Minimal residual disease in non-Hodgkin's lymphoma

Biomed

1996:50:45

& Pharmarother

0 Elsevier,

l-458

Paris

Dossier

Minimal

residual

“Non-Hodgkin’s

disease in non-Hodgkin’s

lymphomas

part II”

lymphoma

JL Schultze, JG Gribben* Department

of Medicine,

Harvard

Medical

School,

44 Binney

Division of Hematologic St, Boston, MA 02115,

Malignancies, USA

Dana-Farber

Cancer

Institute,

Summary - Recent advances in the sensitivity of detection methods have clearly illustrated that patients in complete clinical remission often harbor residual lymphoma cells that are below the limit of detection using standard staging techniques. However, the clinical significance of this detection is by no means clear. Patients have been identified who remain in very long-term clinical remission despite detection of residual lymphoma cells. In contrast, the eradication of lymphoma is associated with improved outcome in patients undergoing autologous bone marrow transplantation. We review the methodologies for the detection of minimal residual lymphoma and discusses the clinical significance of this detection. minimal

residual

disease

/ non-Hodgkin’s

lpmphoma

/ PCR

INTRODUCTION Patients with advanced stage non-Hodgkin’s lymphoma (NHL) often achieve clinical complete remission (CR), but the majority ultimately relapse. Over the past decade new techniques have greatly increased the sensitivity of detection of lymphoma. With the development of these more sensitive techniques, especially by the application of polymerase chain reaction (PCR) technology, the presence of residual neoplastic cells in patients in complete clinical remission, commonly called ‘minimal residual disease’ (MRD), has been demonstrated clearly and these cells are thought to be the source of relapse. It might seem obvious that if residual lymphoma cells can be detected in a patient then additional therapy would be necessary for cure. However, this has never been conclusively established for the minimal residual numbers of neoplastic cells that can now be detected in patients following achievement of a clinical CR. The critical issue now is to determine whether such sensitive detection of residual detectable lymphoma cells by PCR will identify which patients will relapse. If this proves to be the case, then molecular biologic techniques will become a routine part of staging and follow up

* Correspondence

and

reprints

/ clinical

assessment

of patients and redefine our concept of CR, such that our goal should be to aim for a ‘molecular CR’.

TISSUE

SOURCES TO DETECT RESIDUAL DISEASE

MINIMAL

NHL is primarily a disease of the lymph node, but lymph node biopsies are performed at the time of initial diagnosis or at overt relapse and rarely when a patient is in clinical CR. Peripheral blood (PB) and bone marrow (BM) samples provide a readily available tissue source to detect MRD. BM involvement is common in non-Hodgkin’s lymphomas and bilateral BM biopsies are a routine part of initial staging of the disease. The likelihood that BM is infiltrated is determined by a number of clinical variables such as tumor type and stage of disease. In general, the higher the stage of the tumor, the more likely the BM is to be involved. In some subtypes, such as the follicular small and mixed cell lymphomas, BM involvement is almost invariable and may even be the site of the clonogenic lymphoma cell. It is possible therefore that in some subtypes of NHL, relapse will occur at nodal sites with little evi-

452

JL Schultze,

dence of MRD in other tissues such as BM and PB, but in others BM and PB will be as useful as lymph node samples to detect residual disease. METHODS

TO DETECT LYMPHOMA

growth

of follicular

lymphoma

cells

lead to major advances in the use of these assays to detect MRD in lymphoma. MOLECUL.AR BIOLOGIC TECHNIQUES FOR THE DETECTION OF LYMPHOMA

RESIDUAL

The standard technique is visualization and identification of subtype by histological examination. In terms of detecting MRD, this technique has the limitation of detecting lymphoma cells comprising approximately 5% of the population. Application of flow cytometric analysis for the detection of NHL has been hampered by the lack of lymphoma specific monoclonal antibodies (mAbs) since all the cell surface antigens identified to date on the surface of lymphoma cells are also present on normal B cell or B-cell precursor cells. A sensitive method of detecting residual lymphoma cells in normal tissue is the use of clonogenic assays. Clonogenic culture assays detect not only whether a malignant cell is present, but also if the cell has the clonogenic capacity to induce relapse in the patient. A number of studies have demonstrated that clonogenic malignant cells could be grown from morphologically normal BM [6, 12, 281. Not all lymphoma cells can be grown under these culture conditions. Once more is known about the biology of the stroma1 cell/tumor cell interactions, clonogenic culture systems should become more widely applicable. Clonogenic growth of lymphoma cells on a stromal layer is shown in figure 1. The identification of the role of the CD40 ligand in supporting B-cell lymphoma cell growth [5] may

Fig 1. Clonogenic stromal layer.

JG Gribben

on a

The underlying principle for molecular biologic techniques is detection of clonal proliferation of malignant cells. Tumor-specific DNA sequences occur at the sites of non-random chromosomal translocations and are candidates for detection by PCR amplification if the sequences at the sites of the chromosomal breakpoints are known. Because of the specific nature of gene rearrangements occurring at the antigen receptors, the lymphoid malignancies have been studied most extensively. DNA restriction fragment analysis with Southern blot hybridization for immunoglobulin (Ig) and T cell receptor (TCR) probes has demonstrated the presence of the clonal lymphoid populations in the majority of lymphoid neoplasms. This increased the level of detection of MRD to 1% [9] sufficient to demonstrate that residual lymphoma cells could indeed be detected in the PB of patients who were judged to be in complete clinical remission [ 191.

POLYMERASE

CHAIN

REACTION

(PCR)

A number of chromosomal translocations and gene rearrangements associated with NHL are relatively common in specific subtypes. In the lymphoid malignancies these translocations commonly involve the sites of the antigen receptor genes with variety of proto-oncogenes are involved as the reciprocal partner for these translocations as shown in figure 2. These translocations are suitable for PCR amplification if the breakpoints cluster around a region small enough to enable to be amplified as is the case in the t(ll; 14) and t( 14;18), as shown in figure 3. The sensitivity of detection of MRD has been greatly increased with the development of PCR. The principle of PCR amplification of a chromosomal translocation is shown in figure 4. The specificity of the first PCR product must then be confirmed either by performing a secondary or ‘nested PCR’ using amplification primers internal to the original primers or by Southern blotting of the PCR product and hybridization with a labeled oligonucleotide probe.

MRD

in non-Hodgkin’s

I Oncogene

r-l

453

lymphoma

Protein

T,ranslocation

Disease

t(11;14)

Mantle cell NHL

bcl-1

bcl-2 bcl-6

I

Anti-apoptosis

t(14;18)

myc

Zinc finger transcription factor Transcription factor

bcl-3

NF-Kb inhibitor

t(3; 14) t(8;14)

Follicular

NHL

Diffuse large cell NHL Burkitt’s

NHL

t(11;19)

/+14q32 (IgH) Fig 2. Common chromosomal translocations in NHL. A number of these translocations involve the immunoglobulin heavy chain (IgH) locus at 14q32. The reciprocal oncogenes and the association with histologic subtypes of lymphoma are also shown.

bcl-1

+$$= MTC

lg Heavy mtc2

Chain

mtc2

18q21 J’exon

bcl-2

untranslated

lntron

m!m MBR

I

lg Recombinase

mcr

Fig 3. Schematic representation of chromosomal translocations in NHL. In both the t(ll; 14) and t(l4;lS) there is a reciprocal translocation between an oncogene and the immunoglobulin heavy chain locus at 14q32 producing a hybrid chromosome that is a suitable target for PCR amplification. MTC: major translocation cluster: mtc: minor translocation cluster; MBR: major breakpoint region; mcr: minor cluster region.

454 Primer

JL Schultze,

JG Gribben

1

FxEIIIrIl Primer2 \

Primer

PCR 1” L

/

1

-

Primer -

3 -

Probe

PCR 2” ,

Primer4

s!mzl Final

PCR

Southern Blotting + Hybridization -

product

Fig 4. Strategy for PCR amplification of hybrid gene product of chromosomal translocation. PCR amplification using a primer 5’ of the breakpoint region on one chromosome and 3’ on the other chromosome. The specificity product is confirmed using either ‘nested PCR’ using two primers internal to the original primers or by Southern hybridization using a labeled internal oligonucleotide probe.

In the t( 14;18) the hcl-2 proto-oncogene on chromosome 18 is juxtaposed with the Ig heavy chain (IgH) locus on chromosome 14. The breakpoints have been cloned and sequenced 14, 11, 311 and cluster at two main regions 3’ to the bcl-2 coding region. As shown in figure 3, the major breakpoint region (MBR) occurs within the 3’ untranslated region of the bcl-2 gene [ll] and the minor cluster region (mcr) is located some 20 kilobases (kb) downstream [lo]. The clustering of the breakpoints at these two main regions at the bcl-2 gene and the availability of consensus regions of the IgH joining (J) regions make this an ideal candidate for PCR amplification to detect lymphoma cells containing this translocation. A major advantage is that DNA can be used to detect this translocation. The PCR products from different patients are of different sizes and have unique sequences. The size of the PCR product can be assessed by gel electrophoresis and used as confirmation that the expected size fragment is amplified from each patient. This translocation occurs in up to 50% of all patients with NHL, occurring in 85%

is performed of the PCR blotting and

of patients with follicular lymphoma and 30% of patients with diffuse lymphoma [ 1, 361. The t(11;14) is associated with a number of B cell malignancies including B cell chronic lymphocytic leukemia (B-CLL). myeloma, and, in particular, in more than 50% of mantle cell lymphoma (MCL). In this translocation the protooncogene bcl-IIPRAD-1 on chromosome 11 is juxtaposed to the IgH chain locus on chromosome 14 [32]. Although the breakpoints on chromosome 11 have been shown to be widely scattered, the majority are clustered within a restricted fragment known as the major translocation cluster (MTC) (fig 3) [27, 321. This translocation is therefore suitable for amplification by PCR. To date no studies have been published to determine the clinical utility of MRD detection in lymphomas with this translocation, although these studies are underway in a number of institutions. The results of these studies are awaited with interest to determine whether the eradication of PCR detectable lymphoma cells will be necessary for cure in this disease.

MRD

in non-Hodgkin’s

The reciprocal chromosomal translocation t(8;14) is characteristic of the high grade NHL, Burkitt’s lymphoma and a subset of cases of acute lymphocytic leukemia (ALL). This rearrangement juxtaposes coding exons 2 and 3 of the oncogene c-myc on chromosome 8 to the joining regions of the IgH locus on chromosome 14. PCR has been used to detect the site of the translocation and was capable of detecting MRD [29]. However, the breakpoints are highly variable on chromosome 11 and may occur upstream, downstream or at the site of c-myc, which limits the applicability of this technique in MRD detection. Anaplastic large cell NHL is characterized by the expression of CD30 (Ki-1). Around 33% of anaplastic lymphomas express the chromosomal translocation t(2;5) [21, 331. This translocation appears to involve a novel protein tyrosine kinase and nucleophosmin, resulting in a p80 fusion protein. This translocation can be detected by reverse transcriptase (RT) PCR in a subset of patients. It remains to be determined whether this technique will have clinical utility. The hcl-6 oncogene is involved in many cases of diffuse large cell lymphoma [34]. Although the bcl-6 gene has been cloned and sequenced [35], the sites of translocation appear to be widely dispersed making this a difficult translocation to detect by PCR amplification. PCR DETECTION OF ANTIGEN RECEPTOR GENE REARRANGEMENTS Although most lymphomas exhibit specific chromosomal translocations, a significant number of patients with lymphoid malignancies do not demonstrate non-random chromosomal translocations. In these cases, an alternative strategy must be developed to detect MRD. Lymphoid neoplasms usually rearrange either Ig or TCR or both, and their clonal progeny have this identical antigen receptor rearrangement [2, 91. The third complementarity-determining region (CDR III) of the IgH gene is the result of rearrangement of germline variable (V), diversity (D) and joining (J) region elements. In a similar mechanism in both Ig and TCR genes the enzyme terminal deoxynucleotidyl transferase (TdT) inserts random nucleotides (N regions) at two sites: the V-D and D-J junctions, and at the same time random deoxynucleotides are removed by exonucleases [30]. The final V-N-D-N-J sequence is

455

lymphoma

unique to that cell and if the cell expands to form a clone, then this region may act as a unique marker for that leukemic clone. PCR amplification of the CDR III sequence is possible due to the presence of conserved sequences within the V and J regions that are specific to the rearranged. Two strategies have been broadly applied to amplify the CDR III region, as shown in figure 5. Consensus primers to the framework region (FR) 3 are capable of amplifying clonal products in 50% of cases and will result in PCR amplification of a product some loo-120 base pairs (bp) in length. In cases that fail to amplify using consensus primers, V family specific consensusprimers to the FRI region amplify a PCR product of 280-300 bp in an additional 35% of patients. Although these techniques have the advantage of being applicable to a greater number of patients, they are much less sensitive than the detection of chromosomal translocations. These sequences can be cloned and sequenced from diagnostic tissue. Clone specific oligonucleotides can then be constructed against the unique junctional region sequences and used as primers for PCR amplification [8] or as probes for hybridization in that patient. QUANTITATION

USING PCR

Although a number of quantitative methods have been developed, a major drawback of PCR is that it has been extremely difficult to quantitate the tumor cells in the original sample and PCR is usu-

300-350

bp

Fig 5. Strategy to amplify CDR III region of the immunoglobulin heavy chain locus. The CDR III region can be PCR amplified using primers consensus to the framework region (FR) 3 and JH regions producing a product of 100-120 bp. An alternative strategy is to use a series of VH family specific primers to the FRI region and the consensus JH primers yielding a product of 300-350 bp.

456

JL Schultze,

ally read as either positive or negative. Minor differences in efficiency of amplification from tube to tube are accentuated during the logarithmic amplification of DNA samples. However, these variables can be precisely controlled by using an internal standard which controls for amplification efficiency. Since variation in amplification efficiency can also be attributed to primer annealing efficiency, rate of template denaturation, length of template among other variables, the best internal standard is one that can be primed by the same primers as the target DNA, but distinguished either by minor size differences, or by adding or ablating a restriction endonuclease site. These quantitation strategies have been termed ‘competitive’ or ‘quantitative’ PCR [ 131. CLINICAL UTILITY DISEASE DETECTION

OF MINIMAL IN LYMPHOMA

Now that technologies have been developed for the detection of MRD. the important issue is to determine the clinical significance of detection of MRD. The prognostic significance of the achievement of a ‘molecular CR’ remains elusive, and few studies to date have demonstrated the importance of eradicating MRD in the patient to achieve cure. PCR detection procedure

of BM infiltration

as a staging

A number of studies have demonstrated that PCR can detect cells bearing the t( 14; 18) in the BM and peripheral blood at the time of initial presentation patients who had no evidence of marrow infiltration by morphology [ 14, 181. Of great interest are those studies that have evaluated the clinical utility of MRD detection in patients presenting with localized disease [7]. 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 alterations in therapy in those patients currently treated with localized radiotherapy remains to be determined. However, PCR analysis cannot replace morphologic assessment of BM since not all patients have translocation detectable by PCR and these techniques are therefore be viewed as complementary.

JG Gribben

PCR detection

of MRD

after chemotherapy

After combination chemotherapy, PCR detectable lymphoma cells bearing the t( 14; 18) remain detectable in the BM [14]. Long-term analysis after completion of conventional dose chemotherapy has also suggested that conventional dose chemotherapy does not eradicate PCR detectable disease, but that this might not be associated with poor outcome [22, 261. In these studies there was no association between the presence or absence of PCR detectable lymphoma cells and clinical outcome. Since PCR analysis detects residual dose lymphoma cells after conventional chemotherapy in the majority of patients studied, it is hardly surprising that it has not been possible to determine any prognostic significance for the persistence of PCR detectable lymphoma cells. It might be concluded 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 tempo of their disease.

Assessment

of MRD

at and after ABMT

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 myeloablation after high dose therapy can be rescued by infusion of allogeneic or autologous BM. The major obstacle to the use of autologous bone marrow transplantation (ABMT) is that the re-infusion of occult tumor cells harbored within the marrow may result in more rapid relapse of disease. A variety of methods have therefore been developed to ‘purge’ malignant cells from the marrow. PCR has been used to assess the efficacy of immunologic purging in models using lymphoma cell lines [25] and in clinical samples [ 151, demonstrating that PCR is a highly sensitive and efficient method to determine the efficacy of purging residual lymphoma cells. Moreover, the results of these clinical studies suggested that infusion of residual Iymphoma cells was associated with increased likelihood of relapse after ABMT. PCR analysis of serial BM samples obtained after ABMT demonstrated that a subset of patients had eradication of PCR detectable MRD

MRD

III non-Hodgkin’s

after ABMT. Moreover, this was associated with prolonged disease-free survival [ 171. The results of this study suggest that detection of MRD by PCR following ABMT 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. However, these results were not confirmed in a similar study performed in a similar group of patients treated using a similar approach [20]. Whether these differences are due to differences in the patient population being treated or in the level of sensitivity of detection by the different methodologies used in the two studies remains unclear. Of note, although detection of PCR detectable lymphoma cells in the PB was associated with an increased likelihood of subsequent relapse, the BM appears to be a much more sensitive tissue source to detect MRD since 16 patients relapsed with no detectable lymphoma cells in their PB [16]. PROBLEMS

WITH DETECTION

PCR ANALYSIS OF MRD

FOR

A major concern with PCR will always be the fear of false positive results because of the ability of the technique to amplify even minute amounts of contaminating DNA. Unlike cell culture assays, it is not possible to determine whether cells detected by PCR are clonogenic. Cells bearing this translocation might be committed progenitors incapable of further proliferation or might have been sufficiently damaged by previous exposure to chemotherapy or radiotherapy to be already dead or in the process of dying but will still be detectable by PCR analysis. A potential problem with the use of PCR of the hcl-2/IgH translocation is that this translocation may not be specific for lymphoma cells. Cells bearing the translocation have been detected in hyperplastic lymphoid tissue, [3, 231 and rarely in normal B cells [24]. However, t( 14; 18) positive cells within hyperplastic lymph nodes appear to be sufficiently rare that their detection does not interfere with the use of PCR in the clinical management of patients who are being monitored for residual disease. CONCLUSIONS

Methodologies have been developed for the sensitive detection of MRD in lymphoma and are ap-

457

lymphoma

plicable to the majority of patients. The question that now remains to be answered is whether these techniques will have any clinical utility and will predict which patients will relapse. In NHL these studies are most advanced in patients with t( 14;18). In these patients conventional dose chemotherapy does not appear to be capable of depleting PCR-detectable lymphoma cells, although lymphoma cells were detectable in PB in only half of the patients studied. Following ABMT. the persistence or re-appearance of PCR detectable lymphoma cells in the BM was associated with an increased likelihood of relapse. Although detection of lymphoma cells by PCR in peripheral blood sampleswas also associated with an increased risk of relapse, the BM appears to be a more sensitive tissue source to detect minimal residual disease since a significant number of patients relapse who had no evidence of circulating lymphoma cells in their peripheral blood. The clinical significance of detection of lymphoma cells in the PB may well have different clinical implications than the detection of BM infiltration. In lymphomas that do not express the t( 14;18), it is not yet clear whether failure to detect MRD in PB and BM will predict which patients will relapse since other subtypes of lymphoma may relapse in nodal sites without detectable lymphoma cells in the circulation. It is therefore by no means clear whether out goal should really be to attempt to achieve a ‘molecular CR’ in our patients to cure their underlying disease. Further studies in larger patient numbers are underway and shall determine whether PCR detection of lymphoma cells has clinical significance. REFERENCES Aisenberg AC, Wilkes BM, Jacobson JO. The hc/-2 gene is rearranged in many diffuse B-cell lymphomas. Blood 1988;71:969-72 Arnold A, Cossman J, Bakhshi A, Jaffe ES, Waldmann TA, Korsmeyer SJ. Immunoglobulin gene rearrangements as unique clonal markers in human lymphoid neoplasms. New Engl Med 1983;309: 1593-9 Aster JC, Kobayashi Y. Shiota M, Mori S, Sklar J. Detection of the t( 14:18) at similar frequencies in hyperplastic lymphoid tissues from American and Japanese patients. Am .I Parhol 1992;141:291-9 Bakshi A, Jensen JP, Goldman P et al. Cloning the chromosomal breakpoint of t( 14; 18) human lymophomas: clustering around J,, on chromosome 14 and near a transcriptional unit on 18. Cell 1985;41:899-906 Banchereau J, Bazan F, Blanchard D et al. The CD40 antigen and its ligand. Ann Rev Immune/ 1994;12:881-99

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