Hanne Qvist

Detection of Isolated Tumor Cells in Bone Marrow Is an Independent Prognostic Factor in Breast Cancer

PURPOSE This study was performed to disclose the clinical impact of isolated tumor cell (ITC) detection in bone marrow (BM) in breast cancer. PATIENTS AND METHODS BM aspirates were collected from 817 patients at primary surgery. Tumor cells in BM were detected by immunocytochemistry using anticytokeratin antibodies (AE1/AE3). Analyses of the primary tumor included histologic grading, vascular invasion, and immunohistochemical detection of c-erbB-2, cathepsin D, p53, and estrogen receptor (ER)/progesterone receptor (PgR) expression. These analyses were compared with clinical outcome. The median follow-up was 49 months. RESULTS ITC were detected in 13.2% of the patients. The detection rate rose with increasing tumor size (P =.011) and lymph node involvement (P <.001). Systemic relapse and death from breast cancer occurred in 31.7% and 26.9% of the BM-positive patients versus 13.7% and 10.9% of BM-negative patients, respectively (P <.001). Analyzing node-positive and node-negative patients separately, ITC positivity was associated with poor prognosis in the node-positive group and in node-negative patients not receiving adjuvant therapy (T1N0). In multivariate analysis, ITC in BM was an independent prognostic factor together with node, tumor, and ER/PgR status, histologic grade, and vascular invasion. In separate analysis of the T1N0 patients, histologic grade was independently associated with both distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS), ITC detection was associated with BCSS, and vascular invasion was associated with DDFS. CONCLUSION ITC in BM is an independent predictor of DDFS and BCSS. An unfavorable prognosis was observed for node-positive patients and for node-negative patients not receiving systemic therapy. A combination of several independent prognostic factors can classify subgroups of patients into excellent and high-risk prognosis groups.

Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome

Purpose: The aim of the study was to explore the value of analyzing bone marrow (BM) for the presence of isolated tumor cell(s) (ITCs) in disease-free breast cancer patients 3 years after diagnosis. Experimental Design: ITCs in BM at operation was found to be an independent prognostic factor in 817 breast cancer patients. Among these, 356 disease-free patients were analyzed with a second BM after 3 years follow-up (median 40 months, SD 3 months, range 29–52). ITC was detected by immunocytochemistry with anticytokeratine antibodies (AE1/AE3). Results: The population consisted of 70% T1 and 72% node-negative patients. ITC in BM was detected in 15%. At a median of 25 months since the second BM aspiration (66 months since diagnosis), 32 patients had developed relapse, 12 local and 20 systemic. Of the patients with ITC in BM, 21% relapsed compared with 7% of the ITC-negative patients (P < 0.001). Ten patients died of breast cancer. Survival analyses showed that ITC in BM predicted reduced distant disease-free survival (DDFS) and breast cancer specific survival (BCSS; P < 0.001, log-rank test). Uni-and multivariate analyses of the prognostic value of N, T, estrogen receptor/progesterone receptor, and BM status, histological grade, vascular invasion, p53-, c-erb-B2-, and cathepsin D expression were performed. BM status was the only independent prognostic factor for both DDFS and BCSS, whereas c-erbB-2 and N status were independent for BCSS and vascular invasion and T status for DDFS. Conclusions: ITC in BM 3 years after diagnosis in disease-free breast cancer patients is an independent prognostic factor. Detection of residual disease by BM analysis at follow-up may unravel insufficient adjuvant treatment. The clinical implications should be further explored.

Significance of Candida recovered from intraoperative specimens in patients with intra-abdominal perforations.

OBJECTIVE Determine the significance of recovering yeasts from intraoperative specimens from the abdominal cavity and to evaluate the effect of a single intraoperative dose of fluconazole on clinical outcome in patients with intra-abdominal perforations. DESIGN Prospective, randomized, double-blind study. SETTING Multicenter study from 13 hospitals in Norway. PATIENTS One hundred nine patients with intra-abdominal perforations. INTERVENTIONS Patients were randomized to receive either a single 400-mg fluconazole dose or placebo during the operation. MEASUREMENTS AND MAIN RESULTS An intra-abdominal specimen for microbiological culture was obtained at the time of the operation. The primary response variable in the study was death. Secondary response variables were three parameters indicating a complicated postoperative period: mechanical ventilation for > or = 5 days, intensive care treatment for > or = 10 days, and use of a central venous catheter for > or = 10 days. Yeasts were recovered from a intraoperative intra-abdominal specimen from only 1 (3.5%) of 28 patients with perforated appendicitis and from 32 (39.5%) of 81 nonappendicitis patients. Excluding the appendicitis patients, the yeast recovery rate was high both for patients hospitalized at the time of the perforation (45%) and for nonhospitalized patients (32%). The overall mortality was 11% (12 patients). Single-dose intraoperative fluconazole prophylaxis did not reach a statistically significant effect on mortality (4 of 53 patients in the fluconazole group and 8 of 56 patients in the placebo group died [p = .059]). The only two explanatory variables significantly related to death were a intraoperative finding of yeast from an intra-abdominal specimen and the occurrence of a spontaneous perforation in a patient already hospitalized for nonsurgical cancer treatment. Detection of yeast was also a significant explanatory variable for a prolonged period of mechanical ventilation, intensive care treatment, and prolonged use of a central venous catheter. CONCLUSIONS Single-dose intraoperative fluconazole prophylaxis did not have a statistically significant effect on overall mortality (odds ratio = 0.21; 95% confidence interval, 0.04-1.06; p = .059) in patients with intra-abdominal perforation. The recovery rate of yeast from intraoperative specimens from the abdominal cavity was high (>30%) and was associated with death and a complicated postoperative course.

Immunocytochemical detection of isolated epithelial cells in bone marrow: non‐specific staining and contribution by plasma cells directly reactive to alkaline phosphatase

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I–II breast cancer patients were stained with anti‐cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)‐based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti‐CK MAbs AE1/AE3 or A45‐B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n=257), anti‐CK‐positive cells were detected in 26·6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control‐positive cells were detected in 5·4 per cent of patients. Some of the false‐positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly‐reactive cells as CD45‐negative and human Ig kappa/lambda‐positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens.

Increased sensitivity for detection of micrometastases in bone- marrow/peripheral-blood stem-cell products from breast-cancer patients by negative immunomagnetic separation

Immunocytochemical detection (ICC) of isolated tumor cells in bone marrow (BM) is currently the most established method for monitoring early dissemination in epithelial cancer. However, the low sample size that can practically be analyzed restricts the sensitivity and reliability of the ICC method. To be able to analyze larger samples, a negative immunomagnetic separation (IMS) technique, utilising anti‐CD45‐conjugated Dynabeads, has been developed. Tumor‐cell enrichment by depletion of CD45‐expressing mononuclear cells (MNC) is followed by ICC for detection of the cytokeratin (CK)‐positive (+) epithelial cells. In this study, bone‐marrow samples (n = 165) and peripheral‐blood‐progenitor‐cell (PBPC) apheresis products (n = 22) from breast‐cancer patients were analyzed. The negative IMS analysis of 1 to 2 × 107 MNC was compared with ICC analysis of 2 × 106 unseparated MNC. Negative IMS resulted in 85% mean depletion of MNC. The results showed that 11.7% of the samples were positive by ICC analysis of unseparated MNC, as compared with 23.5% after negative IMS. In samples presenting >10 CK+ cells, a 4‐fold higher number of positive cells was detected by the negative IMS technique. Moreover, there was no evidence for general enrichment of false‐positive cells. Altogether our results show that negative IMS is an efficient enrichment method for sensitive detection of CK+ cells in BM/ PBPC products from breast‐cancer patients. This opens the possibility for further characterization of micrometastatic tumor cells. Int. J. Cancer 78:556–560, 1998.

The Prognostic Value of Isolated Tumor Cells in Bone Marrow in Breast Cancer Patients: Evaluation of Morphological Categories and the Number of Clinically Significant Cells

Purpose/Experimental Design: Immunocytochemical detection of isolated tumor cells (ITCs) in the bone marrow (BM) is a prognostic factor in breast cancer. However, hematopoietic cells (HCs) can occasionally be stained by the techniques used. Morphological evaluation improves the specificity of ITC detection, but optimal separation of ITCs from false-positive HCs needs to be determined. Here, predetermined morphological categories of immunocytochemically (ICC)-positive cells in the BM and the number of detected ITCs were analyzed for association with clinical outcome in 817 early-stage breast cancer patients (median 49 months of follow-up). All ICC+ cells detected were categorized into one of the following groups: (a) tumor cell (TC); (b) uninterpretable cell (UIC); (c) probable HC; or (d) HC. Results: Among the TC+ patients, 30.6% and 25.9% experienced systemic relapse (SR) and breast cancer death (BCD), respectively, as compared with 13.3% and 8.5% of patients without TCs in the BM (survival analyses: P < 0.001, log-rank). The SR and BCD rate was 19.7% and 15.8% for TC−/UIC+ patients versus 12.5% and 7.4% for TC−/UIC− patients. Survival analyses confirmed that the UIC+ group contained clinically significant cells (P = 0.018, log-rank). No difference in clinical outcome was observed, regardless of whether probable HCs or HCs were present. Analyzing the number of ITC+ cells, SR and BCD occurred in 12.4% and 7.4% of patients with 0 ITCs present, 21.3% and 18.5% of patients with 1 ITC present, 19.4% and 16.7% of patients with 2 ITCs present, and 42.5% and 32.5% of patients with ≥3 ITCs present. Conclusions: Morphological categorization of ICC+ cells improves the clinical value of ITC detection in the BM. The presence of only one ITC reduces survival, and a greater number of ITCs further aggravates the prognosis.

Cytogenetic comparison of primary tumors and lymph node metastases in breast cancer patients

Chromosome banding analysis of primary tumors and axillary lymph node metastases from 10 breast cancer patients revealed abnormal karyotypes in all samples with cytogenetic similarities between the primary tumor and the metastasis in all informative pairs. Although karyotypically unrelated clones were also found in the lymph node samples, they were less numerous than in the primary tumors, indicating that there was more genetic heterogeneity among the neoplastic cells in the primary than in the secondary tumors. On the other hand, some of the clones had become more complex in the metastases as a result of clonal evolution, and by and large these metastatic breast cancer cases had more karyotypic anomalies than do unselected primary breast carcinomas. Among the aberrations occurring more frequently, and that consequently may predispose to disease spread, were losses of chromosomes 17 and 22 and homogeneously staining regions, a cytogenetic sign of gene amplification. Genes Chromosomes Cancer 22:122–129, 1998.

Cytogenetic analysis shows that carcinosarcomas of the breast are of monoclonal origin

Carcinosarcoma of the breast is a rare biphasic neoplasm composed of a carcinomatous component contiguous or admixed with a pleomorphic spindle cell component. The issues of the histogenesis and clonal composition of carcinosarcomas have long been debated. We present the first cytogenetic characterization of mammary carcinosarcomas by analysis of eight tumor samples from two patients with this disease. In the first case, the same karyotypically complex clone, as well as evidence of clonal evolution, was found in samples from three separate areas of the primary tumor. The analysis of one intramammary and one axillary lymph node metastasis from the same patient, both showing only the sarcomatous tumor component, also revealed the common complex stemline and one of the two sidelines found in the primary tumor. The carcinosarcoma of the second patient contained six complex but karyotypically related clones unevenly distributed among the three samples examined. From this case, cells belonging to the carcinomatous and sarcomatous tumor components were separated by differential sedimentation and culturing in specific growth media. Analysis of both fractions showed largely the same karyotype, although one of the subclones was restricted to the epithelial component. Our findings indicate that the epithelial and mesenchymal components of mammary carcinosarcomas are both part of the neoplastic parenchyma and that they have evolved from a single common stem cell, in agreement with the hypothesis that the tumors are of monoclonal origin. Genes Chromosomes Cancer 22:145–151, 1998.

Discrimination between multicentric and multifocal breast carcinoma by cytogenetic investigation of macroscopically distinct ipsilateral lesions

Whether macroscopically distinct carcinomas in the same breast are clonally related (multifocal breast carcinoma) or unrelated (multicentric breast carcinoma) is no longer only a scientific‐pathological issue but, because different therapeutic strategies may be preferable for cases with intramammary metastatic disease compared with cases of multiple primary breast carcinomas, one that may have profound clinical implications. We studied the evolutionary relationship among macroscopically distinct, ipsilateral breast carcinomas by cytogenetic analysis of 26 tumorous lesions from 12 patients. Sixteen of the 26 foci (62%) were found to contain clonal chromosome abnormalities. Two carcinoma foci were karyotypically abnormal in each of seven patients. Four of these cases had an evolutionarily related, cytogenetically abnormal clone in the two lesions from the same breast, whereas the remaining three cases had completely different clonal karyotypic aberrations in the separate foci. These results, together with our previous findings in five other informative cases, show that multiple, synchronous breast tumors sometimes arise through intramammary spreading of a single primary carcinoma, whereas on other occasions they are the result of the simultaneous emergence of pathogenetically independent carcinomas within the breast. In the total material, an association was seen between the proximity of the foci and the likelihood of them being karyotypically related. Genes Chromosom. Cancer 18:170–174, 1997.

Cytogenetic findings in phyllodes tumors of the breast: Karyotypic complexity differentiates between malignant and benign tumors☆

Clonal karyotypic abnormalities were detected in short-term cell cultures from six phyllodes tumors of the breast. Whereas all five benign tumors had simple chromosomal changes, the highly malignant one had a near-triploid stemline, indicating that karyotypic complexity is a marker of malignancy in phyllodes tumors. Interstitial deletions of the short arm of chromosome 3, del(3)(p12p14) and del(3)(p21p23),were the only aberrations in two benign tumors. Cytogenetic polyclonality was detected in three benign tumors: two had cytogenetically unrelated clones, whereas the third had three different, karyotypically related cell populations as evidence of clonal evolution. The finding of clonal chromosome abnormalities in both the epithelial and connective tissue components of the phyllodes tumors indicates that they are genuinely biphasic, that is, that both components are part of the neoplastic parenchyma.