# Screening for Residual Disease in Pediatric Burkitt Lymphoma Using Consensus Primer Pools

Authors: Melissa Agsalda; Ian Kusao; David Troelstrup; Bruce Shiramizu
Journal: Advances in Hematology
 (2009)

---

## Abstract

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgVH) genes, IgVH primer pools from IgVH1–IgVH7 regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of using IgVH primer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD using IgVH primer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.


---

## Body

## 1. Introduction

The prognosis for
children diagnosed with Burkitt lymphoma (BL) has improved dramatically over
the last decade; however a significant percentage (30–40%) of children with
advanced disease remain unresponsive to or relapse during therapy, particularly
those with B-cell non-Hodgkin lymphoma (NHL) [1–3].
Because the prognosis for children who relapse during therapy is dismal, with
an estimated 2-year overall survival of 10–30%, improvements in early detection
of disease may improve their overall disease-free survival [2].
In such cases, identifying persistent disease or minimal residual disease (PD/MRD)
could be important if the paradigm of early intervention when PD/MRD is found
can be translated from other pediatric cancers such as acute lymphoblastic
leukemia [4].Malignant BL cells
are usually characterized with immunoglobulin heavy chain variable region (IgVH)
gene rearrangements. The IgVH is formed during normal B-cell
ontogeny by an ordered process of Ig gene rearrangement through assembling
distinct variable (V), diversity (D), and joining (J) gene segments known as
VDJ recombination [5]. A single VH gene
is chosen from the available VH repertoire consisting of >50
potentially functional genes that are grouped into 7 structurally related
families (VH1–7)  [6]. Rearrangements of VH genes can be exploited by analyzing the VH1–7 regions using
polymerase chain reaction (PCR). We and others previously used this molecular
fingerprint from NHL primary diagnostic tissue to detect PD/MRD in followup
specimens [7–9].
However, as a clinical tool, identifying patient-specific primers (PSPs) designed
from primary diagnostic tissue is labor-intensive and may not be applicable for
real-time applications [7]. When faced with limited
amount of tissue at the time of diagnosis, particularly from children, there
may not be enough tissue available to design PSP if PD/MRD studies are to be tested
[10–12].
The current study hypothesizes that detection of PD/MRD in BL cases may be
accomplished by more efficient means and not require primary diagnostic tissue to
design unique PSP. Instead, PD/MRD assessment could be achieved with primer
pools made up of IgVH oligomers from respective VH1 to VH7 families. Thus a strategy was designed to detect PD/MRD with consensus primer pools
by real-time PCR [7]. We report data using the
primer pool method and compare results previously reported by our lab using
PSPs [13].


## 2. Methods and Materials

The study focused on the feasibility of using
pools of oligomers comprised of primers from the seven VH regions (VH1–7)  to amplify a clonal immunoglobulin heavy
chain rearrangement that would be characteristic of BL cells by real-time PCR. The
study, which was approved by the Institutional Review Board, analyzed repository specimens and compared against previous
results in which the specimens were characterized using PSPs.


### 2.1. Patients and Specimens

Fourteen
children with Burkitt lymphoma (BL) and known clinical outcomes had specimens available
which were previously analyzed using PSP [7]. The diagnoses and staging
were confirmed through a central review mechanism [7]. DNA from primary diagnostic tumor
tissues and staging/followup specimens (peripheral blood mononuclear cells,
PBMCs; bone marrow aspirate, BMA; cerebrospinal fluid, CSF) were available from paraffin-embedded
diagnostic tissue, unstained peripheral blood smears/bone marrow slides, and
CSF cell pellets;  see Table 1 [7]. DNA was assessed by
ultraviolet spectrophotometry and by amplification with beta-globin primers,
which showed adequate quantity and quality for PCR [13]. Specimens were from entry
(diagnostic) and followup time points (1-month and 2-month postinduction
chemotherapy); see Table 1. From the primary diagnostic tumor tissue, PSPs were designed
and used to assess PD/MRD on followup specimens as previously reported [7].Table 1
Persistent
or minimal residual disease assessment using real-time and PSP assays.
Case   (Dx)Real-time
results/PSP results & clinical status*Entry1 Mo2
Mo3-4 MoClinical statusNHL-1 (BL)Tissue (+/PSP)CR
(End of Rx))Blood (+/+)Blood (−/−)Blood
(+/+)Blood (−/−)NHL-2  (BL)Tissue (+/PSP)CR
(End of Rx)Blood (+/+)Blood (+/+)Blood (−/−)BMA (+/+)NHL-3
(BL)Tissue (+/PSP)Dead (Infection; 3 mo)Blood (+/+)Blood (+/+)BMA (+/+)BMA (−/−)NHL-4 (BL)Tissue (+/PSP)CR ( End of Rx )Blood (+/+)Blood (+/+)Blood (−/−)NHL-5 (BL)Tissue (+/PSP)BMA relapse (6 mo)Blood (+/+)Blood (−/−)BMA (+/+)BMA (+/+)NHL-6 (BL)Tissue (+/PSP)CR ( End of Rx )BMA (+/+)BMA (−/−)NHL-7
(BL)Tissue (+/PSP)BMA relapse (3 mo)BMA (+/+)BMA (+/+)NHL-8
(BL)Tissue (+/PSP)Blood (+/+)Blood (−/−)Blood (−/−)CR (End of Rx)NHL-9 (BL)Tissue (+/PSP)Blood (+/+)Blood (+/+)Blood (−/−)CR (End of Rx)CSF (+/+)CSF (=/=)NHL-10 (BL)Tissue (+/PSP)Blood (+/+)Blood (−/−)CR (End of Rx)NHL-11 (BL)Tissue (+/PSP)Blood (+/+)Blood (+/+)Blood (+/+)Blood (−/−)CR (End of Rx)NHL-12 (BL)Tissue (+/PSP)Blood (+/+)Blood (+/+)Blood (−/−)CR (End of Rx)BMA (Equiv)BMA (+/+)BMA (−/−)CSF (+/+)NHL-13
(BL)Tissue (+/PSP)Blood (+/+)Blood (=/=)Blood (=/=)BMA (+/+)BMA(−/−)CR (End of Rx)CSF(−/−)NHL-14 (BL)Tissue (+/PSP)Blood (+/+)Blood (−/−)Blood (−/−)CR (End of Rx)BMA (+/+)*Specimens were not available from some time points;
Dx: diagnosis; BL: Burkitt lymphoma; BMA: bone marrow aspirate; real-time
results/patient-specific primer (PSPs) results: Positive (+), Negative (−),  or Equivocal (=) for each assay result;  CR: complete remission; End of Rx: End of
therapy.


### 2.2. Real-Time PCR Using VH1–VH7 Primer Pools and Sensitivity Assessment

Groups of5′-primers
from the FR1 region of the variable IgH regions, VH1–VH7 (Operon Biotechnologies, Inc, Huntsville,  AL, USA) were combined to form primer
pools: VH1,  VH2,  VH3,
VH4, VH5, VH6,  and VH7 (Table 2) identified
through VBASE23 (http://vbase.mrc-cpe.cam.ac.uk/) [14]. Primers were designed from the FR1
region because of future plans to use the assay on DNA extracted from fresh or
unstained slides of PBMC, BMA, and CSF. The two consensus 3′-primers were:  LJH and VLJH [7]. Seminested real-time PCR was
performed in triplicate using an iCycler (BioRad Laboratories, Hercules, CA, USA),
and StepOnePlus (Applied Biosystems, Foster City, CA, USA). The PCR parameters were
optimized initially with DNA from a B-cell NHL cell line, Ramos (ATCC,
Manassas, VA, USA) by diluting control human PBMC DNA as noted below with negative and positive controls consisting of peripheral blood mononuclear cells (PBMCs),
water, and BL tumor DNA. The primer pool concentrations of 10 pmol were found
to be optimal with the PCR mix of 2x iQ SYBR Green Supermix (Biorad
Laboratories, Hercules, CA, USA), 10 pmol 5′-IgVH primer pools and 10 pmol
3′-primer (LJH) and parameters:  95C°/3 minutes;
35 cycles 95C°/10 seconds,
60C°/30 seconds;
72C°/3 minutes; final extension of 72C°/3 minutes. PCR products were purified using ExoSAP-it (USB
Corp, Cleveland, Ohio, USA), and recovered in 10 μL.  An aliquot of 1 μL was used for second-round PCR with 10 pmol
5′-primer pools and 10 pmol 3′-primer (VLJH). Following threshold-dependent
cycling, melting was performed from 60 to 95C° at either 0.5C°/s or 0.1C°/s
melt rates with a smooth curve setting averaging 1 point with minor
modifications of a previously described strategy to identify unique amplified
products [9, 15].
Melt curves were plotted as the negative first derivative of decrease in
fluorescence versus temperature (−dF/−dT). Polyclonal products of different
lengths would melt at different temperatures and displayed as broad peaks. A
wide melt curve peak defined as the width at half height ≥ peak height was
considered a negative result modified from previous reports due to different primers
and compensations made in the current real-time methodology versus the plate
reader assay [16, 17].
Thus a positive (peak height ≥ width at half height); negative (peak height  ≤
width at half height) or equivocal (multiple peaks) determination was
established for each melt curve, (Figure 1) with verification by resolving the
amplified products on agarose gels and sequencing, if appropriate. Differentiation
by melt-curve analysis led to distinct melt temperatures for distinct amplified
products if a unique VH region is amplified and represented as a sharp
peak [18, 19].
Following PCR, samples were subjected to two melt runs, with melt rates of
either 0.1 or 0.5C°/s if melt peaks could not be distinguished [20]. As noted by others,
increasing the melt rate can increase the size of the melt peaks due to a more
rapid loss of fluorescence, facilitating more reliable detection of weaker
amplicons [20]. Sharp melt peaks corresponded
to monoclonal bands while widened or flat melt curves suggested polyclonal or no
clonal DNA, Figures 1(b) and 1(c). If a melt curve suggested polyclonality, PCR
products were purified and sequenced. For the current study, polyclonality
determined by the sequence data was interpreted as negative for MRD/PD.Table 2
Primers sequences.
PrimerSequence
(5′–3′)VH1aCAG GT(GT) CAG CTG GTG CAGVH1bCAG GTC CAG CTT GTG CAGVH1c(GC)AG GTC CAG CTG GTA CAGVH1dCA(AG) ATG CAG CTG GTG CAGVH2aCAG ATC ACC TTG AAG GAGVH2bCAG GTC ACC TTG A(AG)G GAGVH3aGA(AG) GTG CAG CTG GTG GAGVH3bCAG GTG CAG CTG GTG GAGVH3cGAG GTG CAG CTG TTG GAGVH4aCAG (CG)TG CAG CTG CAG GAGVH4bCAG (CG)TG CAG CTG CAG GAGVH5aGA(AG) GTG CAG CTG GTG CAGVH6aCAG GTA CAG CTG CAG CAGVH7aCAG GT(CG) CAG CTG GTG CAALJHTGA GGA GAC GGT GAC CVLJHGTG ACC AGG GNC CTT GGC CCC AGB-globin (forward)GAA GAG CCA AGG ACA GGT ACB-globin (reverse)CAA CTT CAT CCA CGT TCA CCExamples of melt curve
analyses with corresponding ethidium-stained amplified products. Melt curves
displayed as −dF/dT
plots:  (a) defined peak with minimal to
no shoulder indicates a positive MRD result; (b) a broad peak indicates a
negative result; (c) a defined peak
with significant shoulders indicates equivocal results. Representative 2.0%
agarose gels are at the left of each of graph. Melt curves of negative
controls: (d) PBMC from normal healthy individual shows a broad undefined peak;
(e) water (no template) shows no peak;  (f) melt curve assessment of PCR products:
melt curve peak shift disparities are noted between diagnostic and followup
specimens, consistent with possible different clonal differentiation over time.An algorithm was
established to screen for VH family usage. In this study, each primary
tumor tissue DNA was subjected to each of the following combinations of primer
pools: VH1 &  VH2;  VH3 & VH4;
VH5, VH6,  &  VH7.  The strategy allowed for combinations
of VH primers to be used to screen-in or screen-out involvement of
variable regions. If particular primer pools were positive, then individual  VH primers were used to individually retest the tumor tissue DNA to identify
the variable region involved. The specific VH primer was then used
to assess PD/MRD on sequential specimens from the same patient. Sensitivity of
the assay was determined by two methods using cell line DNA and BL tumor DNA.
DNA from the B-cell NHL Ramos cell line and control human PBMC were combined in
various dilutions (DNA equivalent of 1 Ramos cell to 102, 103,
105, 107, 108, and 109 PBMC DNA) to test the IgVH primer pools [21]. Dilutions of DNA from tumor DNA were
prepared with control human PBMC to the DNA equivalent of 1 malignant cell to 103,
105, 106, 108, and 109 PBMC DNA.
Each dilution was then assayed using VH4 primer, which was a characteristic of that particular tumor specimen.


## 2.1. Patients and Specimens

Fourteen
children with Burkitt lymphoma (BL) and known clinical outcomes had specimens available
which were previously analyzed using PSP [7]. The diagnoses and staging
were confirmed through a central review mechanism [7]. DNA from primary diagnostic tumor
tissues and staging/followup specimens (peripheral blood mononuclear cells,
PBMCs; bone marrow aspirate, BMA; cerebrospinal fluid, CSF) were available from paraffin-embedded
diagnostic tissue, unstained peripheral blood smears/bone marrow slides, and
CSF cell pellets;  see Table 1 [7]. DNA was assessed by
ultraviolet spectrophotometry and by amplification with beta-globin primers,
which showed adequate quantity and quality for PCR [13]. Specimens were from entry
(diagnostic) and followup time points (1-month and 2-month postinduction
chemotherapy); see Table 1. From the primary diagnostic tumor tissue, PSPs were designed
and used to assess PD/MRD on followup specimens as previously reported [7].Table 1
Persistent
or minimal residual disease assessment using real-time and PSP assays.
Case   (Dx)Real-time
results/PSP results & clinical status*Entry1 Mo2
Mo3-4 MoClinical statusNHL-1 (BL)Tissue (+/PSP)CR
(End of Rx))Blood (+/+)Blood (−/−)Blood
(+/+)Blood (−/−)NHL-2  (BL)Tissue (+/PSP)CR
(End of Rx)Blood (+/+)Blood (+/+)Blood (−/−)BMA (+/+)NHL-3
(BL)Tissue (+/PSP)Dead (Infection; 3 mo)Blood (+/+)Blood (+/+)BMA (+/+)BMA (−/−)NHL-4 (BL)Tissue (+/PSP)CR ( End of Rx )Blood (+/+)Blood (+/+)Blood (−/−)NHL-5 (BL)Tissue (+/PSP)BMA relapse (6 mo)Blood (+/+)Blood (−/−)BMA (+/+)BMA (+/+)NHL-6 (BL)Tissue (+/PSP)CR ( End of Rx )BMA (+/+)BMA (−/−)NHL-7
(BL)Tissue (+/PSP)BMA relapse (3 mo)BMA (+/+)BMA (+/+)NHL-8
(BL)Tissue (+/PSP)Blood (+/+)Blood (−/−)Blood (−/−)CR (End of Rx)NHL-9 (BL)Tissue (+/PSP)Blood (+/+)Blood (+/+)Blood (−/−)CR (End of Rx)CSF (+/+)CSF (=/=)NHL-10 (BL)Tissue (+/PSP)Blood (+/+)Blood (−/−)CR (End of Rx)NHL-11 (BL)Tissue (+/PSP)Blood (+/+)Blood (+/+)Blood (+/+)Blood (−/−)CR (End of Rx)NHL-12 (BL)Tissue (+/PSP)Blood (+/+)Blood (+/+)Blood (−/−)CR (End of Rx)BMA (Equiv)BMA (+/+)BMA (−/−)CSF (+/+)NHL-13
(BL)Tissue (+/PSP)Blood (+/+)Blood (=/=)Blood (=/=)BMA (+/+)BMA(−/−)CR (End of Rx)CSF(−/−)NHL-14 (BL)Tissue (+/PSP)Blood (+/+)Blood (−/−)Blood (−/−)CR (End of Rx)BMA (+/+)*Specimens were not available from some time points;
Dx: diagnosis; BL: Burkitt lymphoma; BMA: bone marrow aspirate; real-time
results/patient-specific primer (PSPs) results: Positive (+), Negative (−),  or Equivocal (=) for each assay result;  CR: complete remission; End of Rx: End of
therapy.


## 2.2. Real-Time PCR Using VH1–VH7 Primer Pools and Sensitivity Assessment

Groups of5′-primers
from the FR1 region of the variable IgH regions, VH1–VH7 (Operon Biotechnologies, Inc, Huntsville,  AL, USA) were combined to form primer
pools: VH1,  VH2,  VH3,
VH4, VH5, VH6,  and VH7 (Table 2) identified
through VBASE23 (http://vbase.mrc-cpe.cam.ac.uk/) [14]. Primers were designed from the FR1
region because of future plans to use the assay on DNA extracted from fresh or
unstained slides of PBMC, BMA, and CSF. The two consensus 3′-primers were:  LJH and VLJH [7]. Seminested real-time PCR was
performed in triplicate using an iCycler (BioRad Laboratories, Hercules, CA, USA),
and StepOnePlus (Applied Biosystems, Foster City, CA, USA). The PCR parameters were
optimized initially with DNA from a B-cell NHL cell line, Ramos (ATCC,
Manassas, VA, USA) by diluting control human PBMC DNA as noted below with negative and positive controls consisting of peripheral blood mononuclear cells (PBMCs),
water, and BL tumor DNA. The primer pool concentrations of 10 pmol were found
to be optimal with the PCR mix of 2x iQ SYBR Green Supermix (Biorad
Laboratories, Hercules, CA, USA), 10 pmol 5′-IgVH primer pools and 10 pmol
3′-primer (LJH) and parameters:  95C°/3 minutes;
35 cycles 95C°/10 seconds,
60C°/30 seconds;
72C°/3 minutes; final extension of 72C°/3 minutes. PCR products were purified using ExoSAP-it (USB
Corp, Cleveland, Ohio, USA), and recovered in 10 μL.  An aliquot of 1 μL was used for second-round PCR with 10 pmol
5′-primer pools and 10 pmol 3′-primer (VLJH). Following threshold-dependent
cycling, melting was performed from 60 to 95C° at either 0.5C°/s or 0.1C°/s
melt rates with a smooth curve setting averaging 1 point with minor
modifications of a previously described strategy to identify unique amplified
products [9, 15].
Melt curves were plotted as the negative first derivative of decrease in
fluorescence versus temperature (−dF/−dT). Polyclonal products of different
lengths would melt at different temperatures and displayed as broad peaks. A
wide melt curve peak defined as the width at half height ≥ peak height was
considered a negative result modified from previous reports due to different primers
and compensations made in the current real-time methodology versus the plate
reader assay [16, 17].
Thus a positive (peak height ≥ width at half height); negative (peak height  ≤
width at half height) or equivocal (multiple peaks) determination was
established for each melt curve, (Figure 1) with verification by resolving the
amplified products on agarose gels and sequencing, if appropriate. Differentiation
by melt-curve analysis led to distinct melt temperatures for distinct amplified
products if a unique VH region is amplified and represented as a sharp
peak [18, 19].
Following PCR, samples were subjected to two melt runs, with melt rates of
either 0.1 or 0.5C°/s if melt peaks could not be distinguished [20]. As noted by others,
increasing the melt rate can increase the size of the melt peaks due to a more
rapid loss of fluorescence, facilitating more reliable detection of weaker
amplicons [20]. Sharp melt peaks corresponded
to monoclonal bands while widened or flat melt curves suggested polyclonal or no
clonal DNA, Figures 1(b) and 1(c). If a melt curve suggested polyclonality, PCR
products were purified and sequenced. For the current study, polyclonality
determined by the sequence data was interpreted as negative for MRD/PD.Table 2
Primers sequences.
PrimerSequence
(5′–3′)VH1aCAG GT(GT) CAG CTG GTG CAGVH1bCAG GTC CAG CTT GTG CAGVH1c(GC)AG GTC CAG CTG GTA CAGVH1dCA(AG) ATG CAG CTG GTG CAGVH2aCAG ATC ACC TTG AAG GAGVH2bCAG GTC ACC TTG A(AG)G GAGVH3aGA(AG) GTG CAG CTG GTG GAGVH3bCAG GTG CAG CTG GTG GAGVH3cGAG GTG CAG CTG TTG GAGVH4aCAG (CG)TG CAG CTG CAG GAGVH4bCAG (CG)TG CAG CTG CAG GAGVH5aGA(AG) GTG CAG CTG GTG CAGVH6aCAG GTA CAG CTG CAG CAGVH7aCAG GT(CG) CAG CTG GTG CAALJHTGA GGA GAC GGT GAC CVLJHGTG ACC AGG GNC CTT GGC CCC AGB-globin (forward)GAA GAG CCA AGG ACA GGT ACB-globin (reverse)CAA CTT CAT CCA CGT TCA CCExamples of melt curve
analyses with corresponding ethidium-stained amplified products. Melt curves
displayed as −dF/dT
plots:  (a) defined peak with minimal to
no shoulder indicates a positive MRD result; (b) a broad peak indicates a
negative result; (c) a defined peak
with significant shoulders indicates equivocal results. Representative 2.0%
agarose gels are at the left of each of graph. Melt curves of negative
controls: (d) PBMC from normal healthy individual shows a broad undefined peak;
(e) water (no template) shows no peak;  (f) melt curve assessment of PCR products:
melt curve peak shift disparities are noted between diagnostic and followup
specimens, consistent with possible different clonal differentiation over time.An algorithm was
established to screen for VH family usage. In this study, each primary
tumor tissue DNA was subjected to each of the following combinations of primer
pools: VH1 &  VH2;  VH3 & VH4;
VH5, VH6,  &  VH7.  The strategy allowed for combinations
of VH primers to be used to screen-in or screen-out involvement of
variable regions. If particular primer pools were positive, then individual  VH primers were used to individually retest the tumor tissue DNA to identify
the variable region involved. The specific VH primer was then used
to assess PD/MRD on sequential specimens from the same patient. Sensitivity of
the assay was determined by two methods using cell line DNA and BL tumor DNA.
DNA from the B-cell NHL Ramos cell line and control human PBMC were combined in
various dilutions (DNA equivalent of 1 Ramos cell to 102, 103,
105, 107, 108, and 109 PBMC DNA) to test the IgVH primer pools [21]. Dilutions of DNA from tumor DNA were
prepared with control human PBMC to the DNA equivalent of 1 malignant cell to 103,
105, 106, 108, and 109 PBMC DNA.
Each dilution was then assayed using VH4 primer, which was a characteristic of that particular tumor specimen.


## 3. Results

### 3.1. Real-Time Pcr Using VH1–VH7 Primer Pools and Sensitivity Assessment

Dilutions of DNA from the control Ramos cell
line were assayed using the IgVH primer pools [22]. Melt curve
analysis of each reaction was plotted as the negative first derivative of
decrease in fluorescence with respect to temperature (−dF/dT) and interpreted:
Positive (distinct peak with minimal or no shoulder); Negative (broad peak); or
Equivocal (multiple peaks with significant shoulder); see Figure 1. The dilution
experiments demonstrated that Ramos cells could be detected on the order of 1
in 105 to 1 in 106 PBMC. Using patient tumor DNA, the
sensitivity of the assay could detect malignant cells as low as 1 in 105 PBMC (Figure 2).Figure 2
MRD sensitivity with
patient primary tumor DNA. Primary diagnostic tumor DNA (100% infiltrated with
malignant cells) was diluted with PBMC DNA to represent the equivalent of 1
malignant cell in 103, 105, 107, 108,
and 109 PBMC. Each representative melt curve has a distinct peak
corresponding to tumor DNA or PBMC DNA, which are designated by the respective
arrows.PD/MRD assays were
performed on clinical specimens from the 14 subjects in which primary diagnostic
tissues were available. The results were compared to the analyses from the PSP
method and found 100% concordance in that when PD/MRD was positive via PSP
method, the same result was obtained using IgVH primer pools (Table 1) [7]. The amplified products from
the positive PD/MRD specimens were sequenced, confirming the monoclonality of
the specimens.Patients, NHL-5
and NHL-7, continued to have PD/MRD in followup BMA specimens 2 months after
starting chemotherapy, and subsequently developed clinically detectable relapse
at 6 months and 3 months, respectively. The amplified products from the BMA
from these two subjects were purified and sequenced, which confirmed the same
clone as the original primary diagnostic tumor. The other 12 cases demonstrated
clearance of their followup blood and/or bone marrow specimens and at the end
of therapy, remained in clinical remission. While no followup specimens were
available from patients NHL-5 and NHL-7, the specimens which were positive for
PD/MRD did not have evidence of disease by standard microscopic pathology.To determine the
utility of the methodology in assessing PD/MRD in specimens when primary diagnostic
tissues were not available, staging (Entry) specimens from each of the cases were
assayed using IgVH primer pools using the same strategy noted above.
Each subject had staging specimens which were positive for PD/MRD by a primer
pool (Table 1). This was important to verify, to validate the use of the strategy
in cases in which primary tumor tissue was not available.


### 4. Discussion

The current study
was designed to assess PD/MRD on followup specimens from pediatric B-NHL
cases regardless of whether primary diagnostic tissue was available from the
time of diagnosis. We hypothesized that PD/MRD could be screened in specimens using
primer pools made up of IgVH oligomers from respective VH1 to VH7 families. The method was compared to a previous study, which
used the PSP-based assay, and the results demonstrated 100% concordance [7]. Sequencing of the amplified
products from primary diagnostic tumor DNA confirmed the results from the VH oligomer amplified product demonstrating that monoclonal results were
represented by both methods.The use of IgVH primer pools to screen for PD/MRD in clinical specimens was shown to have
potential and feasibility. The current study is limited by the number of
subjects and specimens available; however, as a feasibility study, the data
support future consideration of the tool to determine the clinical significance
of PD/MRD in pediatric BL and possibly B-cell NHL therapy trials. A potential
limitation of the approach describe in this paper is that the BL clone needs to
have rearrangement of an IgH gene [23]. Thus in BL cases involving
immunoglobulin light chain rearrangements, the primer pools will need to be
redesigned [23, 24].
There is precedence for potential utility of real-time assessment of PD/MRD. PCR-based
MRD assays have been tested clinically in childhood acute lymphoblastic
leukemia (ALL) by targeting IgH gene rearrangements with sensitivities on the
order of one leukemic cell in 104–106 normal cells. Rapid
reduction of MRD following induction therapy was associated with a 3-year
relapse rate less than 5%, while children with detectable MRD had a 3-year
relapse rate of 23%–75% [25].
In ALL, MRD was the most powerful prognostic factor of outcome, independent of
other risk factors (age, leukocyte count, cytogenetic abnormalities, prednisone
response), with similar results in children undergoing BM transplantation [26, 27].
To date, translating the utility of MRD assessment in ALL to pediatric lymphoma
has been challenging, in part due to access to primary diagnostic tissue in
order to identify PSPs as a marker to be used to assess followup specimens. Other
challenges include presence or absence of cytogenetic abnormalities such as 13q− and 7q+, which could be useful for prognosis, but there remains a
lack of consistent cytogenetic abnormalities other than c-myc in
childhood NHL for use in FISH or PCR [28].
Thus, the current study could potentially be applicable in assessing PD/MRD through
IgVH oligomer pools in a PCR assay that does not rely on primary diagnostic
tissue for a specific tumor marker. However there are currently no
universally accepted or recognized standard methods to assess PD/MRD in
pediatric BL for which to compare. Because the current study is preliminary, emphasis
should be placed on the fact that the current assay cannot substitute for the
use of PSP-based assay and in the absence of primary diagnostic tissue until
further studies are completed.Other
investigators have studied the IgVH region in NHL and found
preferential VH gene usage with  VH3 and VH4 families [8].
For the current study, we screened the primary diagnostic specimens for all of
the VH gene families (VH1 to  VH7) with the VH primer sets and found that, for the limited number of cases, the majority
included VH3 or  VH4 families. The data suggest that the
PCR assay using IgVH primer pools is able to identify B-NHL DNA in
clinical specimens, with the limitations as noted above. Clinical translational
studies with larger number of patients are needed to assess the utility of this
assay to detect PD/MRD and determine the clinical significance. If the assay is
validated in larger studies, one of the potential advantages of the tool would
be the ease of assessing PD/MRD without the need of primary diagnostic tissue.


## 3.1. Real-Time Pcr Using VH1–VH7 Primer Pools and Sensitivity Assessment

Dilutions of DNA from the control Ramos cell
line were assayed using the IgVH primer pools [22]. Melt curve
analysis of each reaction was plotted as the negative first derivative of
decrease in fluorescence with respect to temperature (−dF/dT) and interpreted:
Positive (distinct peak with minimal or no shoulder); Negative (broad peak); or
Equivocal (multiple peaks with significant shoulder); see Figure 1. The dilution
experiments demonstrated that Ramos cells could be detected on the order of 1
in 105 to 1 in 106 PBMC. Using patient tumor DNA, the
sensitivity of the assay could detect malignant cells as low as 1 in 105 PBMC (Figure 2).Figure 2
MRD sensitivity with
patient primary tumor DNA. Primary diagnostic tumor DNA (100% infiltrated with
malignant cells) was diluted with PBMC DNA to represent the equivalent of 1
malignant cell in 103, 105, 107, 108,
and 109 PBMC. Each representative melt curve has a distinct peak
corresponding to tumor DNA or PBMC DNA, which are designated by the respective
arrows.PD/MRD assays were
performed on clinical specimens from the 14 subjects in which primary diagnostic
tissues were available. The results were compared to the analyses from the PSP
method and found 100% concordance in that when PD/MRD was positive via PSP
method, the same result was obtained using IgVH primer pools (Table 1) [7]. The amplified products from
the positive PD/MRD specimens were sequenced, confirming the monoclonality of
the specimens.Patients, NHL-5
and NHL-7, continued to have PD/MRD in followup BMA specimens 2 months after
starting chemotherapy, and subsequently developed clinically detectable relapse
at 6 months and 3 months, respectively. The amplified products from the BMA
from these two subjects were purified and sequenced, which confirmed the same
clone as the original primary diagnostic tumor. The other 12 cases demonstrated
clearance of their followup blood and/or bone marrow specimens and at the end
of therapy, remained in clinical remission. While no followup specimens were
available from patients NHL-5 and NHL-7, the specimens which were positive for
PD/MRD did not have evidence of disease by standard microscopic pathology.To determine the
utility of the methodology in assessing PD/MRD in specimens when primary diagnostic
tissues were not available, staging (Entry) specimens from each of the cases were
assayed using IgVH primer pools using the same strategy noted above.
Each subject had staging specimens which were positive for PD/MRD by a primer
pool (Table 1). This was important to verify, to validate the use of the strategy
in cases in which primary tumor tissue was not available.


## 4. Discussion

The current study
was designed to assess PD/MRD on followup specimens from pediatric B-NHL
cases regardless of whether primary diagnostic tissue was available from the
time of diagnosis. We hypothesized that PD/MRD could be screened in specimens using
primer pools made up of IgVH oligomers from respective VH1 to VH7 families. The method was compared to a previous study, which
used the PSP-based assay, and the results demonstrated 100% concordance [7]. Sequencing of the amplified
products from primary diagnostic tumor DNA confirmed the results from the VH oligomer amplified product demonstrating that monoclonal results were
represented by both methods.The use of IgVH primer pools to screen for PD/MRD in clinical specimens was shown to have
potential and feasibility. The current study is limited by the number of
subjects and specimens available; however, as a feasibility study, the data
support future consideration of the tool to determine the clinical significance
of PD/MRD in pediatric BL and possibly B-cell NHL therapy trials. A potential
limitation of the approach describe in this paper is that the BL clone needs to
have rearrangement of an IgH gene [23]. Thus in BL cases involving
immunoglobulin light chain rearrangements, the primer pools will need to be
redesigned [23, 24].
There is precedence for potential utility of real-time assessment of PD/MRD. PCR-based
MRD assays have been tested clinically in childhood acute lymphoblastic
leukemia (ALL) by targeting IgH gene rearrangements with sensitivities on the
order of one leukemic cell in 104–106 normal cells. Rapid
reduction of MRD following induction therapy was associated with a 3-year
relapse rate less than 5%, while children with detectable MRD had a 3-year
relapse rate of 23%–75% [25].
In ALL, MRD was the most powerful prognostic factor of outcome, independent of
other risk factors (age, leukocyte count, cytogenetic abnormalities, prednisone
response), with similar results in children undergoing BM transplantation [26, 27].
To date, translating the utility of MRD assessment in ALL to pediatric lymphoma
has been challenging, in part due to access to primary diagnostic tissue in
order to identify PSPs as a marker to be used to assess followup specimens. Other
challenges include presence or absence of cytogenetic abnormalities such as 13q− and 7q+, which could be useful for prognosis, but there remains a
lack of consistent cytogenetic abnormalities other than c-myc in
childhood NHL for use in FISH or PCR [28].
Thus, the current study could potentially be applicable in assessing PD/MRD through
IgVH oligomer pools in a PCR assay that does not rely on primary diagnostic
tissue for a specific tumor marker. However there are currently no
universally accepted or recognized standard methods to assess PD/MRD in
pediatric BL for which to compare. Because the current study is preliminary, emphasis
should be placed on the fact that the current assay cannot substitute for the
use of PSP-based assay and in the absence of primary diagnostic tissue until
further studies are completed.Other
investigators have studied the IgVH region in NHL and found
preferential VH gene usage with  VH3 and VH4 families [8].
For the current study, we screened the primary diagnostic specimens for all of
the VH gene families (VH1 to  VH7) with the VH primer sets and found that, for the limited number of cases, the majority
included VH3 or  VH4 families. The data suggest that the
PCR assay using IgVH primer pools is able to identify B-NHL DNA in
clinical specimens, with the limitations as noted above. Clinical translational
studies with larger number of patients are needed to assess the utility of this
assay to detect PD/MRD and determine the clinical significance. If the assay is
validated in larger studies, one of the potential advantages of the tool would
be the ease of assessing PD/MRD without the need of primary diagnostic tissue.



---

*Source: 412163-2009-04-15.xml*