Presence Of Occult Tumor Cells Research Articles

Occult Tumour Cells in Lymph Nodes from Gastric Cancer Patients: Should Isolated Tumour Cells Also Be Considered?

Regional lymph node metastasis is an important prognostic factor for patients with gastric cancer. Occult tumour cells (OTCs), including either micrometastases (MMs) or isolated tumour cells (ITCs), may be a key factor in the development of cancer recurrence in pN0 patients. We aimed to determine the frequency and prognostic significance for disease recurrence of OTCs. This retrospective cohort study included all consecutive patients with pN0 gastric adenocarcinoma between January 2000 and December 2011 (n = 73). Immunohistochemistry using the pan-cytokeratin antibody AE1/AE3 was used to detect OTCs in 1257 isolated lymph nodes. OTCs were identified in 30 patients (41%), including 20 cases with MMs (27%) and 10 cases with ITCs (14%). Disease recurrence and cancer-related death were observed in 24 (33%) and 20 patients (27%), respectively, and both were significantly associated with the detection of OTCs. A significant difference was also observed for the mean survival time between patients with OTCs and those without OTCs [100 vs 158 months (p = 0.015)]. The presence of OTCs was statistically significantly associated with the Lauren classification, tumour size and lymphatic permeation. Multivariate analyses revealed that only age, T stage and the presence of ITCs in lymph nodes were independent factors for recurrence. The presence of ITCs increased the risk for recurrence by 11.1-fold. In a significant proportion of patients diagnosed as stage pN0, OTCs may be identified in lymph nodes if carefully searched for, which can negatively affect their prognosis. The presence of ITCs was found to be an independent factor for recurrence and after proper validation should be considered during lymph node assessment for prognosis definition.

DETECTION OF OCCULT LYMPH NODE TUMOR CELLS IN NODE-NEGATIVE GASTRIC CANCER PATIENTS

ABSTRACTBackground: The presence of lymph nodes metastasis is one of the most important prognostic indicators in gastric cancer. The micrometastases have been studied as prognostic factor in gastric cancer, which are related to decrease overall survival and increased risk of recurrence. However, their identification is limited by conventional methodology, since they can be overlooked after routine staining. Aim: To investigate the presence of occult tumor cells using cytokeratin (CK) AE1/AE3 immunostaining in gastric cancer patients histologically lymph node negative (pN0) by H&E. Methods: Forty patients (T1-T4N0) submitted to a potentially curative gastrectomy with D2 lymphadenectomy were evaluated. The results for metastases, micrometastases and isolated tumor cells were also associated to clinicopathological characteristics and their impact on stage grouping. Tumor deposits within lymph nodes were defined according to the tumor-node-metastases guidelines (7th TNM). Results: A total of 1439 lymph nodes were obtained (~36 per patient). Tumor cells were detected by immunohistochemistry in 24 lymph nodes from 12 patients (30%). Neoplasic cells were detected as a single or cluster tumor cells. Tumor (p=0.002), venous (p=0.016), lymphatic (p=0.006) and perineural invasions (p=0.04), as well as peritumoral lymphocytic response (p=0.012) were correlated to CK-positive immunostaining tumor cells in originally negative lymph nodes by H&E. The histologic stage of two patients was upstaged from stage IB to stage IIA. Four of the 28 CK-negative patients (14.3%) and three among 12 CK-positive patients (25%) had disease recurrence (p=0.65). Conclusion: The CK-immunostaining is an effective method for detecting occult tumor cells in lymph nodes and may be recommended to precisely determine tumor stage. It may be useful as supplement to H&E routine to provide better pathological staging.

Occult tumour cells in peritoneal lavage are a negative prognostic factor in pancreatic cancer

The aim of this study was to test the hypothesis that occult tumour cells in peritoneal lavage are a negative prognostic factor in pancreatic adenocarcinoma. Real-time RT-PCR analysis of CEA, EGFR and hTERT transcript levels was used to identify occult tumour cells in peritoneal lavage samples from 96 pancreatic cancer patients. We found significant association between CEA expression levels in peritoneal lavage and clinical stage. We also found that EGFR transcript levels were higher in peritoneal lavage samples from patients with high grade tumours than in samples from patients with low grade tumours. Detection of CEA and/or EGFR occult tumour cell markers in the peritoneal lavage was associated with significantly shorter overall survival and increased hazard ratio for disease recurrence. The results show that the presence of occult tumour cells in peritoneal lavage is a negative prognostic factor for survival in pancreatic cancer patients, and that detection of occult tumour cells using PCR-based methods can identify patients with advanced disease for whom radical surgery is likely to have little benefit.

Occult tumor cells in peritoneal lavage in pancreatic carcinoma patients.

e14556 Background: Pancreatic carcinoma is one of the most aggressive malignancies with a poor prognosis. Assessment of occult tumor cells could prevent aggressive surgery in patients with metastatic spread. The aim of this study was to investigate the prognostic significance of occult tumor cells in peritoneal lavage of patients with pancreatic carcinoma. Methods: This was a prospective study to assess the presence of occult tumor cells in peritoneal lavage of 96 pancreatic carcinoma patients at the time of surgery using real-time RT-PCR for carcinoembryonic antigen (CEA), epidermal growth factor receptor 1 (EGFR1) and human telomerase (hTERT). Gene expression of tested markers was correlated with clinical/pathological characteristics using Kruskal-Wallis Anova and Mann-Whitney tests. Kaplan-Meier methods and Gehan-Wilcoxon tests were used for overall survival analyses. Results: Overall, 65 of 96 (69.9%) pancreatic carcinoma patients died, the overall survival median was 10.3 months. A significant association between CEA expression in peritoneal lavage and the clinical stage was found (p<0.03). In addition, patients with high grade tumors had significantly higher expression of EGFR in peritoneal lavage compared to patients with low grade tumors (p<0.04). The patients with the presence of occult cancer cells detected in the peritoneal lavage using CEA and/or EGFR had significantly shorter overall survival (p<0.001; HR=2.14 [95% CI: 1.30-3.55]) with overall survival median 6.8 months. Conclusions: These data demonstrate that the presence of occult tumor cells in peritoneal lavage is a negative prognostic factor in pancreatic carcinoma patients. Occult tumor cells detection using PCR based methods can identify patients with advanced disease for whom radical surgery is of no benefit. Prospective studies should confirm usefulness of this method for the selection of patients for radical surgery. Acknowledgements: Supported by grants IGA MZ NS 9937‑4, MPOTIP FR-TI1/525, IGA UP LF_2011_018 and CZ.1.05/2.1.00/01.0030.

Abstract LB-438: Circulating tumor cells in pancreatic carcinoma: negative prognostic factor

Abstract Background: The aim of this ongoing study was to evaluate the hypothesis that circulating tumor cells (CTCs) are negative prognostic factor in pancreatic carcinoma. Pancreatic carcinoma is one of the most aggressive malignancies with a poor prognosis. Assessment of CTCs could prevent aggressive surgery in patients with advanced systemic dissemination. Patients and methods: This was a prospective study to test the presence of CTCs in systemic blood, tumor draining blood, bone marrow and peritoneal lavage of 136 pancreatic carcinoma patients (53 females, 83 males, mean age 63 years, ranging from 37 - 84 years old) at the time of surgery using real-time RT-PCR for carcinoembryonic antigen (CEA), epidermal growth factor receptor 1 (EGFR1) and human telomerase (hTERT). Gene expression of tested markers was correlated with clinical/pathological characteristics and overall survival. Results: Overall, 92 of 136 (67.6%) pancreatic carcinoma patients died, the overall survival median was 10.3 months. 66 of 136 (48.5%) of patients underwent curative surgery (R0 surgery). A significant association between EGFR and hTERT expression levels in the tumor draining blood and clinical stage was found, where patients with advanced disease have a higher expression of EGFR or hTERT in the tumor draining blood, than patients with lower clinical stage (p<0.004, resp. p<0.001). In addition, significant association between CEA expression levels in the peritoneal lavage and the clinical stage was found (p<0.03). The pancreatic carcinoma patients with the presence of CTCs/DTCs in tumor draining blood and bone marrow using hTERT had significantly shorter overall survival (p<0.042; HR=1.44 [95% CI: 0.85-2.45], resp. p<0.045; HR=1.55 [95% CI: 1.01-2.34]). In addition, patients with the presence of occult tumor cells detected in the peritoneal lavage using CEA and/or EGFR had significantly shorter overall survival (p<0.001; HR=2.14 [95% CI: 1.30-3.55]). Interestingly, the patients with presence of CTCs detected in either peripheral blood, tumor draining blood or peritoneal lavage using combination of CEA and/or EGFR and/or hTERT had also significantly shorter overall survival (p<0.07; HR=1.42 [95% CI: 0.94-2.16], resp. p<0.0006; HR=1.95 [95% CI: 1.20-3.18], resp. p<0.01; HR=1.62 [95% CI: 0.98-2.67]). Conclusion: The present ongoing study demonstrates that the presence of CTCs is a negative prognostic factor in pancreatic carcinoma patients. CTCs detection using PCR based methods can identify patients with advance disease for whom radical surgery is of small benefit. Prospective studies should confirm usefulness of this method for the selection of patients for radical surgery Acknowledgements: Supported by grants IGA MZ NS 9937-4, MPOTIP FR-TI1/525, IGA UP LF_2011_018 and CZ.1.05/2.1.00/01.0030 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-438. doi:1538-7445.AM2012-LB-438

Decreased incidence of isolated tumor cells in lymph nodes after laparoscopic resection for colorectal cancer

BackgroundLaparoscopic surgery has potential for less tumor cell spread because of the no-touch technique. We assessed the effect of the surgical approach (open versus no-touch laparoscopic) on the presence of tumor cells in sentinel lymph nodes (SN) of patients with stage I and II colorectal cancer.MethodsA single-center consecutive prospective series of patients operated on for colorectal cancer was analyzed. After conventional hematoxylin and eosin (H&E) staining, 107 patients without lymphatic metastases were included; 59 patients had open surgery, and 48 patients underwent laparoscopic resection. Patients in the laparoscopic group underwent a no-touch medial to lateral approach, whereas the conventional lateral to medial approach was applied in open surgery. A SN procedure was performed in all patients. The SNs were immunohistochemically analyzed for presence of occult tumor cells (OTC). According to the American Joint Committee on Cancer (AJCC) these tumor cells were divided into micrometastases (0.2–2 mm) or isolated tumor cells (ITC, < 0.2 mm).ResultsIn ten patients micrometastases were found, equally distributed between the two groups. However, ITC were more often found after open surgery (18 versus 5 patients, p = 0.03). Presence of OTC was related to depth of tumor invasion and tumor diameter > 3.5 cm. Logistic regression analysis identified lymphovascular invasion as a predictor for micrometastases [odds ratio (OR) 18.4], whereas open resection was predictive for presence of ITC (OR 3.3).ConclusionsNo-touch medial to lateral laparoscopic surgery results in less isolated tumor cells in lymph nodes compared with open lateral to medial surgery in patients with stage I and II colorectal cancer.

Lymphatic micrometastases in patients with early esophageal adenocarcinoma

Both endoscopic and surgical treatments are recommended for m3- or sm1-adenocarcinomas of the esophagus, depending on patients' lymph nodal status. Lymphatic dissemination is related to tumor infiltration depth, but varying incidences have been reported in m3- and sm1-adenocarcinomas. The study aim was to investigate whether the presence of occult tumor cells in lymph nodes could explain this variation. Sixty-three node-negative (N0) patients with early esophageal adenocarcinoma (m2/m3/sm1-tumors) were included. Multilevel-sectioning of lymph nodes was performed; sections were stained by means of immunohistochemistry with cytokeratin marker CAM5.2. Two pathologists searched for micrometastases (0.2-2.0 mm) and isolated tumor cells (ITCs, <0.2 mm). Positive CAM5.2 staining in lymph nodes was not seen in any of the 18 m2-patients. In 2/25 m3-tumors (8.0%) an ITC was found, but no micrometastases. Tumor cells were identified in 4/20 sm1-tumors (20.0%): three micrometastases and one ITC. Median follow-up was 121 months. Two m3-patients (3.2%) died due to disease recurrence, including one patient in whom an ITC was detected. Lymphatic migration of tumor cells was found in node-negative m3- and sm1-adenocarcinomas of the esophagus (8.0% and 20.0%, respectively). However, the clinical relevance of these occult tumor cells should become apparent from large series of endoscopically treated patients.

Diagnosing occult tumour cells and their predictive value in sentinel nodes of histologically negative patients with colorectal cancer

Most studies on the sentinel node (SN) procedure in patients with colorectal cancer include immunohistochemical analysis of the SN only. To evaluate the real diagnostic accuracy of the SN procedure with immunohistochemical analysis, the presence of occult tumour cells in all histologically negative lymph nodes was compared to the presence of these cells in SNs. Also the reproducibility of diagnosing occult tumour cells (OTC) and the sensitivity of three different antibodies was assessed. Between November 2006 en July 2007, an ex vivo SN procedure was performed in 58 histologically N0 patients with colorectal cancer. All lymph nodes (n = 908, mean 15.7) were step-sectioned and immunohistochemistry was performed using two antibodies against cytokeratins (Cam5.2, and CK 20) and one antibody against BerEp-4. OTC were identified in 19 of 58 patients, with micrometastases (0.2-2 mm) in 7 and isolated tumour cells (ITC)(<0.2 mm) in 12 patients. The overall agreement in diagnosing OTC between two independent pathologists was 86%. An SN was identified in 53 of 58 patients. All micrometastases were found in SNs. In two patients with negative SNs, ITC's were demonstrated in non-SNs (sensitivity 88%, and overall accuracy 96%). Additional immunohistochemical analysis of histologically negative lymph nodes demonstrates occult tumour cells in 33% of the patients resulting in an upstaging rate of 12%. Occult tumour cells are predominantly found in the SN, therefore SN mapping has the potential to refine the staging system for patients with colorectal cancer.

Lobular phenotype related to occult-metastatic spread in axillary sentinel node and/or bone marrow in breast carcinoma

To determine whether any histological trait was associated with regional and/or systemic spread of occult tumour cells (OTCs) in small size invasive breast cancer, we compared tumour characteristics, axillary sentinel lymph node (SN) and bone marrow (BM) status in a series of 287 pT1T2 cases. Surgery was the first step of treatment, associated with SN procedure and with BM aspiration for the detection of OTC. SN was histologically classified as negative, metastatic (>2 mm), micro-metastatic (>0.2 mm and ⩽2 mm) or involved by OTC detected by immunohistochemistry (Ni+, ⩽0.2 mm). BM specimens were analysed after immunocytochemistry and classified as negative or positive with atypical cytokeratin-positive OTC. Metastasis and micro-metastasis in the SN were correlated with size, grade and vascular invasion. In contrast, presence of OTC in both SN and BM was independent of these parameters but positively associated with lobular type. This correlation was also observed for BM status, which was similarly independent of the tumour characteristics. No association was found between SN status and BM status. Our data indicate that, in the course of breast cancer, OTC spreading is frequent and could be an early event, related to lobular histological type but independent of classical histoprognostic parameters, and that the loco-regional metastatic spread of OTC is not a prerequisite for systemic involvement.

Quantitative RT-PCR Detection of Tumor Cells in Sentinel Lymph Nodes Isolated From Colon Cancer Patients with an Ex Vivo Approach

To investigate quantitative RT-PCR-based detection of tumor cells in lymph nodes (LNs) isolated from colon cancer patients by ex vivo sentinel lymph node (SLN) mapping. Although lymph node status is among the strongest prognostic factors in colon cancer patients, 20% to 30% of node negative patients experience disease recurrence. These patients may have LN metastases that are not detected by routine examination. Ex vivo SLN mapping was applied to 131 prospectively recruited patients undergoing curative surgery for primary colon cancer. The SLNs were analyzed for the presence of tumor cells by routine histology and real-time RT-PCR quantitation of cytokeratin 20 (CK20) and mucin 2(MUC2) mRNA. SLNs were identified in 125 (95%) of the 131 patients included.Routine histologic analysis of SLNs and other regional lymph nodes revealed LN metastases in 42 patients (N+), of which 29 (69%) had metastases detected in 1 or more SLNs (sensitivity, 69%; false negative rate, 31%).When analyzing the SLNs by quantitative RT-PCR, the sensitivity, compared with routine LN examination, was 37/42 (88%) for both the CK20 and the MUC2 mRNA markers. In addition, 46% and 27% of the patients' node negative by routine LN examination (N0) were positive for the CK20 and MUC2 mRNA markers, respectively, possibly reflecting the presence of occult tumor cells in their SLNs. Quantitative RT-PCR analysis of SLNs identified N+ patients with high sensitivity and revealed a subgroup of N0 patients with potential occult LN disease.

Immunocytochemical Detection of Occult Tumor Cells in the Bone Marrow: Prognostic Impact on Early Stages of Lung Cancer

Objectives: This study was designed to verify the prognostic impact of occult tumor cells in the bone marrow of stage I and II non-small-cell lung cancer patients using cytokeratin as a micrometastatic marker. Methods: One hundred and fifty-two patients with stage I and II non-small-cell lung cancer, who underwent radical surgery by pulmonary lobectomy, were entered into the study. Bone marrow from fragments of resected ribs, and primary tumors were stained by anti-cytokeratin 18 antibody. Fourteen bone marrow specimens from patients without malignancy were used as a control group. Cancer recurrence was the study end point. Results: All the primary tumors were positive for cytokeratin; occult tumor cells were detected in 38 bone marrow specimens (25%). The prevalence of the occult tumor cells was not related to age, gender, tumor stage, histological differentiation or grade. The mean follow-up time was 35.3 months; 68 patients developed recurrence; the mean time for recurrence was 21.2 months. The general disease-free interval was not related to the presence of occult tumor cells in the bone marrow. This result did not change when grouping the patients by tumor stage. The stage was the best predictor of cancer recurrence (Cox proportional hazards model ratio: 2.09; p = 0.0026). Conclusions: This study confirms that immunocytochemical analysis detects occult tumor cells in the bone marrow of at least 25% of patients surgically treated for stage I and II non-small-cell lung cancer. These occult tumor cells do not have any impact on the disease-free interval.

Clinical Relevance of Occult Tumor Cells in Lymph Nodes From Gastric Cancer Patients

The current method for staging in gastric cancer is not sufficient as even after a complete primary tumor resection, patients with node-negative gastric cancer suffer from disease recurrence. In this study, the relation between disease recurrence and the presence of occult tumor cells (OTC) in lymph nodes from gastric cancer patients was evaluated. In a case-control design, lymph nodes from 40 cases (disease recurrence) and 41 controls (no disease recurrence and followed for at least five years) with gastric cancer were examined for the presence of OTC, that comprised micrometastases (MM; >0.2 mm and < or =2.0 mm) and isolated tumor cells (ITC; < or =0.2 mm). The original hematoxylin and eosin-stained sections of all lymph nodes from cases and controls were previously considered as tumor-negative by the local pathologist. Fresh hematoxylin and eosin-stained sections were screened by conventional microscopy. Histologic sections stained by immunohistochemistry with anticytokeratin antibodies CAM5.2 were screened by conventional and automated microscopy. Tumor cells were detected in lymph nodes from 40 of 81 (49%) patients. There was no significant difference in the presence of OTC, MM, or ITC between the case and control groups (P = 0.658, P = 0.691, P = 0.887, respectively). However, significantly more cases presented with 20% or more OTC-positive lymph nodes (P = 0.015). A multivariate logistic regression analysis showed that examination of less than five lymph nodes (odds ratio, 13.8; 95% confidence interval, 1.6-120.6, P = 0.018) was the only significant independent risk factor for disease recurrence, especially for locoregional disease recurrence (odds ratio, 20.4; 95% confidence interval, 2.2-190.8, P = 0.008). A similar analysis for distant disease recurrence showed a percentage of 20% or more OTC-positive lymph nodes to be the only significant independent risk factor (odds ratio, 15.6, 95% confidence interval, 1.6-151.4, P = 0.018). The sensitivity of immunohistochemistry evaluated by microscopy to identify cases with 20% or more OTC-positive lymph nodes increased from 8% for conventional microscopy to 22% for automated microscopy (McNemar's test, P = 0.063). The mere presence of OTC-positive lymph nodes in gastric cancer patients did not predict disease recurrence. However, the number of examined lymph nodes and the percentage of OTC-positive lymph nodes were independent risk factors for locoregional disease recurrence and distant disease recurrence, respectively. Automated microscopy was essential in identifying patients with 20% or more OTC-positive lymph nodes. Therefore, a maximum number of lymph nodes should be removed and meticulously examined for OTC to identify high-risk patients. These patients should be considered for additional treatment.

Neoadjuvant chemotherapy does not effect presence of micrometastases in bone marrow of breast cancer patients

867 Background: The idea of adjuvant systemic therapy in breast cancer patients is to eliminate occult tumor cells which may already disseminate at a very early stage. The presence of occult tumor cells in bone marrow of breast cancer patients without evidence for clinically relevant metastatic disease is associated with worse overall and disease-free survival. However, it is unclear whether cytostatics may eliminate those occult tumor cells. Methods: Bone marrow aspirates obtained during surgery in breast cancer patients with locally resticed disease without evidence of a metastatic tumor cell spread were immunohistochemically stained with anti-cytokeratin antibodies in order to detect disseminated tumor cells. In a matched pair analysis bone marrow results of patients receiving neoadjuvant chemotherapy were matched with patients undergoing surgery first followed by adjuvant therapy. Results: Thirtyfour patients receiving neoadjuvant CTx were included into the analysis and matched for clinical stage and age with 34 breast cancer patients undergoing primary surgery. Detection rate of micrometastases was 23 out of 34 in the neoadjuvant group compared to 13 out of 34 in patients receiving surgery first followd by adjuvant therapy. Both groups did not significantly differ in clincal stage, age or bone marrow status. Interestingly, in 2 patients receiving trastuzumab in a neoadjuvant setting, no disseminated tumor cells were detected after systemic therapy. Conclusions: Our results suggest, that bone marrow status - which is highly correlated to survival - is not affected by cytostatic therapy. Therefore, adjuvant therapies in patients with micrometastases in bone marrow may include additional strategies in order to specifically target cells of a minimal residual disease. No significant financial relationships to disclose.

Detection of occult tumor cells in lymph nodes from bladder cancer patients by MUC7 nested RT-PCR.

Systemic progression is the prevalent form of bladder tumor recurrence after radical cystectomy. The detection of occult bladder tumor cells in histopathologically normal lymph nodes could be of prognostic value. We examined the possibility that mucin 7 (MUC7) RNA might reflect the presence of occult tumor cells in lymph nodes from bladder cancer patients. We used the polymerase chain reaction (RT-PCR), a highly sensitive assay, to monitor MUC7 RNA. We collected 240 pelvic lymph nodes from 25 bladder cancer patients undergoing radical cystectomy. We also obtained 20 lymph nodes from patients with prostate cancer and interstitial cystitis to use as negative controls. Each lymph node was divided in two parts to provide tissue for both histopathological and PCR analysis. 166/240 lymph nodes from bladder cancer patients were usable for MUC7 RT-PCR. By conventional histopathology, six of these nodes contained metastases. MUC7 RT-PCR analysis was positive for five of the six histologically proven lymph node metastases. Histopathological reevaluation of the sixth node revealed tumor in an adjacent vein, not in the lymph node, itself. In contrast, 46/160 (29%) histologically classified normal lymph nodes (pN0) from 17 bladder cancer patients were positive for MUC7. All 20 lymph nodes from control patients were MUC7-negative. MUC7 RT-PCR is a specific and sensitive method for the detection of occult tumor cells in lymph nodes from bladder cancer patients. Long-term observation will be necessary to evaluate the clinical value of MUC7 as a prognostic indicator of lymph node metastasis and disease progression.

P32 Presence of occult tumour cells in peripheral blood and bone marrow in patients with malignant melanoma

Faye, R.1.2; Aamdal, S.1; Hoifodt, H. K.1; Jacobsen, E.1; Kvalheim, G.1; Skovlund, E.1; Fodstad, O.1 Author Information

Minimal residual disease in breast cancer.

The incidence of breast cancer is increasing dramatically in the developed countries. Although attention is being paid to diagnose breast cancer early through screening programs, still a significant number of patients classified to have a localised disease at diagnosis, later experience a systemic recurrence. Therefore, more sensitive and reliable methods to detect the early spread of the disease, permitting the early use of additional systemic therapy, seem justified. New treatment strategies, such as immunotherapy employing monoclonal antibodies against breast cancer cells, is promising. Since such a treatment is most efficient on patients with limited disease, sensitive methods to monitor the therapeutic effect are needed. Immunocytochemistry using tumour associated monoclonal antibodies and reverse transcription polymerase chain reaction assays (RT-PCR), that screen for carcinoma-specific expression of mRNA in bone marrow and blood, have been developed. Many standardisation problems with the detection methods currently in use are unsolved. Despite this, we discuss here recent data showing that the presence of occult tumour cells in the bone marrow is a prognostic factor in patients with breast cancer.

Detection of occult tumour cells in bone marrow and blood in breast cancer patients--methods and clinical significance.

Immunocytochemistry using tumour-associated monoclonal antibodies has led to improvements in the ability to detect occult breast cancer cells in bone marrow aspirates and peripheral blood. Nevertheless, the immunocytochemistry method needs to be further developed before it can be used routinely in the clinic. Reverse transcription polymerase chain reaction assays (RT-PCR) that screen for carcinoma-specific expression of mRNA in bone marrow and blood have been developed. However, it is not yet clear whether the most frequently employed RT-PCR assay for cytokeratin 19 has the specificity required to be safely used in the clinic. In spite of many unsolved standardization problems with micrometastatic detection methods, recent data show that the presence of occult tumour cells in the bone marrow at diagnosis and in the reinfused autograft after high-dose therapy appears to increase the rate of recurrence in the patients.

Autologous bone marrow transplantation in acute leukemia and non-Hodgkin's lymphoma: a phase I study of 4-hydroperoxycyclophosphamide (4HC) incubation of marrow prior to cryopreservation.

The presence of occult tumor cells in remission marrow is the initial obstacle to the use of autologous bone marrow transplantation in acute leukemia. In vitro treatment of tumor-marrow mixture, with heterologous antiserum and complement, has been shown to be capable of eliminating all clonogenic tumor in a number of animal models, and this approach forms the basis for a number of ongoing clinical trials [1]. An alternative procedure, utilizing in vitro drug treatment of marrow, was first investigated at The Johns Hopkins Oncology Center using 4-hydroperoxycyclophosphamide (4HC). In aqueous solution, this congener of cyclophosphamide (CY) has the same alkylating and cytotoxic effects and the same immunoreactive products as the microsomally activated parent compound.