Over the past decade a number of methods capable of detecting MRD have been developed. These techniques have clearly illustrated that patients in clinical complete remission often harbor malignant cells in low numbers. The clinical significance of the detection of such MRD is still being evaluated and remains unclear. The results of these studies will likely have great impact on the clinical management of patients as we understand more about the contribution of minimal disease to subsequent relapse. The prognostic significance of the achievement of molecular complete remission remains elusive, and few studies to date have demonstrated the importance of eradicating MRD in the patient to achieve cure. The majority of studies have been performed using as a target the t(14;18) in follicular lymphoma, but more recently studies have examined other translocations as well as Ig or TCR rearrangements and have been reporting similar results. These studies have suggested that the goal of therapy should be to eradicate the malignant clone and achieve molecular complete remission.

PCR detection of bone marrow infiltration as a staging procedure

Lymphomas generally originate in lymphoid tissue, but as the disease progresses there may be spread to other sites, such as bone marrow and blood. At initial presentation all patients undergo staging investigations to determine the extent of disease as a means of planning treatment. A number of studies have examined the use of PCR detection of t(14;18) as a staging procedure to detect lymphoma cells in the bone marrow and peripheral blood at the time of initial presenta tion. However, PCR analysis cannot replace morphological assessment of bone marrow (BM) since not all patients have translocations detectable by PCR, and these techniques are essentially complementary. These PCR studies have all detected lymphoma cells in the BM in a number of patients who had no overt evidence of marrow infiltration by morphology. Of great interest are those studies that have evaluated the clinical utility of MRD detection in those patients presenting with localized disease. Although the patient numbers studied are small, a significant number of patients can be found who would be upstaged from early stage to advanced stage disease by the results of PCR analysis. Whether PCR detection of minimal marrow infiltration will eventually lead to modifications in therapy in those patients currently treated with localized radiotherapy remains to be determined.

PCR detection of MRD following chemotherapy

In follicular lymphoma, long-term analysis of patients after completion of conventional chemotherapy has shown that conventional-dose chemotherapy does not eradicate PCR-detectable disease, but this may not be associated with poor outcome. One study has shown no association between the presence or absence of PCR-detectable lymphoma cells and clinical outcome. Moreover, this confirms the previous observation that some patients can indeed remain in long-term continuous complete remission despite the presence of PCR-detectable lymphoma cells, strongly suggesting that the detection of residual lymphoma cells has no prognostic significance. Cells containing t(14;18) might not always represent residual lymphoma cells, but may be cells with this translocation but without the additional necessary cellular changes required for malignant transformation. However, an alternative explanation is that conventional chemotherapy might not cure any patients with advanced stage follicular lymphoma and that all patients with persistent lymphoma cells are destined to relapse. The long-term remission status of these small numbers of patients might therefore represent merely the very long duration of their disease course.

These studies suggest that conventional-dose chemotherapy did not result in molecular remission. More novel treatment approaches, including more aggressive induction therapy and combinations of monoclonal antibody therapy with chemotherapy and the use of stem cell transplantation, have all been reported to be capable of eradicating PCR-detectable disease, achieving so-called molecular complete remission. In all of these circumstances, eradication of PCR-detectable disease has been shown to be associated with improved outcome in follicular lymphoma, strongly suggesting that eradication of MRD may be required for cure. With longer follow-up this question should be answered.

Detection of circulating lymphoma cells in peripheral blood

Blood is less frequently involved than marrow at presentation, but becomes more frequent as disease progresses. Studies at the time of initial presentation have suggested a high level of concordance between the detection of lymphoma cells in the peripheral blood (PB) and BM when assessed by PCR. However, other studies have found that the BM is more likely than PB to contain infiltrating lymphoma cells in previously untreated patients. The presence of residual lymphoma in the BM but not in the PB argues strongly that the marrow is indeed infiltrated with lymphoma in these patients and does not simply represent contamination from the PB. The findings of PB contamination with NHL when assessed by PCR are likely to have profound implications since there is now increasing interest in the use of PB stem cells, rather than BM, as a source of hematopoietic progenitors. A number of studies have demonstrated that PB stem cell collections may also be contaminated with lymphoma cells when assessed by PCR techniques. In addition, much work is being performed to monitor the effects of chemotherapy and the growth factors that are used to mobilize hematopoietic progenitor cells, to ensure that these agents do not also mobilize lymphoma cells.

Contribution of re-infused lymphoma cells to relapse after autologous stem cell transplantation

In low-grade NHL there has been increasing interest in the use of high-dose therapy as salvage therapy for patients who have failed conventional-dose chemotherapy regimens. The resulting ablation of a patient's marrow after high-dose therapy can be rescued by infusion of allogeneic or autologous stem cells. Autologous stem cell transplantation (ASCT) has several potential advantages over allogeneic stem cell transplantation for marrow rescue: there is no need for a histocompatible donor and there is no risk of graft-versus-host disease. ASCT can therefore be performed more safely, and in older patients, and has become a major treatment option for an increasing number of patients with hematological malignancies.

The major obstacle to the use of ASCT is that the infusion of occult tumor cells harbored within the stem cell collection may result in more rapid relapse of disease. To minimize the effects of the infusion of significant numbers of malignant cells, stem cells are collected when the patient either is in complete remission or has no evidence of lymphoma in the blood. In addition, a variety of methods have been developed to purge malignant cells from the stem cell collection to attempt to eliminate any contaminating malignant cells and leave intact the hematopoietic stem cells that are necessary for engraft-ment. The development of purging techniques has led to a number of studies of ASCT in patients with either a previous history of BM infiltration or even overt marrow infiltration at the time of BM harvest. Because of their specificity, monoclonal antibodies are ideal agents for the selective elimination of malignant cells. Clinical studies have demonstrated that immunological purging can deplete malignant cells in vitro without significantly impairing hematological engraftment.

Assessing purging efficacy by PCR

PCR has been used to assess the efficacy of immunological purging in models using lymphoma cell lines, demonstrating that PCR is a highly sensitive and efficient method to determine the efficacy of purging residual lymphoma cells. The efficacy of purging varies between the cell lines studied, making it likely that there would also be variability between patient samples.

PCR amplifications of the t(14;18), t(11;14) and IgH rearrangements have all been used to detect residual lymphoma cells in the BM before and after purging in patients undergoing autologous BM transplantation to assess whether the efficiency of purging had any impact on disease-free survival. In one study, 114 patients with B-cell NHL and the BCL-2 translocation were studied. Residual lymphoma cells were detected by PCR analysis in the harvested autologous BM of all patients. Following three cycles of immunological purging using anti-B-cell monoclonal antibodies and complement-mediated lysis, PCR amplification detected residual lymphoma cells in 50% of these patients. The incidence of relapse was significantly increased in the patients who had residual detectable lymphoma cells compared with those in whom no lymphoma cells were detectable after purging.

Detection of residual lymphoma cells in the marrow after transplantation is associated with increased incidence of subsequent relapse

Since PCR analysis detected residual lymphoma cells after conventional-dose chemotherapy in the majority of patients studied, it is not surprising that it has not been possible to determine any prognostic significance for the persistence of PCR-detectable lymphoma cells. At the Dana-Farber Cancer Institute, PCR analysis was performed on serial BM samples obtained after ASCT to assess whether high-dose therapy might be capable of depleting PCR-detectable lymphoma cells. The persistence or reappearance of residual detectable lymphoma cells had a great adverse influence on the disease-free survival of patients in this study after high-dose therapy. In contrast to previous findings that all patients had BM infiltration following conventional-dose therapy, no PCR-detect-able lymphoma cells could be detected in the most recent BM sample obtained from more than 50% of patients following high-dose chemoradiotherapy and ASCT. A number of stud ies have now demonstrated that persistent detection of MRD by PCR following ASCT in patients with lymphoma identifies those patients who require additional treatment for cure, and also suggest that our therapeutic goal should be to eradicate all PCR-detectable lymphoma cells. In addition, quantitative PCR analysis has further shown that a rising tumor burden is a particularly poor prognosis feature.

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