Clinical relevance of bcl-2(MBR)/J(H) rearrangement detected by polymerase chain reaction in the peripheral blood of patients with follicular lymphoma.

We evaluated the prognostic role of peripheral blood polymerase chain reaction (PCR) assay for detection of the bcl-2(MBR)/J(H) rearrangement in 59 patients with follicular lymphoma (FL) treated at our centre since 1989. Thirty-five (59%) patients were bcl-2/J(H) positive and 24 (41%) were negative in the peripheral blood at diagnosis. Peripheral blood bcl-2/J(H) rearrangement detection at diagnosis had no relation to overall survival (OS) and time to progression (TTP). Peripheral blood PCR assay was performed post treatment in 17 patients who were bcl-2/J(H) positive at diagnosis. Fourteen of the patients (82%, 95% CI 56-96%) became bcl-2/J(H) negative. Nine of these patients were further analysed during follow-up and, after several months, circulating cells carrying the bcl-2/J(H) rearrangement reappeared in five of the nine patients. Peripheral blood clearance of bcl-2/J(H)-positive cells was correlated with better overall survival (log-rank P < 0.05) but not with TTP. Our data confirmed that bcl-2(MBR)/J(H) rearrangement detection by PCR at diagnosis is not a prognostic factor in follicular lymphoma. In our series, clearance of circulating bcl-2/J(H)-positive cells appeared to correlate with better overall survival. Post-treatment examination of the peripheral blood by PCR may have clinical relevance for prediction of the survival pattern of the patients.

The majority of follicular lymphomas (FL) and 20-30% of diffuse large B-cell lymphomas carry the t(14;18) (q32;q21) chromosomal translocation which juxtaposes the bcl-2 gene on chromosome 18q21 to the joining region (JH) of the immunoglobulin heavy chain gene on chromosome 14q32. This leads to overexpression of a functionally normal bcl-2 protein, conferring a growth advantage on the neoplastic cells (Yunis et al, 1987;Corbally et al, 1992; Croce, 1993; Mrozek & Bloomfield, 1993). The breakpoint regions on chromosome 18 are clustered in two areas, major breakpoint region (MBR) in 60% and minor cluster region (mcr) in 10-20% (Weiss et al, 1987;Cotter et al, 1990;Cotter et al, 1991) and are readily detectable by polymerase chain reaction (PCR) using the consensus JH primers and primers specific to one or more breakpoint regions. This makes possible a sensitive and reproducible assay for detection of the bcl-2/JH rearrangement at a level of 1 in 105 cells. The clinical significance of detecting the t(14;18) translocation in patients with FL remains controversial (Pezzella et al, 1992;Tilly et al, 1994;Johnson et al, 1995;Whang-Peng et al, 1995). Furthermore, the same rearrangement can be detected, although at very low levels, in healthy people, in whom it does not predispose to malignancy (Liu et al, 1994;Bell et al, 1995;Ji et al, 1995;Limpens et al, 1995;Dolken et al, 1996).
The peripheral blood is readily accessible and it has been suggested that it has a good degree of concordance with bone marrow (Hickish et al, 1991;Yuan et al, 1993) and with lymph node tissue (Lopez-Guillermo et al, 1996). We decided to evaluate prospectively the prognostic significance of bcl-2/JH rearrangement MATERIALS AND METHODS Peripheral blood and/or bone marrow samples from fifty-nine HIV-negative patients with FL, histologically diagnosed by the International Working Formulation (IWF) (The Non-Hodgkin's Lymphoma Pathologic Classification Project, 1982) and the Revised European-American Classification of Lymphoid Neoplasms (Harris et al, 1994), were analysed in our department for the presence of the bcl-2(MBR)/JH rearrangement using PCR assay during the period from January 1989 until December 1995.
All of the patients underwent a complete staging procedure, including physical examination, chest radiography, abdominal computerized tomography (CT) scan, routine haematology with bone marrow biopsy and aspiration and peripheral blood and bone marrow sample collection for assessment by PCR of the presence of the bcl-2(MBR)/JH rearrangement.
Clinical stage was defined according to the Ann Arbor classification. Performance status (PS) was evaluated by the Eastern Cooperative Oncology Group (ECOG) scale. Bulky disease was defined as a mass > 10 cm. The International Prognostic Index (IPI) was calculated according to the original paper (The International Non-Hodgkin's Lymphoma Prognostic Factors Project, 1993).
Peripheral blood samples were collected during the initial evaluation in all patients and in 17 patients also after treatment.

PCR methodology
One and a half micrograms of phenol-chloroform-extracted DNA or 5 ,ul of digestion buffer-extracted DNA were analysed. PCR reactions were performed in 50-il volumes including 1 x PCR buffer (50 mm potassium chloride, 10 mM Tris-HCl pH 8.4, 2.5 mm magnesium chloride), 200 jM of each deoxynucleotide (Pharmacia, Uppsala, Sweden), 25 pmol of each primer and 1.25 U of Taq polymerase (Gibco, Gaithersburg, MD, USA).
The first round of the nested PCR amplification was performed for 30 cycles in a Perkin Elmer Cetus Thermal Cycler 480 with the outer primers 5'-ACCTGAGGAGACGGTGACC-3' for the JH region and 5'-CAGCCTTGAAACATTGATGG-3' for MBR (Pharmacia Biotech, Roosendaal, The Netherlands). Each cycle included 45 s of denaturation at 95°C, 1 min of annealing at 60°C and 2 min of extension at 72°C. A touch-down PCR technique was used starting from an annealing temperature of 60°C, which was decreased every other cycle by 1°C for the first eight cycles.
Five microlitres of the first-round DNA product was reamplified for 30 cycles with inner primers 5'-CAGGGTTCCTTGGC-CCCAG-3' for the JH region and 5'-AGTTGCTTTACGTGGC-CTGT-3' for the MBR (Pharmacia Biotech). Each cycle included 45 s of denaturation at 94°C, 1 min of annealing at 57°C and 1 min of extension at 720C. DNA amplification was always started with 10 min of initial denaturation at 950C and finished with 10 min of final extension at 720C. Patient samples were always analysed together with the human B-cell lymphoma cell line DoHH2 (provided by Dr FE Cotter, London, UK) as positive control and the reaction mixture with no DNA as negative control. The appropriate procedures to avoid contamination were always followed.
Ten microlitres of the second-round product were loaded in 2% agarose electrophoresis gel containing ethidium bromide and visualized under ultraviolet light.

Statistical methods
Overall survival (OS) was defined as the time from the day of diagnosis until death from any cause or until the last follow-up. Time to progression (TTP) was measured as the time from the diagnosis until progression, relapse after response or death from lymphoma. Actuarial survival probabilities were calculated using the life-table method. Survival curves were estimated using the Kaplan-Meier method and differences were evaluated using the log-rank test. The STATA software package (Computing Resource Center, Santa Monica, CA, USA) was used for all statistical procedures.

RESULTS
After PCR analysis of peripheral blood samples, 35 of 59 (59%) cases of FL were bcl-2(MBR)/JH positive and 24 (41 %) were negative at diagnosis. Patient characteristics are listed in Table 1. The two groups had similar clinical, biological and therapeutic characteristics with the exception of the histological subtypes: the number of bcl-2(MBR)/JH-positive patients was significantly higher in the follicular predominantly small cleaved cell lymphoma subgroup (IWF-B).
The presence of the bcl-2/JH rearrangement in the peripheral blood at diagnosis was associated neither with a significantly worse OS (P = 0.75 1) nor with shorter TTP (P = 0.867).
Among the prognostic factors analysed, ECOG PS > 1 was significantly associated with reduced OS (P < 0.0001) and TTP (P < 0.0001); age > 60 years and liver involvement were significantly associated with worse OS (P = 0.0003 and P = 0.0352 respectively). High-risk according to the IPI was associated with worse OS and TTP (both P<0.0001).
For 17 patients who had a bcl-2/JH rearrangement detectable at diagnosis in both peripheral blood and bone marrow, peripheral blood PCR assay was performed after treatment. Fourteen patients (82%; 95% CI 56-96%) became bcl-2/JH negative; 9 of these 14 patients were further analysed during follow-up and circulating cells carrying the bcl-2/JH rearrangement reappeared in five of them after several months (median 16, range 7-28 months). Five patients remained negative after a median follow-up of 23 months (range 16-43 months). Clearance of circulating bcl-2/JH-positive cells was correlated with better OS (P = 0.04) but not with longer TTP.

DISCUSSION
Bcl-2 protein is a potent blocker of programmed cell death (Hockenberry et al, 1990).
More than 90% of FL overexpress bcl-2 protein because of the t(14;18) translocation. The clinical significance of the detection of this translocation by PCR is debatable.
Our series of 59 cases confirmed the absence of prognostic significance of bcl-2(MBR)/JH detection by PCR at diagnosis. These data are not surprising as the majority of the patients with FL have circulating t(14;18) B-cells (Gribben et al, 1991;Berinstein et al, 1993;Lambrechts et al, 1993), and even healthy people may harbour translocated clones (Liu et al, 1994;Bell et al, 1995;Ji et al, 1995;Limpens et al, 1995;Dolken et al, 1996).
Even the significance of 'molecular remission', after both conventional or myeloablative chemotherapy, is a matter of debate. Price et al (1991) and Finke et al (1993) report the presence of t(14; 18)-positive circulating cells in patients in long-term complete remission after conventional chemotherapy. Lambrechts et al (1994) show no correlation between the presence or absence of peripheral blood positive cells and clinical outcome. Gribben et al (1994) report that the reappearance of circulating positive cells is strongly associated with relapse after myeloablative therapy. Similar results are shown by Hardingham et al (1995). Recently, 50-70% of cases with peripheral blood clearance have been reported after non-myeloablative therapy, both in responders and in non-responders (Betticher et al, 1996;Cabanillas et al, 1996).
In our series, positive cases with clearance of circulating bcl-2/Jpositive cells appeared to have a better OS but not a longer TTP. However, because of the prolonged natural course of the disease, a longer follow-up is necessary before any definitive conclusion. Peripheral blood is the most easily accessible tissue for followup analysis of bcl-2/JH rearrangements. Our study suggests that the loss of t(14;18) detection by PCR in the blood bodes well for survival. It may be concluded that post-treatment examination of the peripheral blood in FL by PCR may have clinical relevance in predicting the survival pattern of patients and for monitoring response to treatment. However, the use of a standardized PCR method would be needed in the future to allow comparison of results from different institutions, as current data on the clinical value of molecular remission are still controversial.