Tumor volume, nodal status, and metastasis in breast cancer in women.

The relationship between primary tumor volume at detection, number of positive nodes, and probability of and time until first distant metastasis was examined for a group of 2,663 women with breast cancer. Time until metastasis was shown to decrease and probability of metastasis to increase as tumor volume and number of nodes increased. Either factor remained significant after correction for the other. Simple proportional hazards models were shown to be inadequate to describe the data. Graphic techniques were used to obtain nonparametric estimates of the forms of the relationships between tumor volume, nodal status, and the time course of the occurrence of metastasis. A simple calculation demonstrated that the average contribution per cell to the probability of metastasis decreased with increasing volume.

Lack of concordance of growth rates of primary and recurrent breast cancer.

The degree of concordance of growth rates of primary tumors with corresponding recurrences was investigated by using data from 184 patients with breast cancer with measurable recurrences. For conduction of this examination, the detection processes of both the primary tumor and the recurrence were explored. The probability of detection of a recurrence per unit time was found to be nearly proportional to the tumor's diameter. Approximately 60,000 cells initiated the recurrence, and the distribution of doubling times of the recurrences was exponential, with a mean of 2.1 months. The probability of detection of the primary tumor per unit time was approximately proportional to its volume. The distribution of doubling times of primary tumors was nearly exponential; from other evidence, we inferred that the mean doubling time was also close to 2.1 months. Several techniques to measure growth rate agreement between the primary and recurrent tumors within individuals were developed, and all of them yielded the result that the growth rates are nearly unrelated.

Estimation of human tumor growth rate from distribution of tumor size at detection.

Methods are presented for estimating the growth curve of a tumor (up to an unknown scale factor of time) from the distribution of volumes at detection on the basis of two assumptions: 1) the existence of a common growth curve and 2) the assumption that the probability of detecting a tumor in a period of time is proportional to the tumor volume. These methods can accommodate variation between individuals in speed of traversal of the growth curve. The methods are applied to volumes of tumor at detection of a large series of breast cancers. The simplest, adequate description is exponential growth with great individual-to-individual variation in tumor doubling time. The data are consistent with bounded growth (Gompertzian or logistic form) as well as exponential growth. However, there is no evidence that the bound on growth is within the range of the data. The shape of the distribution of volumes does not yield an effective lower limit on such a bound.

An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo.

Low levels of gene expression following systemic delivery have impaired the effectiveness of tumor suppressor gene replacement in treating metastases. We asked whether combined treatment with 2-methoxyestradiol (2-Me), which increases levels of wild-type p53 protein in cancer cells, and the systemic administration of an adenoviral vector expressing wild-type p53 (Ad-p53) would inhibit the growth of human metastatic lung cancer cells in vivo. The simultaneous administration of p53 and 2-Me resulted in a greater than additive reduction with the lung colony count reduced to 33% of its control value. These results suggest that the synergistic effect of 2-Me and Ad-p53 in combination treatment may have application in the systemic treatment of cancer.

Improved prediction of survival in advanced adenocarcinoma of the ovary by immunocytochemical analysis and the composition adjusted receptor level of the estrogen receptor.

Conventional cytosol estrogen receptor analysis is not a significant prognostic variable in serous ovarian carcinoma. Although the use of immunocytochemical receptor analysis for estrogen does provide prognostically useful information in enhanced accuracy of predicting survival in patients with ovarian cancer, its usefulness can still be improved. Surgical samples from ovarian carcinomas are heterogeneous in tissue composition. Immunocytochemical receptor analysis allows for the specific assessment of the tumorous portions of a histological specimen. However, it is limited by its dependence on staining intensity as the determining factor. Biochemical receptor analysis does provide objective information concerning the number of receptor molecules present in a given sample, but the value is not adjusted for histological composition of the tumor section. Therefore, we have attempted to combine the advantages of both methods. By adjusting the conventional receptor analysis for the percentage of tumor present in the specimen, we have eliminated the tissue heterogeneity as a confounding variable. The resulting value is named Composition Adjusted Receptor Level or CARL. A prospective study was performed on the estrogen receptor concentrations in 61 ovarian cancers. Minimum follow-up was 8 years. For the percentage of tumor in the specimen, a highly significant correlation of the assessment of the two pathologists was observed. Stage (P < 0.05) and grade (P < 0.05) as well as cell type (P < 0.05) were found to be significant prognostic variables. In an attempt to eliminate the confounding influences of these variables, the CARL of the estrogen receptor was assessed with regard to its prognostic significance in 32 grade 2 and 3 serous carcinomas of the ovary, stage III and IV. A linear correlation between CARL and survival was found above a threshold estrogen receptor concentration of 15 fmol/mg cytosol protein using a correlation of the Cox proportional hazards model (P < 0.02). Our data suggest that (a) the assessment of the percentage of tumor in a given sample is not significantly observer dependent, (b) CARL is a significant predictor of survival in serous ovarian carcinoma, and (c) a CARL should be determined for the analysis of any cytosol receptor in solid tumors.

Metastatic potential of human colorectal carcinomas implanted into nude mice: prediction of clinical outcome in patients operated upon for cure.

To determine whether the production of experimental hepatic metastases in athymic nude mice by human colorectal carcinomas (HCC) correlated with the clinical outcome in patients, we harvested colorectal carcinomas from 82 patients, dissociated the tumors with collagenase and DNase, and injected them into groups of nude mice, either in the flank to assess experimental tumorigenicity or into the spleen to produce experimental metastasis in the liver. Growth in mice was then associated with clinicopathological factors and clinical outcome. Growth of HCC in either the flanks or the livers of nude mice was associated with the time to recurrence in a Wilcoxon analysis. Analysis of the outcome data in a Cox proportional hazards model suggested that there was an interaction between tumorigenicity and metastatic potential of HCC in nude mice and serum CEA concentration in the patient and stage of disease. A univariate analysis indicated that both tumorigenicity and metastatic potential of HCC in nude mice were significantly associated with the serum CEA concentration of the patient but not with the other variables of stage of disease, mucin production, local tissue invasion, state of differentiation, or sex. A subset of 57 patients was operated upon for cure and followed prospectively for up to 61 months. Tumorigenicity and, to a lesser extent, experimental metastatic potential were associated with disease recurrence in 23 of these patients. Seventy-eight % of the subset of patients who were operated upon for cure developed liver metastasis as one site of their progressive disease. Thus, the ability of HCC cells isolated from surgical specimens to grow in athymic nude mice correlates with the development of advanced disease in patients.

Docetaxel for patients with paclitaxel-resistant Müllerian carcinoma.

PURPOSE: To determine the efficacy and toxicity of docetaxel in patients with müllerian carcinoma resistant to paclitaxel. PATIENTS AND METHODS: Thirty-two patients with epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer who failed paclitaxel-based chemotherapy received either 100 or 75 mg/m(2) of docetaxel every 3 weeks. Resistance to paclitaxel was defined as either progression of disease during treatment, failure to achieve regression of disease after at least four courses, or rapid recurrence (within 6 months) after completion of therapy. RESULTS: Eighteen patients were treated on a formal protocol and fourteen with the commercially available docetaxel. Thirty were assessable for response. Toxicities were thoroughly evaluated in the 18 patients on protocol. Twenty-seven patients (85%) had epithelial ovarian cancer. The overall response rate was 23% (one complete and six partial responses), with a median survival time of 44 weeks (9.5 months). Nine patients had stable disease and 14 progressive disease. Among 19 patients who progressed during prior paclitaxel treatment, two (11%) responded to docetaxel, compared with five (45%) of 11 patients in other paclitaxel-resistance categories. The responders had a median taxane-free interval (ie, the time between the last paclitaxel and first docetaxel treatment) of 73 weeks, compared with 19 weeks for the nonresponder group. Toxic effects were as expected. CONCLUSION: Docetaxel is an active chemotherapeutic agent in patients with müllerian carcinoma previously treated with paclitaxel-based chemotherapy, especially in the patients who had a long taxane-free interval after a previous short response to paclitaxel.

Phase II study of vinorelbine in advanced and recurrent squamous cell carcinoma of the cervix.

PURPOSE: To evaluate the efficacy and toxicity of intravenous vinorelbine as single-agent chemotherapy in patients with advanced or recurrent squamous cell carcinoma of the cervix. PATIENTS AND METHODS: Between August 1993 and July 1995, 35 patients with advanced or recurrent squamous cell carcinoma of the cervix were entered onto this study. Patients had received no prior therapeutic chemotherapy. The initial dose of vinorelbine 30 mg/m2 was administered as a weekly intravenous infusion. Subsequent doses were unchanged, reduced, escalated, or omitted according to observed toxicity. Patients were evaluated for response and toxicity using standard Gynecologic Oncology Group (GOG) and World Health Organization criteria, respectively. RESULTS: Thirty-three of 35 patients were assessable for response and 35 of 35 for toxicity. The overall response rate was 18% (one complete response [CR], five partial responses [PR]). The mean response duration was 5.2 months and the median survival from treatment for all patients was 11.0 months. The major toxicity was leukopenia, with 61% of patients who had grade 3 or 4. Gastrointestinal and neurotoxicity were infrequent and mild. CONCLUSION: Vinorelbine has moderate activity in advanced or recurrent squamous cell carcinoma of the cervix. Further studies of combination regimens with this agent are justified in this patient population.

Relative value of physical examination, mammography, and breast sonography in evaluating the size of the primary tumor and regional lymph node metastases in women receiving neoadjuvant chemotherapy for locally advanced breast carcinoma.

The purpose of this study was to correlate physical examination and sonographic and mammographic measurements of breast tumors and regional lymph nodes with pathological findings and to evaluate the effect of neoadjuvant chemotherapy on clinical Tumor-Node-Metastasis stage by noninvasive methods. This was a retrospective analysis of 100 patients with locally advanced breast cancer registered and treated in prospective trials of neoadjuvant chemotherapy. All patients received four cycles of a doxorubicin-containing regimen and had noninvasive evaluation of the primary tumor and regional lymph nodes before and after neoadjuvant chemotherapy by physical examination, sonography, and mammography and underwent breast surgery and axillary dissection within 5 weeks after completion of neoadjuvant chemotherapy. The correlations between clinical and pathological measurements were determined by Spearman rank correlation analysis. A proportional odds model was used to examine predictive values. Eighty-three patients had both a clinically detectable primary tumor and lymph node metastases. Sixty-four patients had a decrease in Tumor-Node-Metastasis stage after chemotherapy. For 54% of patients, there was concordance in clinical response between the primary tumor and lymph node compartment; for the rest, results were discordant. Physical examination correlated best with pathological findings in the measurement of the primary tumor (P = 0.0003), whereas sonography was the most accurate predictor of size for axillary lymph nodes (P = 0.0005). The combination of physical examination and mammography worked best for assessment of the primary tumor (P = 0.003), whereas combining physical examination with sonography gave optimal evaluation of regional lymph nodes (P = 0.0001). In conclusion, physical examination is the best noninvasive predictor of the real size of locally advanced primary breast cancer, whereas sonography correlates better with the real dimensions of axillary lymph nodes. The combination of physical examination with either mammography or sonography significantly improves the accuracy of noninvasive assessment of tumor dimensions.

Synergistic inhibition of human lung cancer cell growth by adenovirus-mediated wild-type p53 gene transfer in combination with docetaxel and radiation therapeutics in vitro and in vivo.

Chemotherapy given sequentially or concurrently with external beam radiation therapy has emerged as a standard for the treatment of locally advanced lung cancer. Gene therapy by adenovirus-mediated wild-type p53 gene transfer has been shown to inhibit lung cancer growth in vitro, in animal models, and in human clinical trials. However, no information is available on the combined effects of p53 gene transfer, chemotherapy, and radiation therapy on lung cancer growth in vitro and in vivo. Therefore, we developed two-dimensional and three-dimensional isobologram modeling and statistical methods to evaluate the synergistic, additive, or antagonistic efficacy among these therapeutic agents in human non-small cell lung cancer cell lines A549, H460, H322, and H1299, at the ID50 and ID80 levels. The combination of these three therapeutic agents exhibited synergistic inhibitory effects on tumor cell growth in all four cell lines at both the ID50 and the ID80 levels in vitro. In mouse models with H1299 and A549 xenografts, combined treatment synergistically inhibited tumor growth in the absence of any apparent increase in toxicity, when compared with other treatment and control groups. Together, our findings suggest that a combination of gene therapy, chemotherapy, and radiation therapy may be an effective strategy for human cancer treatment.

A randomized clinical trial of 4-hydroxyphenylretinamide for high-grade squamous intraepithelial lesions of the cervix.

PURPOSE: Previous trials of topical trans-retinoic acid treatment of cervical intraepithelial neoplasia (CIN) grades 2 and 3 led to a statistically significant regression of CIN 2, but not CIN 3. We tested N-(4-hydroxyphenyl)retinamide (4-HPR), a promising oral retinoid that has been shown to induce apoptosis through nonretinoic receptor acid-mediated pathways, for its toxicity and efficacy against CIN 2/3. EXPERIMENTAL DESIGN: In a blinded randomized trial, 4-HPR at 200 mg/day for 6 months (with a 3-day/month drug holiday) was compared with placebo in patients with biopsy-proven CIN-2/3 [high-grade squamous intraepithelial lesions (HGSILs)]. Patients were treated with placebo or 4-HPR for 6 months, biopsied, and then followed for an additional 6 months. At the 12-month end point, they underwent either loop excision if a histological lesion was present or a biopsy from the original area of the lesion if no lesion was present. RESULTS: An interim analysis of blinded data showed a significantly worse prognosis at 12 months for one group. When the code was broken because of the poorer outcomes, we discovered that the 4-HPR treatment arm was performing more poorly than was the placebo at 6 and 12 months (25 versus 44% response rates at 6 months; 14 versus 50% at 12 months). Toxicity was not significant in either arm. CONCLUSIONS: 4-HPR at 200 mg/day with a 3-day/month drug holiday is not active compared with placebo in the treatment of HGSIL. Because 4-HPR is active in the laboratory, the lack of effect in our trial may indicate that higher doses are needed in patients to achieve comparable results.

Quantitative analysis of transforming growth factor beta 1 and 2 in ovarian carcinoma.

Transforming growth factor beta (TGF-beta) is an important family of cytokines that may promote tumor growth in vivo through several mechanisms including interference with antitumor T-cell immune responses, alteration of factors in the stroma and matrix, and the promotion of angiogenesis. TGF-beta isotypes have been detected in malignant and normal ovarian tissues. We have determined by quantitative immunohistochemistry the density of TGF-beta1, TGF-beta2, and human leukocyte antigen (HLA) Class I and Class II antigens on malignant cells in paired primary and metastatic specimens from 10 patients with ovarian carcinoma. Cryostat sections of specimens from the carcinomas and from normal ovaries of three women of similar age without ovarian cancer were stained respectively with specific antibodies to TGF-beta1, TGF-beta2, and HLA Class I and II antigens, and with isotype-matched control antibodies. Antigen density was quantitated blindly as mean absorbance on a SAMBA 4000 image analyzer. TGF-beta1 and TGF-beta2 were overexpressed in both primary and metastatic tumor specimens in comparison with normal ovarian tissue. No statistical correlation was found between the expression of TGF-beta1 or TGF-beta2 and HLA class I or HLA class II, which suggests that TGF-beta isotypes could have effects on the immune system other than down-modulation of these HLA molecules. Furthermore, the lack of association between levels of TGF-beta expression and the reduced expression of HLA molecules could suggest that tumor cells expressing both HLA and TGF-beta may be suitable targets for adaptive immunotherapy. Additional studies are necessary to determine whether TGF-beta expressed by ovarian cancer cells merits evaluation as a therapeutic target.

Clinical and biological effects of intraperitoneal injections of recombinant interferon-gamma and recombinant interleukin 2 with or without tumor-infiltrating lymphocytes in patients with ovarian or peritoneal carcinoma.

To identify strategies that enhance tumor-specific immunity in patients with ovarian carcinoma, 22 patients received four to six doses of i.p. recombinant IFN-gamma (rIFN-gamma), 200 microg/m2 on days 1, 3, 5, 8, 10, and 12, and i.p. recombinant interleukin 2 (rIL-2), either 6.0 x 10(5) IU/m2 (group A) or 1.0 x 10(5) IU/m2 (group B), on days 9, 10, and 11. Two patients in group A also received T-cell lines expanded from peritoneal tumor-infiltrating lymphocytes (TILs) obtained after i.p. rIFN-gamma/rIL-2 administration. Toxicity was manageable and included five nonhematological grade 3 or 4 events in 22 patients (23%). A patient had normalization of CA-125 values and a progression-free interval of 18 months, after receiving i.p. rIFN-gamma/rIL-2 without TILs. Another patient who received i.p. rIFN-gamma/rIL-2 plus TILs had stabilization of ascites and intra-abdominal tumors and >50% reduction in serum CA-125 values over 6 months. A third patient who received i.p. rIFN-gamma/rIL-2 had stabilization of intra-abdominal tumors and ascites accompanied by CA-125 values of 50 to 100 units over 6 months. T-cell lines for adoptive immunotherapy were developed for only 3 of 20 patients who were treated with rIFN-gamma/rIL-2. Large numbers of CD3- CD56+ adherent cells were expanded in rIL-2 in the remaining patients, precluding the development of T-cell lines. i.p. rIFN-gamma, either alone or followed by rIL-2, increased proportions of human leukocyte antigen (HLA) class I+ and class II+ tumor cells and increased HLA class I staining intensity on peritoneal carcinoma cells. i.p. rIFN-gamma plus rIL-2 also enhanced cytotoxic activity against Daudi and K562 cells and against allogeneic ovarian tumor cells. Increased cytotoxic activity was associated with an increase in the proportion of CD56+ cells. IFN-gamma and IL-2 transcripts were expressed more frequently after rIFN-gamma and rIL-2 treatment. In addition, the proportions of CD45RA+ (naive lymphocytes) were increased, and CD8+ DR+ lymphocytes were increased relative to CD8+ CD69+ cells, which were decreased. IL-10 concentrations in peritoneal fluids were increased after treatment with rIFN-gamma and the higher rIL-2 dosing (group A) but not in those treated with rIFN-gamma and the lower rIL-2 dosing (group B). These results demonstrated that patients with ovarian carcinoma can tolerate treatment with rIFN-gamma and rIL-2 and that rIFN-gamma alone or rIFN-gamma combined with rIL-2 enhances the expression of HLA class I and class II antigens on ovarian tumor cells, although immunosuppressive cytokines, such as transforming growth factor-beta and IL-10, may persist. Treatment with rIFN-gamma/rIL-2 i.p. did not facilitate the production of TIL-derived T-cell lines ex vivo.

DNA image cytometric measurement as a surrogate end point biomarker in a phase I trial of alpha-difluoromethylornithine for cervical intraepithelial neoplasia.

Cervical intraepithelial neoplasia grade 3 (CIN 3) is considered a high-risk precursor of invasive cervical cancer. alpha-Difluoromethylornithine (DFMO) is a promising antiproliferative chemopreventive agent. The purpose of this study was to evaluate image cytometric measurement of nuclear DNA (ICM-DNA) as a surrogate end point biomarker (SEB) in a Phase I trial of DFMO for CIN. Thirty patients with CIN 3 were treated with DFMO at five doses, ranging from 0.0625 to 1.0 g/m2/day, for 1 month. Half of the patients had histological responses. Twenty-five pre- and posttreatment cervical biopsy specimens (from 11 responders and 14 nonresponders) were available for this analysis. ICM-DNA was performed on 4-micron sections cut from formalin-fixed tissue blocks and stained with a thionin-SO2 Feulgen reaction. ICM-DNAs for each case were expressed as normalized measurements (against the nuclear modal absorbance of lymphocytes) of the absorbance of each cell of interest and were presented in bar histograms. The mean normalized summed absorbance (sigma ODn) was obtained as a mean histogram of the cell population of interest. Nineteen (76%) of 25 patients had a significant decrease in sigma ODn after DFMO treatment. Posttreatment values were significantly lower than pretreatment values in a paired analysis, and responders had significantly lower values than nonresponders. Analyses of different ICM-DNA references, including percentile values of sigma ODn distribution, DNA malignancy grade, and 5c exceeding rate, showed a decrease of mean sigma ODn during DFMO treatment. In addition, the summed posttreatment sigma ODn histograms also showed progressively shorter right shoulders compared with pretreatment histograms in both responders and nonresponders. We concluded that the modulation of sigma ODn reflected the chemoprevention effect of DFMO even before morphological changes appeared, and thus, ICM-DNA may be useful as a SEB in chemoprevention trials of DFMO. Additional reasons for using ICM-DNA as a SEB are the relative simplicity of its use, the high accuracy of the results, the low cost of the reagents, the ability to use small tissue samples, and the objectivity and reproducibility of the procedure.

Quantitative nuclear morphometry by image analysis for prediction of recurrence of ductal carcinoma in situ of the breast.

Clinical management of ductal carcinoma in situ (DCIS) remains a challenge because significant proportions of patients experience recurrence after conservative surgical treatment. Unfortunately, it is difficult to prospectively identify, using objective criteria, patients who are at high risk of recurrence and might benefit from additional treatment. We conducted a multi-institutional, collaborative case-control study to identify nuclear morphometric features that would be useful for identifying women with DCIS at the highest risk of recurrence. Tissue sections of archival breast tissue of 29 women with recurrent and 73 matched women with nonrecurrent DCIS were stained for DNA, and nuclei in the DCIS lesions were evaluated by image analysis. A clear correlation between mean fractal2_area (FA2) and nuclear grade was observed (P < 0.001), allowing an objective determination of nuclear grade. Several nuclear morphometric features, including mean and variance of variation of radius, mean area, mean and variance of frequency of high boundary harmonics (FQH), and variance in sphericity, were found to be useful in discriminating recurrent from nonrecurrent DCIS subjects. However, the nuclear features associated with recurrence differed between high- and low-grade lesions. For lesions with high FA2 (nuclear grade 3), mean variation of radius, mean FQH, and mean area alone yielded recurrence odds ratios of 4.55 [95% confidence interval (CI) 0.45-45.96], 3.86 (95% CI, 0.88-16.98), 2.90 (95% CI, 0.31-27.2), respectively. Using a summed feature model, high-FA2 lesions showing three poor prognostic features had an odds ratio of 15.63 (95% CI, 1.22-200), compared with those with zero or one poor prognostic feature. Lesions with low mean FA2 (nuclear grade 1 or 2) showing high variances in sphericity and FQH had an odds ratio of 7.71 (95% CI, 1.77-33.60). Addition of other features did not enhance the odds ratio or its significance. These results suggest that nuclear image analysis of DCIS lesions may provide an adjunctive tool to conventional pathological analysis, both for the objective assessment of nuclear grade and for the identification of features that predict patient outcome.

Large-scale expansion in interleukin-2 of tumor-infiltrating lymphocytes from patients with ovarian carcinoma for adoptive immunotherapy.

Tumor infiltrating lymphocytes (TIL) from malignant ascites or solid tumor specimens obtained from patients with ovarian carcinoma were expanded to large numbers in vitro (10(10)-10(11)) by a four-step method using AIM V medium and low concentrations of recombinant interleukin-2 (rIL-2). The expansion procedure employed 24-well culture plates, T-flasks, polyolefin gas-permeable bags (PGPB), and an artificial capillary culture system (ACCS). The mean number of mononuclear leukocytes introduced into the 24-well plates was 16.5 +/- 4.2 x 10(6) cells. TIL from a total of 16 patients were expanded only through the first three steps of the process (24-well-plates, T-flasks, and PGPB) with an overall expansion of 255 +/- 99 fold and mean duration of 27.4 +/- 2.2 days. TIL from 9 of 16 patients were expanded further through the fourth step (ACCS) of the expansion method. The cumulative fold-expansion in nine patients was 8044 +/- 4807 (mean +/- SEM), the median was 2876 and the mean expansion time was 47.1 +/- 4.7 days. TIL from seven additional patients did not grow in rIL-2. Six of these 7 patients received chemotherapy at least four weeks before the specimens were collected. Two ACCS were used in parallel to facilitate expansion of TIL. Viable rIL-2-expanded TIL in the range of 1 x 10(10)-1 x 10(11) were recovered from the two ACCS, a number sufficient for adoptive immunotherapy of patients with ovarian carcinoma. The rIL-2-expanded TIL were predominantly CD3+ CD4+ CD8- alpha beta TCR+, although CD3+ CD4- CD8+ alpha beta TCR+ T cell lines were obtained from certain patients. An increase (43 +/- 8 vs 75 +/- 13; P = 0.05) in the proportion of CD4+ cells was observed over the duration of the four expansion steps. However, CD8+ TIL-derived T cells lines were also expanded in the ACCS. The four-step expansion method described here has several significant advantages over existing techniques. It requires substantially less personnel, equipment and space and the risk of contamination during expansion of the cultures is decreased. These results demonstrate that the four-step method described here can be effectively used for the large-scale expansion of ovarian TIL for the treatment of patients with ovarian carcinoma by adoptive immunotherapy.

The correlation of two argyrophilic nucleolar organizer region counting methods with bromodeoxyuridine-labeling index: a study of metastatic tumors of the brain.

Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into cellular DNA in the synthetic phase. The BrdU-labeling index (BLI) thus reflects the S-phase fraction. The argyrophilic nucleolar organizer regions (AgNORs) are silver-stained granules that have been correlated with ploidy and/or S-phase fraction. Two AgNOR counting methods have been proposed to distinguish between ploidy and S-phase fraction: the mean AgNOR (mAgNOR) count (the mean number of AgNOR granules in 100 cells), which is believed to reflect ploidy, and the AgNOR proliferative index (pAgNOR) (the percentage of cells exhibiting five or more AgNOR granules per nucleus), which is believed to reflect S-phase fraction. To evaluate the latter hypothesis we studied 19 tumors metastatic to the brain in patients who had received preoperative low-dose BrdU injections. Formalin-fixed, paraffin-embedded sections of the resected tumors were stained using the indirect immunoperoxidase technique and a monoclonal antibody to BrdU. The BLI was determined by counting 1,000 tumor cells in each case; AgNOR silver staining was performed on sections of the same blocks studied for BLI. The correlation between pAgNOR counts and BLI was statistically significant (r = .649, P = .003). No statistically significant correlation could be obtained between mAgNOR counts and BLI (r = .421, P = .17). We conclude that the pAgNOR count can reliably reflect the proliferative activity of cells. The method can prove beneficial in situations in which cell kinetic analysis is needed and in which invasive procedures, such as BrdU injection, are not feasible or are contraindicated.

Ductal carcinoma in situ treated with lumpectomy and irradiation: histopathological analysis of 49 specimens with emphasis on risk factors and long term results.

Forty-nine women with ductal carcinoma in situ (DCIS) treated with lumpectomy and irradiation were studied retrospectively. The median age was 50 years (range, 29 to 73 years) and the median follow-up time from initiation of therapy was 86 months (range, 17 to 230 months). Twelve patients presented with palpable masses (0.4 to 4 cm), three with breast thickening, and three with nipple discharge. In 31 patients the tumors were detected by mammography. Intraoperatively, excision of lesions was confirmed by specimen x-ray (38 specimens) or gross inspection (five specimens) and was recorded to be complete. No record was available in the other six patients. Margins of excision free of DCIS were microscopically confirmed in 25 specimens. The size of impalpable DCIS lesions recorded in 25 patients ranged from 0.4 to 5.0 cm (mean, 1.5 cm). Using Lagios' classification system, there were 18 classic comedocarcinomas, high nuclear grade (NG) with necrosis; seven cribriform/papillary, high NG with necrosis; 17 cribriform/micropapillary, intermediate NG with or without necrosis; and seven cribriform/micropapillary, low NG without necrosis. In two patients residual malignant calcifications were present on the postoperative mammogram. Disease recurred in the treated breast at the site of incision in five patients at 18 months and 8, 11, and 12 (two patients) years from initial therapy. The rate of local disease recurrence was 2% at 5 years and 6% at 10 years; three recurrences showed invasive ductal carcinoma and two were DCIS. To evaluate risk factors the following characteristics were considered: necrosis, NG, histological type, periductal fibrosis, periductal lymphoid infiltrate, margin status, age, and method of tumor detection. The end points chosen were recurrence and death from any cause (because only one patient died of disease). Although the recurrences were attributed to residual disease in two patients, of the clinical and pathological parameters evaluated, only periductal fibrosis showed a significant relationship with outcome, with a P value < or = .05 by the Wilcoxon test. On the other hand, using the proportional hazards model, necrosis was a significant predictor for recurrence (P = .02), as was the pair fibrosis and tumor detection when taken together (P = .05). Fibrosis significantly associated with high NG, Lagios' histological subtypes I and II, periductal lymphoid infiltrate, and necrosis (P < or = .0006).(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term follow-up of patients enrolled in a randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small-cell lung cancer.

Our previously reported randomized study of patients with untreated, potentially resectable clinical stage IIIA non-small-cell lung cancer found that patients treated with perioperative chemotherapy and surgery had a significant increase in median survival compared to patients treated with surgery alone. We have now re-analyzed the results of the study with a median time from random allocation to analysis for all patients of 82 months. The increase in survival conferred by perioperative chemotherapy was maintained during the period of extended observation.

Immunotyping of lymphoma by fine-needle aspiration. A comparative study of cytospin preparations and flow cytometry.

Immunotyping is an essential adjunct to cytomorphology for the diagnosis of lymphoma by fine-needle aspiration (FNA). Two independent techniques, cytospin preparations and flow cytometry, were used for immunotyping studies on 71 patients with histologically confirmed non-Hodgkin's lymphoma (63 B-cell lymphomas and 8 T-cell lymphomas). Diagnostic concordance between the two methods was obtained in 69 patients (97%). kappa, lambda, and CD3 (Leu-4) markers were routinely measured on all cytospins, and additional markers were requested when indicated. The standard panel measured by flow cytometry included 14 markers. In general, mean values of light-chain (kappa and lambda) immunoglobulins were consistently slightly higher by cytospin analysis than by flow cytometry. Light-chain immunoglobulin ratios (kappa/lambda or lambda/kappa) obtained by both methods proved to be reliable independent predictors of polyclonality or monoclonality. Correlation studies using the Spearman rank coefficient revealed good concordance among values of kappa, alpha, CD3, and CD5 obtained by the two techniques, suggesting that subjective quantitation by cytospins yields similar results to objective quantitation by flow cytometry. Cytospin analysis and flow cytometry appear equally capable of immunotyping aspirated lymphoid samples reliably. The advantages of each method are discussed.