The independent prognostic significance of isolated tumour cells in bone marrow is still a matter of debate. This study evaluated the possible association of bone marrow micrometastases with tumour progression and prognosis in patients affected by gastric cancer. Bone marrow aspirates from both iliac crests were obtained from 114 consecutive patients operated on for gastric cancer. The specimens were stained with monoclonal antibody CAM 5.2 which reacts predominantly with cytokeratin filaments 8 and 19. Among 114 cases analysed, 33 cases (29%) had cytokeratine-positive cells in the bone marrow. There was no significant relationship between the presence of bone marrow micrometastases and site, depth of tumour invasion, lymph node metastases, presence of metastases. Patients with cytokeratine-positive cells had a trend towards a diffuse type histology ( P =0.06). Among the 88 curatively resected patients, median survivals were 40 months and 36 months for cytokeratine-negative and cytokeratine-positive subsets respectively ( P =0.9). Recurrence of the disease was observed in 39 cases (44.3%); 11 of 24 (45.8%) in the cytokeratine-positive subset and 28 of 64 (43.7%) in the cytokeratine-negative subset. In conclusion in our experience the presence of cytokeratine-positive cells in the bone marrow of curatively resected gastric cancer patients did not affect outcome and its independent prognostic significance remains to be proven before its official acceptance in the TNM classification.

Recent reports have suggested that tumour cell immunodetection in bone marrow of small-cell lung cancer patients is by far more frequent than found cytohistologically and may have clinical relevance. This study evaluates primarily the efficacy of chemotherapy as method of in vivo purging, but also the relationship of marrow involvement with survival. A total of 112 bone marrow aspirates from 30 chemo-naïve patients were stained twice using anti-NCAM antibodies, first at diagnosis and then after chemotherapy (24 patients) or at disease progression (six patients). Marrow contamination was associated with lower survival (P = 0.002), and was also detected in 7/17 patients conventionally staged as having limited disease. At multivariate analysis, marrow involvement was an independent factor of unfavourable prognosis (P = 0.033). The amount of tumour contamination, before and after chemotherapy, remained unchanged also in responders and even in the subset of patients with apparent limited disease. Following chemotherapy, bone marrow became tumour negative only in 25% of initially positive responders and in none of non-responders. Our results indicate that (i) chemotherapy is not effective in purging bone marrow even in chemo-responsive patients and (ii) a subset of patients with limited disease and negative bone marrow aspirates might have a more favourable prognosis.

Recent reports have suggested that the immunodetection of tumor cells in bone marrow of small-cell lung cancer (SCLC) patients is by far more effective than traditional cytohistological methods and that this may be clinically relevant. This study aimed to evaluate whether the level of detection of tumor cells in bone marrow is affected by different immunostaining methods. Using two anti-NCAM monoclonal antibodies (MAbs), we compared four different "sandwich" methods on cytospin preparations of the N592 human SCLC cell line and of bone marrow aspirates from 37 SCLC patients. Our data indicate that the combination of the alkaline phosphatase-anti-alkaline phosphatase and streptavidin-biotin-alkaline phosphatase complex methods provides the best results in terms of sensitivity and specificity, and of intensity of immunoreaction and absence of staining background. Moreover, bone marrow micrometastases detected by this method were prognostically relevant and identified, among patients with apparently limited disease according to conventional staging procedures, a subgroup with shorter survival. We suggest that the choice of a sensitive immunostaining technique may significantly increase the detection rate of SCLC cells in bone marrow, mirroring the biological aggressiveness of the disease.

Pasini, Felice M.D.; Pelosi, Giuseppe M.D.; Pavanel, Flavia; Bresaola, Enrica CT(IAC), CMIAC; Bassetto, Maria Antonietta M.D. Author Information

The presence of epithelial cells in bone marrow may be a prognostic factor in breast cancer, and so we evaluated their evolution in treated and untreated patients. A first bone marrow aspirate was obtained from 125 stage I/II breast cancer patients at diagnosis and repeated every 6–8 months; the samples were processed for leukocyte separation, used to prepare cytospin slides, stained with a pool of monoclonal antibodies (MoAb) recognising epithelial antigens, and immunocytochemically processed. The median follow-up was 48 months (range 15–82); 23 patients relapsed, and 14 died. MoAb positive cells were observed in 31.2% of first, 24.3% of second, and 27.8% of third aspirates. In 68/100 pairs of successive aspirates, bone marrow status remained unchanged; in 20 it became negative, and in 12 positive (not statistically significant even after adjusting for adjuvant therapy). An analysis based on Mantel and Byar's approach to time-dependent covariates using all 225 aspirates found no statistically significant prognostic difference between the patients with negative and positive bone marrow. Bone marrow status changed over time in about 1/3 of the patients; adjuvant therapy did not affect the probability of its becoming negative or positive. No significant association was found between bone marrow evolution and relapse or death, but the relatively high probability of a change in status over time cannot exclude the possibility that a positive aspirate during the course of breast cancer may be a negative prognostic factor.

Background. Immunocytochemistry has been proven able to identify tumor cells in bone marrow aspirate (BMA) of patients with SCLC. However, few data exist about the clinical significance of the procedure.Patients and methods: 108 BMA taken from 60 patients were incubated with the MoAb MLuCl (cluster 6) and stained by the APAAP (alkaline phosphatase-antialkaline phosphatase) method. The serum levels of LDH, TPA, NSE and CEA were also studied in relation to bone marrow involvement by means of discriminant analysis.Results. Immunocytochemistry of the aspirate with MLuCl detected positive cells in 23 patients (38%) (38 of 108 samples) vs, 13% of the conventional biopsies studied without MLuCl (P < 0.001). With respect to bone marrow positivity, three groups of patients were identified: those with no positive cells in the aspirate and negative biopsy (group A); those with less than 10 positive cells in the aspirate and negative biopsy (group El); and those with more than 10 positive cells or clumps in the aspirate or positive biopsy (group C). Group C patients had Floorer median survivals than those in the other two groups (5.5 vs, 11 months, respectively, P=0.01). Discriminant analysis showed that the four serum markers were poor discriminators cf the degree of bone marrow involvement, with only 55% of grouped cases being correctly classified.Conclusions. These results show that detection of bone marrow involvement i) can be improved by the use of MLuCl ii) is not predictable by conventional tumor markers, and iii) is related to poor outcome.

This study evaluated the efficacy of chemotherapy in 37 chemonaive patients as method of in vivo purging bone marrow. Bone marrow aspirates were taken at diagnosis (all patients), after chemotherapy (31) or at disease progression (6). The slides were stained using anti-NCAM MoAbs and revealed with a modified APAAP method. The number of tumor cells, assessed before and after chemotherapy on 10(5) mononuclear cells, remained statistically unchanged also in the group of responders and only 19% of the patients became tumor-negative as a result of chemotherapy. These results challenge the opinion that chemotherapy is an effective purging method even in chemo-responsive patients.

Background: The presence in bone marrow of cells which react with monoclonal antibodies against tumor-associated antigens has been proposed over the last few years as a new prognostic factor in breast cancer patients. Patients and methods: Bone marrow aspirates were obtained from 109 stage I and II breast cancer patients during or 2–4 weeks after primary surgery. The samples were processed for leukocyte separation on a Ficoll-Hypaque gradient and then used to prepare cytospin slides for immunocytochemical analysis. The slides were stained with a pool of monoclonal antibodies (MoAbs) which recognize tumor associated antigens, using the alkaline phosphatase anti-alkaline phosphatase method. The median follow-up was 36 months (range 15–62); 22 patients relapsed and 7 died. Results: Thirty-four of the 109 patients (31.1%) had MoAb positive bone marrow cells. The bone marrow was positive in 28/74 (37.9%) patients who had the aspirate taken during surgery and in 6/35 (17.1%) who had it taken after surgery (p = 0.055). No association was found between bone marrow positivity and tumour size, nodal status, menopausal status, estrogen receptor positivity or the proliferative index. No association was found between bone marrow and prognosis: the log-rank test was 0.291 (p > 0.5) for OS and 0.023 for DFS; the hazard ratio (positive vs negative) was 1.51 for OS (95% CI: 0.33–6.86) and 0.93 for DFS (95% CI: 0.35–2.45). Conclusions: In our series, bone marrow positivity did not correlate with prognostic parameters or prognosis. Of interest is the relative excess of positivity when the bone marrow was obtained during surgery.

The immunodetection of breast-cancer cells in bone marrow may be a useful technique for monitoring the efficiency of chemotherapy in selected clinical settings. We used monoclonal antibodies against cytokeratins and an epithelial membrane antigen pool to detect microscopic metastatic deposits in bone marrow aspirates from a high-risk patient before and after high-dose sequential chemotherapy. In the slides done before therapy, 250 immunostained cells were found, but only 20 immunostained cells were found in slides made after chemotherapy. Moreover, double-staining procedures showed consistent colocalization of the two markers on the same cancer cells. A fraction of the micrometastatic cells were additionally immunostained for Ki-67, a well-established marker of cell proliferation. These data suggest that the immunodetection of breast cancer cells in bone marrow may have potential clinical implications in the management of high-risk breast cancer.

Platinum-DNA adducts can be assayed in peripheral blood leukocytes by means of atomic absorption spectroscopy and ELISA, and high adduct levels have been correlated previously with favorable clinical response to platinum-based chemotherapy. Our purpose was to study adduct formation in peripheral blood leukocytes by means of a new method, inductively coupled plasma mass spectroscopy (ICP-MS), and to correlate adduct formation with clinical response and toxicity. Platinum (Pt)-DNA adducts were measured by means of ICP-MS in leukocytes of 66 patients receiving a cisplatin- or carboplatin-based chemotherapy, collected either before the beginning of treatment and incubated in vitro with cisplatin or 1 and 24 h after the administration of drug to the patient. The Pt-DNA adduct level in leukocytes from patients exposed to drug in vitro was 14.33 +/- 14.71 fmol/microgram DNA (mean +/- SD), which was not significantly different from the value of 23.4 +/- 19.53 fmol/microgram DNA observed in leukocytes from nine healthy volunteers. In samples collected after the administration of chemotherapy, Pt-DNA adducts ranged from 1.91 +/- 3.59 fmol/microgram DNA (mean +/- SD) at the 1-h time point to 2.61 +/- 3.35 fmol/microgram DNA at 24 h (P > 0.05). Adduct levels in leukocytes exposed in vitro did not correlate with adduct levels from patients treated with cisplatin-based chemotherapy (r = 0.085 and 0.011 at 1 and 24 h, respectively). At 24 h, adduct levels in patients receiving cisplatin (3.15 +/- 3.64 fmol/microgram DNA, mean +/- SD) were significantly higher (P = 0.02) than those observed in patients treated with standard dose carboplatin (0.57 +/- 0.73 fmol/microgram DNA) and also higher than those in patients receiving high-dose carboplatin (1.18 +/- 1.06 fmol/microgram DNA), although the latter difference did not reach statistical significance (P = 0.071). No differences in adduct levels (mean +/- SD) were evident between patients responsive (3.23 +/- 3.51 fmol/microgram DNA) and nonresponsive (2.34 +/- 3.01 fmol/microgram DNA) to chemotherapy. In the homogeneous group of patients treated with combination of cisplatin and 5FU, received dose intensity, hemoglobin decrease, and posttreatment creatinine could not be linked with the extent of leukocyte adduct formation. The data presented here demonstrate that ICP-MS allows the detection of adducts in patients treated with cisplatin or carboplatin and suggest that adduct formation in leukocytes is not a major determinant of response or toxicity.

Platinum (Pt)-DNA adducts by means of Inductively Coupled Plasma Mass Spectroscopy (ICPMS), were determined in peripheral blood leukocytes of 46 patients treated with Cisplatin- or Carboplatin-based chemotherapy, before the beginning of chemotherapy, and at 1 h and 24 h after the end of Cisplatin (or Carboplatin) infusion on each 1st and 3rd chemotherapy cycle. In basal samples Pt-DNA adducts were never detected. In 33 patients no correlation was found between the response to chemotherapy and the Pt-DNA adducts levels. In 20 patients, Pt-DNA adducts were determined in peripheral blood leukocytes withdrawn before the beginning of chemotherapy and incubated in vitro with Cisplatin (15 μg/ml in RPMI medium). In these leukocytes median Pt-DNA adducts was 8.64 fmol/ug of DNA (range = 1.6–25.4). A significant correlation was found between adducts formation in vitro and, in vivo at the 1 h after chemotherapy time point (r = 0.664; P = 0.0113); it was lost at the 24 h after chemotherapy time point (r = 0.241; P = 0.305). No correlation was found between in vitro adducts formation and response. Platinum (Pt)-DNA adducts by means of Inductively Coupled Plasma Mass Spectroscopy (ICPMS), were determined in peripheral blood leukocytes of 46 patients treated with Cisplatin- or Carboplatin-based chemotherapy, before the beginning of chemotherapy, and at 1 h and 24 h after the end of Cisplatin (or Carboplatin) infusion on each 1st and 3rd chemotherapy cycle. In basal samples Pt-DNA adducts were never detected. In 33 patients no correlation was found between the response to chemotherapy and the Pt-DNA adducts levels. In 20 patients, Pt-DNA adducts were determined in peripheral blood leukocytes withdrawn before the beginning of chemotherapy and incubated in vitro with Cisplatin (15 μg/ml in RPMI medium). In these leukocytes median Pt-DNA adducts was 8.64 fmol/ug of DNA (range = 1.6–25.4). A significant correlation was found between adducts formation in vitro and, in vivo at the 1 h after chemotherapy time point (r = 0.664; P = 0.0113); it was lost at the 24 h after chemotherapy time point (r = 0.241; P = 0.305). No correlation was found between in vitro adducts formation and response.