0.41, respectively). There were no significant differences in tumor stage and grade between the two arms. Seven patients presented a second neoplasm during follow-up: six in the nihil arm, but only one patient in the Cisplatin arm. Death in these patients was due to the second neoplasm and not to progression of ovarian cancer.Conclusion. Three courses of additional platinum+FU treatment after five cycles of first-line chemotherapy without FU produced no increase in overall survival or disease-free survival.

Vindesine in the treatment of squamous cell carcinoma (WHO I), adenocarcinoma (WHO III), and large cell carcinoma (WHO IV) of the lung.

The antineoplastic activity of vindesine was evaluated in 57 patients with non-small-cell carcinoma of the lung, 53 patients were fully evaluable for response and toxicity. Twenty-seven patients had squamous cell carcinoma (WHO I), 14 had adenocarcinoma (WHO III), and 12 had large cell carcinoma (WHO IV). Forty percent of patients were previously treated. Vindesine was administered at a weekly i.v. dose of 3 mg/m2. Partial remissions were observed in 2 of 12 patients with large cell carcinoma and in 1 of 27 patients with squamous cell carcinoma. Among 14 patients with adenocarcinoma, 3 minor responses were observed. Drug-related toxic effects (mainly leukopenia with manageable and reversible neurotoxicity) required modification of dose in 41% of patients: this finding and previous treatment may have adversely affected the response rate. It is concluded that vindesine as a single agent has some activity in large cell carcinoma. Activity in the other histologic types was minimal but not totally absent and deserves further evaluation, possibly in non-pretreated patients.

Phase II study of 4'epi-doxorubicin.

Sixty-five patients with advanced solid tumors were treated with 4'epi-doxorubicin, a new analogue of doxorubicin (DXR). Forty-three of 61 evaluable patients had not received previous chemotherapy and/or hormonal treatment. 4'Epi-doxorubicin has been administered at the dose of 75 mg/m2 i.v. once every 21 days, for a minimum of 2 courses. The pattern of acute toxicity was similar to that of DXR. Transient electrocardiographic abnormalities were found in about 50% of patients. The ratio of pre-ejection period to the left ventricular ejection time (PEP/LVET) increased within 1 h after drug injection and returned to near basal values after 24 h. Three patients received a total dose of more than 550 mg/m2, still maintaining a baseline PEP/LVET ratio near to pretreatment values. Up to now, no patient has developed clinical signs of heart failure. Partial responses were seen in patients with tumors generally sensitive to DXR such as breast carcinoma (6 of 14) and soft tissue sarcomas (2 of 6), and in patients with tumors generally resistant to DXR such as melanoma (1 of 9), colorectal carcinoma (3 of 18) and pancreatic carcinoma (1 of 2). These data suggest that 4'epi-doxorubicin may have a broader spectrum of antitumor activity than DXR.

Phase II evaluation of 4'epi-doxorubicin in patients with metastatic colorectal carcinoma.

Thirty-four evaluable patients with metastatic colorectal carcinoma (13 rectal primary and 21 colonic primary, 4 pretreated and 30 untreated) received 4'epi-doxorubicin at the dose of 75 mg/m2 i.v. once every 21 days, for a minimum of 2 courses. Symptomatic toxicity (mainly confined to gastrointestinal complaints) was short-lived and easily managed. Hematologic toxicity was mild. Transient electrocardiographic abnormalities were found in 50% of patients, without signs of significant cumulative cardiotoxicity. Three previously untreated patients achieved a partial response (lasting 16, 12 and 12 weeks, respectively) with a response rate of 9% (3%-23%, 95% confidence interval). More interestingly, all responsive patients had rectal cancer: further studies of 4'epi-doxorubicin confined to the rectal localization seem warranted.

Phase II evaluation of vindesine in mycosis fungoides, extraosseous plasmacytoma and other hematologic malignancies.

We conducted a phase II trial of Vindesine in 24 patients, mostly pretreated (23/24 fully evaluable for therapeutic response) with advanced hematologic malignancies. The drug was administered at weekly bolus doses of 3 mg/m2 i.v. Overall, objective tumor regressions were seen in 9 of 23 patients. The drug appeared effective in extraosseous plasmacytoma (1 complete response, 1 partial response and 1 minor response in 6 patients). Further phase II trials in these 2 diseases are justified. In addition, 3 partial responses in 7 patients with advanced lymphoma were also obtained. Previous vinca-alkaloid exposure did not adversely affect the response rate: 8 of 9 responsive patients had previously received vincristine and/or vinblastine. Drug-related toxic effects were mainly represented by manageable and reversible neurotoxicity and by moderate leukopenia with apparent lack of thrombocytopenia. In heavily pretreated patients, leukopenia may be occasionally severe: in these conditions a starting dose of 2 mg/m2 seems more appropriate.

Cisplatin plus vindesine versus cisplatin plus VP16 versus doxorubicin plus cytoxan in non-small-cell carcinoma of the lung. A randomized study.

From March 1981 to January 1984, 116 patients with advanced non-small-cell carcinoma of the lung (NSCCL) were randomly assigned to 3 combinations as follows: CDDP + DVA, CDDP + VP16 and DXR + CTX. 94 patients were evaluable for response, 106 for toxicity and survival. Of 31 patients, 15 (48%; 3 CRs and 12 PRs) responded to CDDP + DVA; of 33 patients, 12 (36%, 2 CRs and 10 PRs) responded to CDDP + VP16; of 30 patients, 3 (10%) obtained a PR with DXR + CTX (CDDP + DVA vs DXR + CTX, P less than 0.005; CDDP + VP16 vs DXR + CTX, P less than 0.05; CDDP + DVA vs CDDP + VP16, P = NS). The median duration of response was 22 weeks in the CDDP-DVA group, 17 weeks in the CDDP-VP16 group, and 16 weeks in the DXR + CTX group. No significant difference in survival was observed among the 3 groups (median: 43, 47, 41 weeks, respectively). Hematologic and neurologic toxicities were significantly higher in the DVA-containing regimen. Despite the lack of improvement of overall survival with the CDDP-containing combinations over the DXR + CTX control group, the good response rate makes them suitable to be used in combined therapeutic strategies.

Long-term survival in a randomized study of nonplatinum therapy versus platinum in advanced epithelial ovarian cancer.

The purpose of this study was to compare long-term survival in first-line chemotherapy with and without platinum in advanced-stage ovarian cancer. From July 1987 to November 1992, 161 untreated patients with FIGO stage III-IV epithelial ovarian cancer were randomized: 81 patients received no platinum and 80 received platinum combination. Residual disease after surgery was <2 cm in 61 patients without platinum, 59 with platinum. Median age was 58 years in nonplatinum arm and 55 years in platinum arm (range: 15-73). Complete and partial responses were 51% and 10% for nonplatinum arm and 51% and 8% for platinum arm, respectively (P= 0.7960). Stable disease was observed in 18% of patients in nonplatinum arm and 15% of patients in platinum arm and progression in 20% of nonplatinum- and 21% of platinum-treated cases. Ten-year disease-free survival was 37% for therapy without platinum and 31% for platinum combination (P= 0.5679); 10-year overall survival was 23% without platinum and 31% with platinum combination (P= 0.2545). Fifteen-year overall survival showed a trend of short duration in favor of platinum (P= 0.0678). Relapses occurred after 60 months in ten patients (seven with and three without platinum). The overall and disease-free survivals at 5, 10, and 15 years show no statistically significant long-term advantage from the addition of cisplatin; however, there is a slight trend in its favor.

Oestrogen-dependent expression of the SM2 smooth muscle-type myosin isoform in rabbit myometrium.

Ovarectomized rabbits displayed a decreased SM1 to SM2 ratio of smooth muscle-type myosin heavy chain isoforms compared to unoperated, virgin females which was reversed after 17beta-oestradiol administration to a value similar to that of control animals. When this steroid was given to sexually immature animals or to adult virgin rabbits, SM2 expression was not induced, as also happened with proliferating myometrial smooth muscle cells grown in vitro. In growing rabbit, the 17beta-oestradiol administration induced the formation of the circular and the longitudinal muscle layers, characteristics of sexually competent females. The SM2 isoform was up-regulated during postnatal development and the SM1 to SM2 ratio changed during pregnancy and post-partum period but not with human gonadotropin treatment which increases the level of circulating progesterone. Immunofluorescence staining of adult myometrium with anti-SM2 antibody indicated that this isoform is localized to the longitudinal layer exclusively and, in contrast to the circular layer, its expression was independent of oestrogen level. Difference in oestrogen sensitivity between the two layers was also detected for the expression of the intermediate filament protein vimentin and the thin filament protein calponin. Changes of SM2 expression in the myometrium correlated with variations in the oestrogen receptor density as also confirmed by decreased SM2 content/oestrogen receptor density in the circular layer when ovarectomized females were treated with the oestrogen antagonist ICI 182,780. Our results indicate that: (1) a specific distribution of myosin heavy chain exists within rabbit myometrium, and (2) SM2 myosin expression in this smooth muscle is under oestrogen control.

Expression of non-muscle myosin isoforms in rabbit myometrium is estrogen-dependent.

The putatative effects of different estrogen levels on the expression of non-muscle myosin isoforms in rabbit myometrium have been investigated using three monoclonal anti-platelet myosin heavy chain (MyHC) antibodies (NM-F6, NM-G2, and NM-A9). Western blotting analysis of proteolytic digests of human platelet actomyosin indicates that these antibodies are specific for three distinct epitopes. Comparative immunofluorescence tests on cultered human fibroblasts with polyclonal sequence-specific anti-MyHCA antibody suggest that the patterns of NM-F6, NM-.G2 and NM-A9, although similar, do not overlap with that of type-A MyHC. Distribution of NM myosin isoforms has been studied in indirect immunofluorescence assays using cryosections of tissues from rabbits at various stages of development, pregnancy, or from ovariectomized, 17beta-estradiol-treated ovariectomized, and human chorionic gonadotropin-treated animals. Non-muscle myosin antigenicity is still present in the myometrium when the female becomes sexually competent. The immunoreactivity of non-muscle myosin for NM-F6 is steroid-independent, since it does not change with pregnancy or ovariectomy, but that of NM-G2 is estrogen-dependent; the latter disappears during pregnancy and in ovariectomized animals treated with estradiol, whereas it is expressed in ovariectomized rabbits. Although non-muscle myosin immunoreactivity for NM-A9 is detectable under all the experimental conditions, it can assume different patterns of intracellular distribution in vitro (punctate vs filamentous), depending on culture conditions and the presence of estrogens.