ENCUESTA

Tumour Cells

Response to Adjuvant Chemotherapy

4.1 Introduction

Many previously unmanageable cancers can now be cured by modern treatment methods, provided that a diagnosis is made early. Most current methods of cancer diagnosis and detection of metastasis rely on biopsy analysis and imaging principles, including radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone scintigraphy, and sonography. These techniques require a degree of subjective interpretation and may be subject to error. Furthermore, the lower limit of tumour size detection of these techniques is ~1cm, which represents 10® cells or a mass of 1g. A more sensitive detection method would help early diagnosis and could improve the rate of survival.

Metastatic spread, via the blood stream is probably the single most important factor affecting the prognosis of patients with cancer. Patients with primary tumours such as breast, colon, or lung carcinomas who have undergone radical, potentially curative surgery have a recurrence rate in the range of 20-60%. Currently, lymph node involvement is the most important prognostic factor for tumour recurrence in these patients. However, 30-50% of patients with cancer who show no evidence of disease in the locoregional lymph nodes will still have a recurrence at a distant site. Therefore, novel diagnostic methods that separate patients into low-risk and high-risk prognostic groups for recurrence, and need for adjuvant therapy, are required.

The metastatic process is a complex cascade of events: tumour cells in the primary site erode the endothelial basement membrane; penetrate blood vessels; and spread to distant sites or via lymphatics. Thus, detection of cancer cells in the blood could be important to identify patients at high risk of relapse. Immunocytochemical detection of micrometastases in the BM of patients with breast, colon, and gastric cancer has been shown to correlate with early disease relapse (Schlimok et al, 1991; Diel et al, 1992; Lindemann ef a/, 1992). Several markers have been used to detect circulating cancer cells. In particular, CKs and CEA have been proposed as useful markers to detect

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circulating tumour cells of the gastrointostfno; breast, and/or lung (Datta et al, 1994; Gerhard et al, 1994; Soeth et al, 1996; Castaldo et al, 1997). Few reports have suggested that a correlation between expression of these markers in the PB and/or in the BM and patient outcome might exist (Fields et al, 1996; Castaldo et al, 1997; Soeth et al, 1997; Mori et al, 1998). The identification of circulating tumour cells in the PB of patients with BrCa has the potential to provide an important prognostic indicator for survival, since early dissemination of tumour cells is one of the main causes for disease progression (Frost and Levin, 1992).

Death from carcinoma of the breast is principally caused by distant métastasés. More than 95% of patients with breast cancer will have no evidence of metastatic disease on clinical, radiologic, and biochemical examination at presentation. The presence of BM métastasés has been correlated with early recurrence and shorter overall survival. However, a proportion of patients relapse at these sites in the absence of histologic or immunohistochemical evidence of BM micrometastases after resection of the primary tumour (Mansi et al, 1991). In advanced BrCa, the rationale for

adjuvant therapy is based on the assumption that clinically undetectable haematogenous dissemination of viable tumour cells has already occurred, as indicated by a number of risk factors, including tumour size greater than 2cm, cutaneous lymphangioitis carcinomatosa, and regional lymph node métastasés. Unfortunately, little is known about the natural history of micrometastases, and it has been suggested by some studies that the monitoring of such “minimal residual disease" (MRD) could be used to improve disease staging, as a marker for evaluating new therapeutic strategies, and to assess treatment response in individual patients. Most current methods are not sensitive enough to detect circulating cells in significant numbers of patients with early-stage carcinomas (Redding et al, 1983; Leather et ai, 1993; Datta et al, 1994). However, this may be due not only to the inadequate sensitivity of the assays used but also to the possibility that such cells may only appear periodically in the circulation during early tumour development. There may be intermittent shedding of tumour cells into the circulation corresponding with microinvasive events within the tumour. Previous studies have shown detection rates of MRD in the PB of patients with solid tumours in the order of 0% to 27% using PCR methodology (Datta et al, 1994; Schoenfeld et al, 1997) and 0% to 5% using ICC (Aihara et al, 1997; Schoenfeld et al, 1997) in patients with early-stage disease. Patients with advanced disease have a much greater tumour burden and are more likely to have tumour cells present at blood sampling and as such represent a group of patients who can more readily be studied to determine the effect of therapy on disease.

A reliable indicator of the efficacy of adjuvant therapy requires trials with large numbers of patients observed over several years (Sloane, 1995), especially in BrCa, because residual tumour cells may manifest and impact on

survival many years later (Overgaard et al, 1997). Because adjuvant treatment is usually delivered to patients with clinically occult micrometastatic disease, after the successful resection of the primary tumour, the efficacy of therapy can be only assessed retrospectively from the rate of disease-free survival. Consequently, progress in this form of therapy is extremely slow and cumbersome, and therapy is difficult to tailor to the special needs of an individual patient. The importance of a surrogate marker assay that would permit the immediate assessment of therapy-induced cytotoxic effects on residual cancer cells is therefore apparent.

The ICC detection of haematogenously disseminated tumour cells may be such a surrogate marker assay since numerous studies have demonstrated the prognostic impact of such early tumour cell dissemination in BrCa patients (e.g., reviewed by Braun and Pantel, 1998). In this study, I have therefore applied the MoAb A45-B/B3 directed against the heterodimers CK8/18 and CK8/19 as well as a common epitope of several CK polypeptides (Braun at al, 1998a; Stigbrand at al, 1998), to demonstrate the validity of the Cell-Tak® Cell and Tissue Adhesive immunoassay with the MoAb directed against CK as specific marker antigen of extrinsic epithelial cells in the background of mesenchymal cells (Delsol at al, 1984; Schlimok at al, 1987; Pantel at al, 1994; Braun at al, 1998).

The primary objective of the study was to (a) validate the CK8/18 and CK8/19 ICC approach by quantitating tumour cells in the venous blood of women with BrCa (both metastatic and non-metastatic disease); (b) to correlate tumour cells detected in the venous blood with tumour cells detected in the BM of women with cytologically or histologically confirmed primary BrCa, to elucidate whether primary carcinoma patients are already afflicted with

disseminated disease; (c) to evaluate the impact of therapy (surgery and/or chemotherapy) on the presence of BrCa cells detected in the venous blood (and/or BM); {d) to determine whether the Cell-Tak® Cell and Tissue Adhesive

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immunoassay is a sufficiently powerful detection m ethod^venous blood; (e) to assess the use of venous blood as an alternative to BM; (/) to determine whether CK ICC detection in BM and venous blood have a prognostic value with respect to the survival of the patients.

4.2

4.2.1 Patients

From June 1995 to October 1999, samples of peripheral venous blood and/or BM aspirates taken from the posterior iliac crest, were obtained from 94 BrCa patients admitted to the Department of Clinical Oncology, at the Royal Free Hospital in London, after providing their written informed consent. The procedures were approved by the institutional review board. The stage and grade of the tumour were classified according to the tumour-node-metastasis classification of the Union Internationale contre le Cancer [UlCC (Sobin and Wittekind, 1997)] by investigators unaware of the ICC findings in BM and/or PB. In addition, ICC analysis of the BM and/or PB specimens was performed without knowledge of the histopathological results.

In the preliminary phase of the analysis, samples of PB were first obtained from 25 unselected patients with primary BrCa. All patients had cytologically confirmed primary BrCa and no evidence of distant metastatic disease on chest radiology and bone and liver scanning. In comparison, PB samples from 25 patients being treated for advanced BrCa were also obtained. Inclusion criteria were histologically or cytologically proven progressive

metastatic disease, and clinically or radiologically assessable disease. No patient had received any previous endocrine or other treatment for at least 1 week before the blood samples were taken. All patients were clinieally-