Minimal residual disease (MRD) was evaluated in 30 patients with follicular or mantle cell non-Hodgkin's lymphoma (NHL) undergoing an intensive treatment with high-dose sequential (HDS) chemotherapy and peripheral blood progenitor cell (PBPC) autografting. To minimize the potential tumor cell contamination, PBPC harvests were scheduled at the end of HDS pretransplant phase. All patients had advanced-stage disease and most of them presented with bone marrow (BM) involvement. A tumor marker could be generated in 90% of patients using bcl-2 or Ig heavy-chain genes. MRD was analyzed on PBPC, BM harvests, and after autografting by polymerase chain reaction (PCR). All evaluable follicular and 6 of 9 mantle cell patients achieved clinical complete remission. PCR negativity of PBPC and/or BM harvests was documented in 68% of follicular and 12% of mantle cell lymphomas. Molecular remission of PBPC and/or BM harvests was achieved in 9 of 15 patients with overt marrow involvement and in all patients with only molecular marrow infiltration at onset. Molecular follow-up was conducted on 14 patients: all 7 evaluable patients who received at least one PCR-negative graft maintained the negative status at a median follow-up of 24 months and none of them relapsed so far. Thus, the results show that (1) a molecular marker to monitor MRD can be obtained in most follicular and mantle cell NHL patients, (2) the HDS regimen may provide PCR-negative PBPC and/or BM harvests even from patients with BM disease, and (3) autograft with at least one PCR-negative harvest is associated with a durable clinical and molecular remission.

HIGH-DOSE CHEMORADIOTHERAPY with autologous bone marrow transplantation (ABMT) is a potentially curative modality for patients with relapsed intermediate/high-grade non-Hodgkin's lymphoma (NHL).1-7 However, the use of ABMT in forms with less aggressive histology, such as follicular and mantle cell subtypes, has been viewed with concern.8-10 Patients with advanced stage follicular or mantle cell lymphomas are generally managed with conventional chemotherapy and/or radiotherapy. Despite initial responses achieved with this approach, disease recurrence is the rule and the outcome is ultimately fatal.10-13 A more intensive approach, using high-dose chemotherapy with ABMT, has been investigated in relapsed patients. However, ABMT toxicity has limited the use of such a procedure to restricted study groups.8,14-16 The prompt engraftment offered by mobilized peripheral blood progenitor cells (PBPC) has greatly reduced morbidity and mortality linked to autografting programs, widening their applicability.17-20 Thus, high-dose chemotherapy with PBPC autograft has recently gained growing interest also for the treatment of follicular and mantle cell lymphomas.21-23 

A major concern in autografting programs is that cell harvests contain occult tumor cells that may contribute to disease relapse. This is particularly relevant in follicular and mantle cell lymphomas in which bone marrow (BM) infiltration is a common feature.8 For a long time, it has been speculated that PBPC were less contaminated than BM cells.24 However, several groups have recently shown that PBPC may contain residual tumor cells in hematologic malignancies and some solid tumors.25-27 Hence, programs of PBPC autografting for low- and intermediate-grade NHL should include an adequate tumor reduction before harvesting, along with a close monitoring of minimal residual disease (MRD). Based on these premises, a high-dose sequential (HDS) chemotherapy program, with PBPC harvest at the end of an intensive pretransplant phase, was designed for patients with advanced-stage follicular or mantle cell lymphoma.23 A molecular monitoring of MRD was included in the program.

The strategies currently in use to detect MRD are based on polymerase chain reaction (PCR), thus providing high specificity and sensitivity. The rearrangement of the Bcl-2 oncogene is commonly used for PCR detection of residual tumor cells in NHL.28,29 This rearrangement is found in approximately 75% of follicular and 25% of diffuse lymphomas. The t(14; 18) translocation juxtaposes the Bcl-2 oncogene, normally located on chromosome 18, to the Ig heavy chain (IgH) locus on chromosome 14.30-32 However, in a sizeable fraction of lymphoma patients, the Bcl-2 translocation is not present. For such a reason, we devised an alternative strategy using IgH genes. The rearrangement of variable, diversity, and joining segments (VDJ) of IgH genes generates unique DNA sequences that are specific to each B-cell clone. These unique sequences are called complementarity-determining regions (CDR) and code for the antigen binding site.33 From the CDR sequences, clone-specific primers and probes were derived and used for the PCR detection of residual lymphoma cells.

MRD was evaluated in 30 patients with follicular or mantle cell lymphoma treated with HDS regimen. The aims of the study were (1) to devise a PCR-based strategy able to detect residual disease in most of the cases, (2) to evaluate the possibility of having PCR-negative PBPC and/or BM harvests after an intensive pretransplant treatment, (3) to compare tumor contamination of PBPC versus BM, and (4) to evaluate the clinical relevance of PCR analysis on cell harvests and in vivo after autograft.

Patient characteristics and treatment plan.Since 1990, 30 patients (3 at relapse) with follicular lymphoma, follicular lymphoma with signs of histologic transformation, or mantle cell NHL were enrolled in a pilot HDS chemotherapy program (Table 1). Diagnosis was made by morphologic and immunologic analysis according to the Working Formulation (WF ) for Clinical Usage. Diagnosis of mantle cell lymphoma was made according to criteria of Revised European-American Lymphoma classification.34 All patients had advanced-stage disease and most of them presented with tumor-related symptoms (B symptoms or compression symptoms given by local enlargement of lymph nodes). The HDS program was approved by the hospital ethical committee, and written informed consent was obtained from all patients. The program is organized as follows: 2 cycles of APO (doxorubicin at 75 mg/m2 on days +1 and +22; vincristine at 1.4 mg/m2 on days +1, +8, and +22; and prednisone at 50 mg/m2 on days +1 through day +28) and 1 or 2 courses of DHAP followed by etoposide at 2 g/m2, methotrexate at 8 g/m2 plus vincristine at 1.4 mg/m2, 3 cycles of dexamethasone at 25 mg/m2, and, finally, cyclophosphamide at 7 g/m2 (Fig 1). Granulocyte colony-stimulating factor (G-CSF; 5 μg/kg) was administered after etoposide and cyclophosphamide.23 Dexamethasone cycles before cyclophosphamide were used to allow a complete recovery of BM cellularity to obtain a better PBPC mobilization.35 After cyclophosphamide, a median of three leukophereses were performed to harvest PBPC. Five patients (N44, N45, N66, N103, and N67) had been enrolled in a previous study in which an earlier HDS regimen was used.17 Two of these latter patients (N44 and N103) had PBPC collection after the first high-dose course, whereas the remaining patients had their cells collected after the final high-dose course. PBPC estimation in leukopheresis product was performed in terms of both CD34+ cells as well as myeloid clonogenic cells (colony-forming unit–granulocyte-macrophage [CFU-GM]), as described elsewhere.35 All patients underwent a back-up BM harvest after HDS therapy. The conditioning regimen consisted of mitoxantrone at 60 mg/m2 followed by melphalan at 180 mg/m2, except for the 5 patients treated according to the original HDS protocol, who received total body irradiation (13.2 Gy) and melphalan at 120 mg/m2. Autologous cryopreserved cells were infused the day after melphalan administration. Complete remission (CR) was defined as the disappearance of all clinical evidence of active tumor. Partial remission (PR) was defined as a tumor reduction greater than 50%.

Table 1.

Patient Characteristics

Mantle Cell Lymphoma (n = 9)Follicular Lymphoma
(n = 21)
Sex (M/F) 9/0 9/12 
Median age in years (range) 46 (32-60) 48 (33-59) 
Entry at diagnosis 20 
Entry at relapse 
Stage III-IV 21 
Bulky disease 
BM disease 16 
Median LDH in U/L (range) 350 (246-772) 316 (224-965) 
Tumor-related symptoms 13 
Mantle Cell Lymphoma (n = 9)Follicular Lymphoma
(n = 21)
Sex (M/F) 9/0 9/12 
Median age in years (range) 46 (32-60) 48 (33-59) 
Entry at diagnosis 20 
Entry at relapse 
Stage III-IV 21 
Bulky disease 
BM disease 16 
Median LDH in U/L (range) 350 (246-772) 316 (224-965) 
Tumor-related symptoms 13 
Fig. 1.

Schematic representation of HDS program. APO, see the Materials and Methods; VP16, etoposide; MTX, methotrexate; CTX, cyclophosphamide; MITO, mitoxantrone.

Fig. 1.

Schematic representation of HDS program. APO, see the Materials and Methods; VP16, etoposide; MTX, methotrexate; CTX, cyclophosphamide; MITO, mitoxantrone.

Close modal

Nucleic acid extraction.BM, PB, and lymph node samples were collected during standard diagnostic procedures. Genomic DNA was purified by proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation.36 RNA was isolated using RNAzol B method (Biotecx Laboratories, Houston, TX).

PCR amplification of Bcl-2/IgH translocation.Amplification of major (MBR) and minor (mcr) breakpoints was performed using nested PCR. The oligonucleotide primers were the same originally designed by Gribben et al.28 Briefly, 1 μg of genomic DNA was amplified in 200 μmol/L dNTPs, 1× Taq buffer (50 mmol/L KCl, 10 mmol/L TRIS-HCl, pH 8, 2 mmol/L MgCl2 , 0.1% [wt/vol] gelatin) final concentration, adding 2.5 U of Taq DNA polymerase (Promega, Madison, WI) in a final volume of 50 μL. The amplification was performed as follows: 94°C for 1 minute, 55°C (MBR) or 58°C (mcr) for 1 minute, and 72°C for 1 minute for 25 cycles, followed by 7 minutes of final extension at 72°C. The reamplification for 30 cycles of a 5-μL aliquot of the first reaction was performed using internal primers. Amplified DNAs were analyzed by electrophoresis on 2% agarose gel containing ethidium bromide and were visualized by UV light. Each amplification contained a weak-positive control consisting of 10−5 dilution of the appropriate cell line. Polyclonal DNAs were used as negative controls.

PCR amplification and sequencing of IgH VDJ.The tumor VDJ was amplified starting from genomic DNA as previously described.27 Briefly, 1 μg of DNA was amplified in 200 μmol/L dNTPs, 1× Taq buffer (50 mmol/L KCl, 10 mmol/L TRIS-HCl, pH 8, 1.5 mmol/L MgCl2 , 1% [wt/vol] gelatin) final concentration, adding 2.5 U of Taq DNA polymerase (Promega), 10 pmol of VH.L or VH.D primers, and 10 pmol of JH.D primer (without restriction sites) in a final volume of 50 μL. The amplification was performed as follows: 94°C for 1 minute, 62°C for 30 seconds, and 72°C for 30 seconds for 35 cycles, followed by 7 minutes of final extension at 72°C. The prevention of PCR contamination was performed as previously described.37 Amplified DNA was directly sequenced using VH.L, VH.D, and JH.D primers, as previously described.27 When the sequence quality did not allow a complete reading of CDR regions, DNA was reamplified with primers containing EcoRI and HindIII restriction sites and cloned in Bluescript SK vector (Stratagene, San Diego, CA). Restriction enzyme analysis was performed on plasmid DNAs prepared by the alkaline lysis method, and miniprep plasmid DNAs were sequenced as previously described.38 The analysis of sequencing data was performed using the PC-GENE software (IntelliGenetics, Inc, Mountain View, CA).

Detection of residual lymphoma cells.PBPC, BM cell harvests, and BM and PB samples after autografting were evaluated for the presence of residual lymphoma cells. One microgram of DNA was amplified using the Bcl-2 or IgH assay. When IgH were used as genetic marker, 20% of the PCR product was analyzed by agarose gel electrophoresis, blotted overnight, and hybridized to CDR3 probes end-labeled with [γ-32P] ATP, as described previously.38 To avoid false-negatives, all the DNA samples failing to produce a PCR product were reamplified, and the DNA quality was tested by amplifying the sequence of p53 exon 5 or N-ras exon 2.

Oligonucleotide synthesis.Oligonucleotides were synthesized with a 391 PCR-MATE EP, DNA synthesizer (Applied Biosystems, Foster City, CA) on a 0.2 μmol scale, according to the users' manual.

Treatment feasibility and clinical outcome.PBPC collection was satisfactory in 24 patients with more than 20 × 104 CFU-GM/kg harvested with a median of 3 leukophereses (range, 1 to 4). Four patients (N15, N45, N66, and N67) had low yields (<20 × 104 CFU-GM/kg); in two more patients (N156 and N165), there was virtually no collection due to heavy marrow infiltration. Five patients were not able to undergo the final autografting phase because of (1) the occurrence of esophageal carcinoma immediately before autograft (N60), (2) transplantation refusal (N154), or (3) persistence of overt BM lymphoma cell infiltration after HDS chemotherapy (N165, N193, and N156).

The treatment was well tolerated. There was one treatment-related death from intracranial bleeding secondary to severe thrombocytopenia during the postgraft period (day +21). Of note, this was in the patient (N45) who received a reinfusion of a suboptimal dose of PBPC (<10 × 104 CFU-GM/kg) and BM cells.

The regimen proved to be effective in terms of tumor reduction, although follow-up is short. Clinical outcome before autografting was evaluated in 27 of 30 patients. In follicular lymphomas, 10 CR (55%) and 8 PR (44%; 3 PR had only a residual marrow infiltration) were obtained. In patients with mantle cell lymphoma, 4 CR (44%) and 5 PR (55%; 4 PR had disease persistence at marrow level) were achieved. Sixteen of 21 patients with follicular lymphoma have been autografted so far and are evaluable for clinical results (5 patients are waiting for the final autografting phase). All evaluable patients achieved CR. Fourteen patients (87%) are presently alive in continuous CR (CCR) at a median follow up of 19 months (range, 3 to 66 months). All patients with mantle cell lymphoma are evaluable: 6 achieved CR and 3 (33%) are presently in CCR at a median follow up of 18 months (range, 3 to 40 months); 3 patients achieved PR only. Overall, 28 patients (20 follicular and 8 mantle cell) are alive at a median follow-up of 24 months (range, 3 to 66 months).

Identification of patients with a molecular marker.A molecular marker was obtained from the diagnostic tissue in 27 of 30 patients (90%). A PCR-amplifiable Bcl-2/IgH translocation was detected in 16 of the 21 patients (76%) with follicular lymphoma: 15 involving the MBR and 1 the mcr breakpoint (Tables 2 and 3). To detect residual lymphoma cells, a nested PCR was used. In 5 patients, the Bcl-2 breakpoint amplified with nested PCR was also hybridized to a specific probe (data not shown). As previously reported, no differences in assay sensitivity were found; thus, subsequent cases were analyzed by nested PCR only.29 The assay sensitivity was tested diluting appropriate cell lines (RL and DHL-16; kindly provided from Dr J.G. Gribben, Division of Hematological Malignancies, Dana-Farber Cancer Institute, Boston, MA) with normal BM cells. Nested PCR for Bcl-2 could detect 1 lymphoma cell in 105 normal cells.

Table 2.

PCR Analysis of Residual Tumor Cells in Follicular Lymphomas

UPNHistology (W.F.)Molecular MarkerStatus/StageBM Before TreatmentBM HarvestsPBPC Harvests
HistologyPCR123
N44 Bcl-2/MBR D/IV NA 
N234 Bcl-2/mcr D/IV 
N54 Bcl-2/MBR D/IV 
N14 G (transf) Bcl-2/MBR D/IV − − NA 
N45 Bcl-2/MBR D/IV − − − NA 
N15 F (transf) Bcl-2/MBR D/IV − − − − 
N60 F (transf) Bcl-2/MBR D/IV − − − − 
N245 Bcl-2/MBR D/IV − − − − 
N281 Bcl-2/MBR D/IV − 
N41 IgH D/IV 
N101 D (transf) IgH R/III − − − − 
N404 G (transf) Bcl-2/MBR D/III − − − − − 
N347 D (transf) Bcl-2/MBR D/IV − 
N66 G (transf) NA D/III − NA NA NA NA NA 
N228 NA D/IV NA NA NA NA NA 
N357 Bcl-2/MBR D/IV − 
N103 G (transf) IgH D/III − − 
N360 Bcl-2/MBR D/IV 
N427 Bcl-2/MBR D/IV − 
N373 Bcl-2/MBR D/IV 
N379 Bcl-2/MBR D/III − − − − − 
UPNHistology (W.F.)Molecular MarkerStatus/StageBM Before TreatmentBM HarvestsPBPC Harvests
HistologyPCR123
N44 Bcl-2/MBR D/IV NA 
N234 Bcl-2/mcr D/IV 
N54 Bcl-2/MBR D/IV 
N14 G (transf) Bcl-2/MBR D/IV − − NA 
N45 Bcl-2/MBR D/IV − − − NA 
N15 F (transf) Bcl-2/MBR D/IV − − − − 
N60 F (transf) Bcl-2/MBR D/IV − − − − 
N245 Bcl-2/MBR D/IV − − − − 
N281 Bcl-2/MBR D/IV − 
N41 IgH D/IV 
N101 D (transf) IgH R/III − − − − 
N404 G (transf) Bcl-2/MBR D/III − − − − − 
N347 D (transf) Bcl-2/MBR D/IV − 
N66 G (transf) NA D/III − NA NA NA NA NA 
N228 NA D/IV NA NA NA NA NA 
N357 Bcl-2/MBR D/IV − 
N103 G (transf) IgH D/III − − 
N360 Bcl-2/MBR D/IV 
N427 Bcl-2/MBR D/IV − 
N373 Bcl-2/MBR D/IV 
N379 Bcl-2/MBR D/III − − − − − 

Abbreviations: D, diagnosis; R, relapse; transf, transformed histology; NA, not available.

Table 3.

PCR Analysis of Residual Tumor Cells in Mantle Cell Lymphomas

UPNMolecular MarkerStatus/StageBM Before TreatmentBM HarvestsPBPC Harvests
HistologyPCR123
N67 IgH D/IV 
N154 IgH D/IV 
N140 IgH D/IV 
N165 IgH R/IV NA NA 
N70 IgH D/IV − − − − NA 
N193 IgH R/IV 
N156 IgH D/IV NA NA 
N362 IgH D/IV 
N68 IgH D/IV NA NA NA NA NA 
UPNMolecular MarkerStatus/StageBM Before TreatmentBM HarvestsPBPC Harvests
HistologyPCR123
N67 IgH D/IV 
N154 IgH D/IV 
N140 IgH D/IV 
N165 IgH R/IV NA NA 
N70 IgH D/IV − − − − NA 
N193 IgH R/IV 
N156 IgH D/IV NA NA 
N362 IgH D/IV 
N68 IgH D/IV NA NA NA NA NA 

Abbreviations: D, diagnosis; R, relapse; NA, not available.

Lymphoma VDJ sequence was obtained in 11 of 14 Bcl-2–negative patients (79%). The VDJ was amplified using sense consensus primers derived from the leader (VH.L) or first framework (VH.D) region of IgH genes and an antisense primer from JH region.27 The lack of amplification in 3 patients may be explained either by the presence of somatic mutations or by the use of unknown variable regions that prevent correct primer/template annealing. Amplified VDJ regions were then sequenced. Direct sequencing analysis provided a good quality sequence in 8 cases. For the remaining patients, amplified VDJs were cloned in a plasmid vector. For each patient, 10 clones were sequenced, and, together with a few heterogeneous clones, a predominant clone was present in all cases. Concordant results were obtained by both direct sequencing of amplified DNA and after cloning in a previous study.27 For all patients, a CDR2 sense primer and a CDR3 probe were synthesized and used with a JH region primer to detect residual lymphoma cells. Several polyclonal lymph nodes were used as negative controls, and no false-positive results were detected. Our IgH PCR-based assay could detect 1 tumor cell in 105 normal BM cells. Such a sensitivity was achieved performing a serial dilution of a Burkitt cell line (BJAB) with normal cells (data not shown).

Detection of residual lymphoma cells in PBPC and BM harvests.PCR-based analysis of MRD was performed on PBPC, and BM harvests were performed in 27 patients. All patients had a PCR-positive BM before treatment, and in 22 overt lymphoma, infiltration was present at morphologic examination of trephine BM biopsy. A total of 73 PBPC and 27 BM harvests were analyzed. Twenty-three of 73 PBPC and 13 of 27 BM harvests were PCR negative (Tables 2 and 3). In 7 patients (26%), lymphoma cells were not detected in both PBPC and BM harvests. In 7 patients (26%), either PBPC or BM harvests were PCR negative. In 13 patients (48%), both PBPC and BM harvests contained residual lymphoma cells. Nine of 14 patients (64%) with PCR-negative harvests had an overt BM infiltration at diagnosis. No statistical correlation could be found between the degree of BM infiltration at diagnosis and PCR negativity of cell harvests.

The analysis of MRD showed a marked difference between follicular and mantle cell lymphoma: 13 of 19 (68%) patients with follicular lymphoma had at least one PCR-negative harvest, whereas only 1 of 8 (12%) patients with mantle cell histology had PCR-negative harvests.

Detection of residual lymphoma cells after transplantation.Seventeen of 27 patients having a molecular marker have been autografted so far. Molecular monitoring of BM samples by PCR was possible in 14 patients (Fig 2). Patient N45 died from intracranial bleeding at day +21 after autografting, patient N103 refused restaging examinations, and patient N379 has been just autografted. Median follow-up from autografting was 25.5 months (range, 3 to 61 months). It is noteworthy that the subset of 7 patients who received at least one PCR-negative harvest maintained the negative status during molecular follow-up (longer follow-up of 61 months). Patients receiving only PCR-positive harvests remained positive (longer follow-up of 52 months; Fig 2). None of the PCR-negative patients relapsed so far; 3 relapses (N67, N54, and N140) occurred in the PCR-positive group. Clearly, the follow-up is short and, at the present time, the patient groups are too small for anything other than a descriptive statistical analysis.

Fig. 2.

PCR-based monitoring of MRD after autografting. harv,harvests. (□) PCR-negativity; (▪) PCR-positivity.

Fig. 2.

PCR-based monitoring of MRD after autografting. harv,harvests. (□) PCR-negativity; (▪) PCR-positivity.

Close modal

Follicular and mantle cell lymphomas share some clinical features: (1) an indolent course, with a median survival ranging from 3 to 9 years; (2) incurability with conventional chemotherapy regimens; and (3) frequent marrow involvement at onset.8-13 Because long-term life expectancy is relatively poor, we designed an innovative HDS chemotherapy regimen for young patients with advanced-stage follicular or mantle cell NHL. High tumor reduction and prolonged disease-free survival after intensive chemoradiotherapy and autologous transplantation have been recently reported in high-risk follicular lymphomas.14-16,21 22 At present, these promising results do not allow definite conclusions on disease curability. In fact, clinical studies require a long follow-up as a consequence of the indolent course characterizing follicular lymphomas.

A major concern in autografting programs is the reinfusion of occult tumor cells contained in the graft. PCR-based techniques are essential to evaluate the contamination of residual tumor cell in harvests for autografting purposes.39 In addition, MRD analysis would provide an early end-point to assess the curative potentiality of novel intensive chemotherapy approaches in indolent lymphomas. The predictive value of PCR-based analysis emerged from a recent study in follicular lymphomas in which in vitro-purged BM cells were used for autograft. Patients autografted with PCR-negative harvests seldom relapsed, whereas the opposite occurred in positive cases.29,40 These observations prompted us to develop a strategy aimed to molecular monitoring of NHL patients undergoing PBPC autograft. A two-step experimental strategy was developed: first, patients were screened for the presence of bcl-2 gene rearrangements; second, lymphoma-specific markers were derived from IgH gene rearrangement in bcl-2–negative cases. The PCR assay using IgH genes offered a sensitivity and specificity similar to a translocation marker such as Bcl-2.27 The combined use of bcl-2 and IgH gene rearrangements was successful in providing a molecular marker for 90% of the patients.

The particular feature of our program was PBPC collection after repeated high-dose chemotherapy courses. Previous extensive administration of myelotoxic drugs could impair the subsequent progenitor mobilization.35 However, with the introduction of a chemotherapy-free interval before the final mobilizing course, adequate PBPC collections could be restored in 80% of patients. The result is of particular interest considering that most patients presented with marrow involvement, a feature often associated with poor PBPC mobilization.41 Moreover, the extensive pretransplant treatment provided PCR-negative PBPC and/or BM harvests in 14 of 27 evaluable patients. Therefore, the designed schedule allowed satisfactory PBPC collections in terms of both quantity and quality.

The collection of PCR-negative PBPC in follicular lymphomas treated with high-dose chemotherapy has been recently reported by two groups.42,43 In the study reported by Haas et al,42 the proportion of patients with PCR-negative PBPC was lower compared with our results. The intensive pretransplant phase of our program was probably responsible for the higher rate of PCR negativity. In addition, our molecular analysis was performed also on BM harvests, whereas in the report by Haas et al,42 there is no mention about the possibility of having PCR-negative BM harvests. Hardingham et al43 reported a percentage of PCR-negative PBPC harvests slightly higher than ours. However, some PCR-negative PBPC were collected from patients in PR, and none of them had long-term survival in CR. This unexpected observation could be partially related to a lower sensitivity in MRD detection. In fact, the sensitivity of Hardingham et al's IgH assay was less efficient than ours, because it was not based on the use of lymphoma-specific primers and probes.25 42 

The efficacy of our HDS program was proved by the high rate of CRs documented in all evaluable follicular and in 6 of 9 mantle cell patients. However, the achievement of molecular remission was influenced by marrow histology at diagnosis: 9 of 15 patients with overt marrow involvement had BM and/or PBPC harvests negative at PCR analysis, whereas all patients presenting with only molecular infiltration without histologic evidence of marrow involvement had PCR-negative harvests. This is one more proof that BM lymphoma cells are somehow less susceptible to cytotoxic agents than cells from other sites.44 The achievement of PCR negativity did also positively correlate to follicular histology. PCR negativity of PBPC and/or BM harvests was documented in 68% of follicular patients; on the contrary, only 1 patient with mantle cell lymphoma achieved PCR negativity. The differences between follicular and mantle cell lymphoma cannot be merely explained by a different degree of marrow involvement. They probably reflect an intrinsic low sensitivity of mantle cell subtype to chemotherapy, even when delivered at high doses.45 

PCR negativity was more frequent in BM than in PBPC harvests. In particular, 5 patients had PCR-negative BM harvests despite positive PBPC. This observation seems to contradict the never-proved hypothesis that PBPC are less contaminated by lymphoma cells than BM.24 46 A possible explanation might be that, during growth factor mobilization, all the cells present in the marrow, including lymphoma cells, are driven into circulation; by contrast, during BM harvest, only a restricted area of the bone is tested and the amount of lymphoma cells collected might be below the threshold of PCR detection.

Clearly, the follow-up is short, and a longer period of observation is required to conclusively evaluate the clinical relevance of molecular analyses. However, preliminary data from the molecular follow-up on 14 patients suggest that the reinfusion of some harvests devoid of PCR-detectable lymphoma cells might be critical to mantain a PCR-negative status after transplantation. The collection of PCR-positive and -negative harvests in the same patient is a sign of pronounced tumor reduction, and it may be speculated that the molecular status is close to the threshold of detection of MRD assay. All 7 evaluable patients who received at least one PCR-negative graft maintained the negative status with a follow-up of 24 months. All PCR-negative patients are at present in CCR. The durable PCR-negativity achieved by patients who received also some positive grafts may have different explanations: reinfused cells could have been irreversibly damaged by chemotherapy, losing their clonogenic potentiality; alternatively, the relatively small number of reinfused lymphoma cells could have been controlled in vivo by the immune system. Whatever the explanation, our results are in line with those from Gribben et al29,40 showing that reinfusion of a PCR-negative BM graft, after in vitro purging, correlates with a durable PCR-negativity. Thus, HDS chemotherapy can provide an in vivo purging similar to the one obtained using in vitro immunologic purging. These data suggest that the achievement of PCR-negativity might predict a prolonged disease-free survival, if not even cure, and support the use of in vitro purging when both BM and PBPC are positive for PCR-detectable lymphoma cells.47 48 

In conclusion, our results show that (1) it is possible to obtain a molecular marker to monitor residual disease by PCR in most of follicular and mantle cell NHL patients, (2) the HDS regimen may provide PCR-negative PBPC and/or BM harvests even from patients with BM disease, and (3) autograft with at least one PCR-negative harvest is associated with a durable clinical and molecular remission. Based on these findings, we started a pilot study using the HDS regimen and in vitro immunologic purging for follicular and mantle cell patients having only PCR-positive harvests.

We thank Dr A.M. Gianni for critical reading of the manuscript, Dr P. Omedè and Dr F. Giaretta for CD34 cell analysis, and P. Bondesan for cryopreservation.

Supported by Associazione Italiana Ricerca sul Cancro (AIRC; Milano Italy) and Consiglio Nazionale delle Ricerche (Progetto Finalizzato ACRO No. 95.00544.PF39 and 9500416.PF39). M.A. is a recipient of a fellowship from AIRC.

Address reprint requests to Paolo Corradini, MD, Cattedra di Ematologia, Via Genova 3, 10126 Torino, Italy.

1
Philip
T
Armitage
JO
Spitzer
G
Chauvin
F
Jagannath
S
Cahn
JY
Colombat
P
Goldstone
AH
Gorin
NC
Flesh
M
Laporte
JP
Maraninchi
D
Pico
J
Bosly
A
Anderson
C
Schots
R
Biron
P
Cabanillas
F
Dicke
K
High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade non-Hodgkin's lymphoma.
N Engl J Med
316
1987
1493
2
Takvorian
T
Canellos
G
Ritz
J
Freedman
AS
Anderson
KC
Mauch
P
Tarbell
N
Coral
F
Daley
H
Yeap
B
Schlossman
SF
Nadler
LM
Prolonged disease-free survival after autologous bone marrow transplantation in patients with non-Hodgkin's lymphoma with poor prognosis.
N Engl J Med
316
1987
1499
3
Freedman
AS
Takvorkian
T
Anderson
KC
Mauch
P
Rabinowe
SN
Blake
K
Yeap
B
Soiffer
R
Coral
F
Heflin
L
Rits
J
Nadler
LM
Autologous bone marrow transplantation in B-cell non-Hodgkin's lymphoma: Very low treatment-related mortality in 100 patients in sensitive relapse.
J Clin Oncol
8
1990
784
4
Peterson
FB
Appelbaum
FR
Hill
R
Fisher
LD
Bigelow
CL
Sanders
JE
Sullivan
KM
Bensinger
WI
Witherspoon
RP
Storb
R
Clift
RA
Fefer
A
Press
OW
Weiden
PL
Siger
J
Thomas
ED
Buckner
CD
Autologous marrow transplantation for malignant lymphoma: A report of 101 cases from Seattle.
J Clin Oncol
8
1990
638
5
Caracciolo
D
Gavarotti
P
Aglietta
M
Bondesan
P
Falda
M
Gallo
E
Locatelli
F
Novarino
A
Paolino
F
Sanavio
F
Urgesi
A
Vitolo
U
Tarella
C
High-dose sequential (HDS) chemotherapy with blood and marrow cell autograft as salvage treatment in very poor prognosis, relapsed non-Hodgkin's lymphoma.
Bone Marrow Transplant
12
1993
621
6
Vose
JM
Anderson
JR
Kessinger
A
Bierman
PJ
Coccia
P
Reed
EC
Gordon
B
Armitage
JO
High-dose chemotherapy an autologous hematopoietic stem-cell transplantation for aggressive non-Hodgkin's lymphoma.
J Clin Oncol
11
1993
1846
7
Philip
T
Guglielmi
C
Hagenbeek
A
Somers
R
Van Der Lelie
H
Bron
D
Sonneveld
P
Gisselbrecht
C
Cahn
JY
Harousseau
JL
Coiffier
B
Biron
P
Mandelli
F
Chauvin
F
Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin's lymphoma.
N Engl J Med
333
1995
1540
8
Armitage
JO
Treatment of non-Hodgkin's lymphoma.
N Engl J Med
328
1993
1023
9
Longo
D
What's the deal with follicular lymphomas?
J Clin Oncol
11
1993
202
10
Horning
SJ
Treatment approaches to the low grade lymphomas.
Blood
83
1994
881
11
Shivdasani
RA
Hess
JL
Skarin
AT
Pinkus
GS
Intermediate lymphocytic lymphoma: Clinical and patological features of a recntly characterized subtype of non-Hodgkin's lymphoma.
J Clin Oncol
11
1993
802
12
Zucca
E
Roggero
E
Pinotti
G
Pedrinis
E
Cappella
C
Venco
A
Cavalli
F
Patterns of survival in mantle cell lymphoma.
Ann Oncol
6
1993
257
13
Teodorovic
IT
Pittaluga
S
Kluin-Nelemans
JC
Meerwaldt
JH
Hagenbeek
A
van Glabbek
M
Somers
R
Bijnens
L
Noordijk
EM
De Wolf
Peeters C
Efficacy of four different regimens in 64 mantle-cell lymphoma cases: Clinicopathologic comparison with 498 other non-Hodgkin lymphoma subtypes.
J Clin Oncol
13
1995
2819
14
Freedman
AS
Ritz
J
Neuberg
D
Anderson
KC
Rabinowe
SN
Mauch
P
Takvorian
T
Soiffer
R
Blake
K
Yeap
B
Coral
F
Nadler
LM
Autologous bone marrow transplantation in 69 patients with a history of low-grade B-cell non-Hodgkin's lymphoma.
Blood
77
1991
2524
15
Rohatiner AZS, Johnson PWM, Price CGA, Arnott SJ, Amess JAL, Norton AJ, Dorey E, Adams K, Whelan JS, Matthews J, MacCallum PK, Oza AM, Lister TA: Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. Ann Oncol 5:143, 1994 (suppl 2)
16
Armitage
JO
Bone marrow transplantation for indolent lymphomas.
Semin Oncol
20
1993
136
17
Gianni
AM
Siena
S
Bregni
M
Tarella
C
Stern
AC
Pileri
A
Bonadonna
G
Granulocyte-macrophage colony-stimulating factor to harvest circulating haematopoietic stem cells for autotransplantation.
Lancet
334
1989
580
18
Kessinger
A
Armitage
JO
The evolving role of autologous peripheral stem cell transplantation following high-dose therapy for malignancies.
Blood
77
1991
211
19
Sheridan
WP
Begley
CG
Juttner
C
Szer
J
To
LB
Maher
D
McGrath
KM
Morstyn
G
Fox
RM
Effect of peripheral blood progenitor cells mobilized by filgrastim (G-CSF ) on platelet recovery after high-dose chemotehrapy.
Lancet
1
1992
640
20
Pettengell
R
Nydia
G
Swindell
R
Crowther
D
Dexter
TM
Transplantation potential of hematopoietic cells released into the circulation during routine chemotherapy for non-Hodgkin's lymphoma.
Blood
82
1993
2239
21
Haas
R
Moos
M
Mohle
R
Dohner
H
Witt
B
Goldschmidt
H
Murea
S
Flentje
M
Wannenmacher
M
Hunstein
W
High-dose therapy with peripheral blood progenitor cell transplantation in low-grade non-Hodgkin's lymphoma.
Bone Marrow Transplant
17
1996
149
22
Bastion
Y
Brice
P
Haioun
C
Sonet
A
Salles
G
Marolleau
JP
Espinouse
D
Reyes
F
Gisselbrecht
C
Coiffier
B
Intensive therapy with peripheral blood progenitor cell transplantation in 60 patients with poor-prognosis follicular lymphoma.
Blood
86
1995
3257
23
Tarella
C
Gavarotti
P
Caracciolo
D
Corradini
P
Cherasco
C
Castellino
C
Pileri
A
Haematological support of high-dose sequential chemotherapy: Clinical evidence for reduction of toxicity and high response rates in poor risk lymphomas.
Ann Oncol
6
1995
3
24
Kessinger
A
Vose
JM
Bierman
PJ
Armitage
JO
High-dose therapy and autologous peripheral blood stem cell transplantation for patients with bone marrow metastases and relapsed lymphoma: An alternative to bone marrow purging.
Exp Hematol
19
1991
1013
25
Hardingham
JE
Kotasek
D
Sage
RE
Dobrovic
A
Gooley
T
Dale
BM
Molecular detection of residual lymphoma cells in peripheral blood stem cell harvests and following autologous transplantation.
Bone Marrow Transplant
11
1993
15
26
Rill
DR
Santana
VM
Roberts
WM
Nilson
T
Bowman
LC
Krance
RA
Heslop
HE
Moen
RC
Ihle
JN
Brenner
MK
Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells.
Blood
84
1994
380
27
Corradini
P
Voena
C
Astolfi
M
Ladetto
M
Tarella
C
Boccadoro
M
Pileri
A
High-dose sequential chemoradiotherapy in multiple myeloma: Residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting.
Blood
85
1995
1596
28
Gribben
JG
Freedman
AS
Neuberg
D
Roy
DC
Blake
KW
Woo
SD
Grossbard
ML
Rabinowe
SN
Coral
F
Freeman
GJ
Ritz
J
Nadler
LM
Immunologic purging of marrow assessed by PCR before autologous bone marrow tranplantation for B-cell lymphoma.
N Engl J Med
325
1991
1525
29
Gribben
JG
Freedman
AS
Woo
SD
Blake
K
Shu
RS
Freeman
G
Longtine
JA
Pinkus
GS
Nadler
LM
All advanced stage non-Hodgkin's lymphomas with polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.
Blood
78
1991
3275
30
Tsujimoto
Y
Finger
LR
Yunis
J
Norwell
PC
Croce
CM
Cloning of the chromosome breakpoint of neoplastic B cells with the t(14; 18) chromosome translocation.
Science
226
1984
1097
31
Cleary
ML
Sklar
J
Nucleotide sequence of a t(14; 18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18.
Proc Natl Acad Sci USA
81
1985
593
32
Lee
MS
Chang
KS
Cabanillas
F
Freireich
EJ
Trujillo
JM
Stass
SA
Detection of minimal residual disease carrying the t(14; 18) by DNA sequence amplification.
Science
237
1987
175
33
Alt
FW
Blackwell
TK
Yancopoulos
GD
Development of the primary antibody repertoire.
Science
238
1987
1079
34
Harris
NL
Jaffe
ES
Stein
H
Banks
PM
Chan
JKC
Cleary
ML
Delsol
G
De Wolf-Peeters
C
Falini
B
Gatter
KC
Grogan
TM
Isaacson
PG
Knowles
DM
Mason
DY
Muller-Hermelink
HK
Pileri
SA
Piris
MA
Ralfkiaer
E
Warnke
RA
A revised European-American classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group.
Blood
84
1994
1361
35
Tarella
C
Caracciolo
D
Gavarotti
P
Bondesan
P
Cherasco
C
Omedè
P
Bregni
M
Siena
S
Gianni
AM
Pileri
A
Circulating progenitors following high-dose sequential (HDS) chemotherapy with G-CSF: Short intervals between drug courses severely impair progenitor mobilization.
Bone Marrow Transplant
16
1995
223
36
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989
37
Kwok
S
Higuchi
R
Avoiding false positives with PCR.
Nature
339
1989
237
38
Corradini
P
Boccadoro
M
Voena
C
Pileri
A
Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to Cμ sequence in immunoglobulin (IgG)- and (IgA)-secreting multiple myelomas.
J Exp Med
178
1993
1091
39
Lin
F
Cross
NCP
Molecular methods for monitoring malignant disease after bone marrow transplantation.
Curr Opin Hematol
2
1995
460
40
Gribben
JG
Neuberg
D
Barber
M
Moore
J
Pesek
KW
Freedman
AS
Nadler
LM
Detection of residual lymphoma cells by polymerase chain reaction in peripheral blood is significantly less predictive for relapse than detection in bone marrow.
Blood
83
1994
3800
41
Tarella
C
Boccadoro
M
Omedè
P
Bondesan
P
Caracciolo
D
Frieri
R
Bregni
M
Siena
S
Gianni
AM
Pileri
A
Role of chemotherapy and GM-CSF on hemopoietic progenitor cell mobilization in multiple myeloma.
Bone Marrow Transplant
11
1993
271
42
Haas
R
Moos
M
Karcher
A
Mohle
R
Witt
B
Goldschmidt
H
Fruhauf
S
Flentje
M
Wannenmacher
M
Hunstein
W
Sequential high-dose therapy with peripheral-blood progenitor-cell support in low grade non-Hodgkin's lymphoma.
J Clin Oncol
12
1994
1685
43
Hardingham
JE
Kotasek
D
Sage
RE
Gooley
LT
Mi
JX
Dobrovic
A
Norman
JE
Bolton
AE
Dale
BM
Significance of molecular marker-positive cells after autologous peripheral-blood stem-cell transplantation for non-Hodgkin's lymphoma.
J Clin Oncol
13
1995
1073
44
Yan
Y
Chan
WC
Weisenburger
DD
Anderson
JR
Bast
MA
Vose
JM
Bierman
PJ
Armitage
JO
Clinical and prognostic significance of bone marrow involvement in patients with diffuse aggressive B-cell lymphoma.
J Clin Oncol
13
1995
1336
45
Velders
GA
Kluin-Nelemans
JC
De Boer
CJ
Hermans
J
Noordijk
EM
Schuuring
E
Kramer
MHH
van Deijk
WA
Rahder
JB
Kluin
PM
Van Krieken
JHJM
Mantle-cell lymphoma: A population-based clinical study.
J Clin Oncol
14
1996
1269
46
Janssen
WE
Peripheral blood and bone marrow hematopoietic stem cells: Are they the same?
Semin Oncol
20
1993
19
47
Gorin
NC
Lopez
M
Laporte
JP
Quittet
P
Lesage
S
Lemoine
F
Berenson
RJ
Isnard
F
Grande
M
Stachowiak
J
Labopin
M
Fouillard
L
Morel
P
Jouet
JP
Noel-Walter
MP
Detourmignies
L
Aoudjhane
M
Bauters
F
Najman
A
Douay
L
Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma.
Blood
85
1995
1647
48
Dreger
P
von Neuhoff
N
Suttorp
M
Loffler
H
Schmitz
N
Rapid engraftment of peripheral blood progenitor cell grafts purged with B cell-specific monoclonal antibodies and immunomagnetic beads.
Bone Marrow Transplant
16
1995
627
Sign in via your Institution