References

Beillard E, Pallisgaard N, Van der Velden VH, BI W, Dee R, Van der Schoot E, Delabesse E, Macintyre E, Gottardi E, Saglio G, Watzinger F, Lion T, Van Dongen JJ, Hokland P, Gabert J (2003) Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia 17(12): 2474-2486.

Biondi A, Valsecchi MG, Seriu T, D'Aniello E, Willemse MJ, Fasching K, Pannunzio A, Gadner H, Schrappe M, Kamps WA, Bartram CR, Van Dongen JJ, Panzer-Grumayer ER (2000) Molecular detection of minimal residual disease is a strong predictive factor of relapse in childhood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group. Leukemia 14(11): 1939-1943.

Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV (1998) The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. Blood 92(9): 3362-3367.

Fehse B, Chukhlovin A, Kuhlcke K, Marinetz O, Vorwig O, Renges H, Kruger W, Zabelina T, Dudina O, Finckenstein FG, Kroger N, Kabisch H, Hochhaus A, Zander AR (2001) Real-time quantitative Y chromosome-specific PCR (QYCS-PCR) for monitoring hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation. Journal of Hematotherapy & Stem Cell Research, 10(3): 419-425.

Gabert J, Beillard E, Van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cave H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, Gonzalez M, Viehmann S, Malec M, Saglio G, Van Dongen JJ (2003) Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. [Review]. Leukemia 17(12): 2318-2357.

Hochhaus A, Weisser A, La Rosee P, Emig M, Muller MC, Saussele S, Reiter A, Kuhn C, Berger U, Hehlmann R, Cross NC (2000) Detection and quantification of residual disease in chronic myelogenous leukemia. [Review]. Leukemia 14(6): 998-1005.

Jurlander J, Caligiuri MA, Ruutu T, Baer MR, Strout MP, Oberkircher AR, Hoffmann L, Ball ED, Frei-Lahr DA, Christiansen NP, Block AM, Knuutila S, Herzig GP, Bloomfield CD (1996) Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia. Blood 88(6): 2183-2191.

Langerak AW, Szczepanski T, Van der Burg M, Wolvers-Tettero IL, Van Dongen JJ, (1997) Heteroduplex PCR analysis of rearranged T cell receptor genes for clonal-ity assessment in suspect T cell proliferations. Leukemia 11(12): 2192-2199.

Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG (1999) Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. [Review]. Blood 94(1): 12-22.

Maas F, Schaap N, Kolen S, Zoetbrood A, Buno I, Dolstra H, De Witte T, Schattenberg A, Van de Wiel-van Kemenade E (2003) Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms. Leukemia 17(3): 630-633.

Moppett J, Van der Velden VH, Wijkhuijs AJ, Hancock J, Van Dongen JJ, Goulden N (2003) Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin. Leukemia 17(1): 268-270.

Pongers-Willemse MJ, Seriu T, Stolz F, D'Aniello E, Gameiro P, Pisa P, Gonzalez M, Bartram CR, Panzer-Grumayer ER, Biondi A, San Miguel JF, Van Dongen JJ (1999) Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.[see comment]. Leukemia 13(1): 110-118.

Van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, Van Dongen JJ (2003) Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. [Review]. Leukemia 17(6): 1013-1034.

Van der Velden VH, Wijkhuijs JM, Jacobs DC, Van Wering ER, Van Dongen JJ (2002) T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis. Leukemia 16(7): 1372-1380.

Van der Velden VH, Willemse MJ, Van der Schoot CE, Hahlen K, Van Wering ER, Van Dongen JJ (2002) Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR. Leukemia 16(5): 928-936.

Van Dongen JJ, Seriu T, Panzer-Grumayer ER, Biondi A, Pongers-Willemse MJ, Corral L, Stolz F, Schrappe M, Masera G, Kamps WA, Gadner H, Van Wering ER, Ludwig WD, Basso G, De Bruijn MA, Cazzaniga G, Hettinger K, Van der Does-Van der Berg A, Hop WC, Riehm H, Bartram CR (1998) Prognostic value of minimal residual disease in acute lymphoblastic leukemia in childhood. Lancet 352(9142): 1731-1738.

Verhagen OJ, Willemse MJ, Breunis WB, Wijkhuijs AJ, Jacobs DC, Joosten SA, Van Wering ER, Van Dongen JJ, Van der Schoot CE (2000) Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia. Leukemia 14(8): 1426-1435.

Protocol 16.1: Post-transplant MRD monitoring

OUTLINE_

This is a complex technique involving several steps. The major processes are:

1. Identify an individual patient's monoclonal rearrangements and perform sequencing

2. Design and order clone specific forward primers for at least 2 separate loci

3. Prepare control DNA from the pooled mononuclear cells up at least 10 normal donors

4. Prepare dilution series of patient's diagnostic DNA in control DNA

5. Test clone specific primers for sensitivity and specificity. Re-design and retest if necessary

6. Test remission specimens from the individual patient and calculate the level of MRD relative to the diagnostic specimen using the standard curve

Reagents and consumables

PCR primers for chosen antigen receptor rearrangements

Polyacrylamide gel solution (Sigma, 40% Stock, 29:1 bis: acrylamide) TRIS borate EDTA electrophoresis buffer DNA Sequencing Kit

Germline reverse primers and TaqMan® probes (Verhagen et al., 2000; van der Velden et al., 2002; van der Velden et al., 2002) Control gene TaqMan® assay, e.g ALB (Verhagen et al., 2000)

2 x TaqMan® universal master mix (Applied Biosystems)

TE Buffer (10mM TRIS, 1mM EDTA, pH 8.0) Molecular biology grade H2O 96-well PCR plates

Optical quality adhesive covers or caps

Equipment

Thermal Cycler

Real-time PCR instrument such as ABI 7500

Genespec Micro spectrophotometer (Hitachi)

Vertical Polyacrylamide electrophoresis apparatus

(SE 600 Ruby, GE Healthcare)

PCR micropipettes

Waterbath at 37°C

Microcentrifuge

Benchtop centrifuge with microplate carriers

Specimens required

• DNA from patient at presentation (marrow or blood should contain >90% leukemic cells). Adjust concentration to 100 ng/pl

• DNA from pooled mononuclear cells from at least 10 healthy donors (concentration as above). This will be the non-amplified control (NAC) and will also be used as diluent for dilution series of patient's diagnostic DNA. Leukemic cell DNA in a background of polyclonal lymphocyte DNA mimics the DNA isolated from the patient in remission. It will also be used to prepare dilutions for control gene standard curve

• DNA from patient during treatment at 100 ng/pl

Identify clonal rearrangements

Analyze the patient's diagnostic DNA using PCR assays for IgH, TCR8, TCRy and kappa deleting element (KDE) as described (Pongers-Willemse et al., 1999) and identify suitable rearrangements by heteroduplex analysis (Langerak et al., 1997). Select monoclonal rearrangements with no evidence of heteroduplex formation. If possible at least two rearrangements from different loci should be selected, e.g. IgH or TCR. Where there has been sufficient resolution of clonal bands they may be excised from the gel and sequenced. Analyze the sequence using software available at http://www.ncbi.nlm.nih.gov/igblast, http://imgt.cines.fr/textes/vquest, or http://vbase.mrc-cpe.cam.ac.uk/vbase1/ dnaplot2.php to designate the gene segments involved in the junctional region

Design of clone specific forward primer

Forward patient specific primers are designed (Primer Express®, Applied Biosystems) to lie over the area of clone specific rearrangement. In the case of IgH, this should be the DNJ junction as opposed to the VND junction, to minimize the risk of clonal evolution. For TCRy targets the primer should lie over the VND or DND junction, and for y deleting elements KDE the VNKDE join. For maximal specificity avoid including anymore than 3-5 bases in the germline sequence at the 3' end. In practice this can be very difficult to achieve with some delta and many kappa targets.

Due to the more limited diversity of TCR rearrangements, the germline probe assay sensitivity is dependent upon the rearrangement present and the number of insertions present. Rearrangements containing >12 insertions and deletions provide useful markers whilst those with <12 are unlikely to be specific (van der Velden et al., 2002).

Criteria for an acceptable forward primer (in addition to the rules listed in Chapter 7, Table 1)

• Tm of 57-60°C on Primer Express® using Nearest Neighbor algorithm

• Choose shortest primer with the least number of germline nucleotides

• Check for the presence of 3' forward/forward and forward/reverse primer-dimmers and for possible 3' hairpin formation

• In practice, with some rearrangements it is impossible to meet these criteria, however, primers with >3 G/C at the 3' end do not work well

• If Primer Express® will not identify a primer with an appropriate Tm in the region of interest, (particularly common with IgH), primers may be designed by eye and using the Wallace rule (Tm = 4(G + C) + 2(A + T) aim for Tm = 55 - 60°C), using a 2-3 base overhang into consensus at the 3' end

• Design three candidate primers per locus and order from a reliable supplier

Control PCR A suitable control PCR must be run. The control

PCR is used to confirm the amount and quality of amplifiable DNA in each sample analyzed for MRD. It is thus simultaneously a check for the accuracy of DNA quantitation, accuracy of pipetting and for the presence of PCR inhibitors.

As all test PCRs use 500 ng of test DNA, the control standard curve needs to cover this range only. The UK Childhood Leukaemia Network use ALB, other European groups use B2M, our laboratory uses ALB. Other control genes may be used but their chromosomal location should be considered in order to avoid loci that may be deleted or amplified in a particular leukemia.

A standard curve should be constructed from duplicate analysis of a dilution series made from pooled normal control DNA. Dilute the DNA in TE. Only the 100, 10-1,10-2, and 10-3 points need be analyzed.

Generating standard curves for assessment of probe sensitivity and measurement of MRD

A dilution series in which leukemic DNA is diluted with pooled normal blood mononuclear cell DNA is used to generate the standard curve.

Preparation of patient DNA dilution series for TaqMan® MRD analysis

Dilution point

Quantity of leukemic (target) DNA

Volume of target DNA

Volume of control DNA

100

1000 ng

-

10-1

100 ng

10 pl

90 pl

10-2

10 ng

10 pl

90 p

10-3

1000 pg

10 pl

90 p

10-4

100 pg

10 pl

90 p

10-5

10 pg

10 pl

90 p

10-6

1 pg

10 pl

90 p

1. Adjust concentration of presentation DNA (containing >90% leukemic cells) to 100 ng/ml. Warm in 37°C waterbath for 1 h to ensure that the solution is homogenous, vortex briefly and spin down

2. 10-1 Point: 1000 ng (10 pl of 100 ng/pl solution) of target DNA is added to 90 pl of pooled normal donor peripheral blood mononuclear cell DNA (100 ng/pl), making a final solution of 10 ng/pl target DNA. 5 pl of this solution is then used in TaqMan® PCR representing 50 ng of target (= 10-1 point)

3. 10-2 Point: Leave 10-1 point in water bath for 1 h, vortex and spin down. 10 pl of 10-1

solution is added to 90 |l of pooled normal DNA (100 ng/|jl), to make 10-2 point 4. Repeat step 2 four more times to make 10-3, 10-4, 10-5, and 10-6 points

TaqMan® PCR The following master mix should be used for all

PCR reactions, with 20 |l added to each well, 5 |l of sample DNA (i.e. 500 ng DNA) is added.

Constituent Vol/reaction

TaqMan® Universal Master Mix 12.5 |l

Total volume 25 |l

Initial clone specific PCR and ALB PCR conditions are: 2 min at 50°C 10 min at 95°C

Sensitivity and specificity testing

The clone specific primers must be tested for efficiency and specificity before using to analyze patient specimens. An acceptable primer must reach a sensitivity of 10-4 and not have significant reaction with pooled normal DNA control (NAC).

Method 1. Amplify the 10-2 dilution point of the leukemic (target) DNA in duplicate and 6 NAC (normal mononuclear cell DNA) control wells. Use 500 ng DNA per well

2. Examine Ct of 10-2 point. For an efficient primer the Ct should be less than 30

3. Examine Ct of NAC. Ideally this should 50, or at least 11 greater than Ct of 10-2 if sensitivity of 10-4 is to be achieved

4. Choose the primer that best fits these 2 criteria. If none fit the first, then primer redesign is required

5. If none fit the second, then incremental increases in the annealing temperature can be attempted (63, 66, and 69°C recommended)

6. If these steps do not result in a primer that fits the criteria, then that particular rearrangement is not suitable as an MRD marker

7. The sensitivity of a primer/probe combination is defined as the lowest dilution point that has a Ct value at least 3 less than that for NAC for TCR 8, IgK and TCR y or at least 6 less for IgH. See below for definitions of sensitivity

Reproducible sensitivity Lowest dilution of standard curve with reproducible Ct values (Ct values that differ by less than 1.5) that is at least 3 less than that for the NAC (at least 6 for IgH)

Maximal sensitivity The lowest dilution of standard curve giving specific but non-reproducible Ct values, which differ at least 1.5 Ct from Ct value of previous 10-fold dilution or NAC

Quantitation of MRD in remission specimens

1. Adjust DNA concentration to 100 ng/^l in TE

2. The patient's dilution series is used as standard curve for MRD measurement

3. The dilution series of pooled normal mononuclear cell DNA is used as standard curve for control gene for quantity and quality of test DNA

4. The clone specific forward primer, germline reverse primer and probe are used to amplify; the patient's leukemic dilution series in duplicate (the 10-6 is usually not included as such sensitivity is not achieved); duplicate NAC (pooled normal) 500 ng DNA per well; duplicate NTC controls; the test sample in triplicate with 500 ng DNA per well

5. The control PCR is used to amplify; normal control dilution series in duplicate; duplicate NTC; duplicate test samples

Both assays can be performed on same plate unless the clone specific PCR assay requires an increased annealing temperature as control gene assays may not work at higher temperatures. See Figure 16.4 for plate set-up

6. Perform 50 cycles of PCR and analyze the data

7. The slope of the clone specific standard curves should be in the range -3.3 to -4.0. The correlation coefficient of curves >0.95. The maximum Ct variation between replicates of a point <1.5. This defines the reproducible range

1

2

3

4

5

6

7

8

9

10

11

12

A

L10-1

L10-1

Test 1

Test 1

Test 1

C100

C100

L10-1

L10-1

Test 1

Test 1

Test 1

B

L10-2

L10-2

Test2

Test2

Test2

C10-1

C10-1

L10-2

L10-2

Test2

Test2

Test2

C

L10-3

L10-3

Test 3

Test 3

Test 3

C10-2

C10-2

L10-3

L10-3

Test 3

Test 3

Test 3

D

L10-4

L10-4

C10-3

C10-3

L10-4

L10-4

E

L10-5

L10-5

NTC

NTC

L10-5

L10-5

F

NAC

NAC

Test 1

Test 1

NAC

NAC

G

NAC

NAC

Test2

Test2

NAC

NAC

H

NTC

NTC

Test 3

Test 3

NTC

NTC

Columns 1-5 and 8-12 are the clone specific PCR experiments, columns 6 + 7 the ALBUMIN control.

Columns 1-5 and 8-12 are the clone specific PCR experiments, columns 6 + 7 the ALBUMIN control.

KEY: each well contains 500 ng of DNA except NTC which contain TE

L10 = leukemic dilution series C10 = normal DNA dilution series Test = test samples

NAC = normal mononuclear control DNA NTC = no template control

Adapted with permission of Dr J. Hancock, University of Bristol Figure 16.4

Clone specific TaqMan® MRD plate set-up for 2 markers.

8. Curves that do not fit these criteria are not acceptable, as they will result in inaccurate MRD quantitation

9. DNA concentration (and amplifiability) of the test samples should be determined from the control gene results. If the test sample DNA is within the range 400-600 ng, the MRD result should not be adjusted; however values between 250-400 ng should be adjusted. If test samples give a value significantly lower than the supposed quantity of DNA put into the control PCR, inhibition is the likely cause. Inhibition may be overcome by using BSA in the PCR master mix (Moppett et al., 2003) (a 2% w/v solution is prepared, filter sterilized through a 0.2 ^m filter and 5 ^l is used in each 25 ^l master mix)

10. Quantitative results should be reported: if the replicates are with 1.5 Ct and within the reproducible range of the assay; if at 2/3 wells have a reproducible Ct below the maximal sensitivity of the assay. If both of these conditions are satisfied the results calculated from the standard curve should be reported

11. Positive but unquantifiable results are reported if; there are non-reproducible Ct values outside reproducible range but outside range of NAC; if at least 2/3 wells have a reproducible Ct above the maximal sensitivity but outside the range of NAC. Report the data as <10-X (where X = lower limit of reproducible range)

12. Report as not detected: if Ct values are 50; if Ct values are within the range of NAC

Monitoring post-stem cell transplantation chimerism

The monitoring of patients who have undergone allogeneic stem cell transplantation has relied on semi-quantitative methods, such as short tandem repeat (STR) PCR with fluorescent primers or Southern blotting. Real-time PCR based on the detection of single nucleotide polymorphisms has been developed in some centers and is under evaluation, however encouraging results are emerging and sensitivities of 0.1-0.01% may be possible (Maas et al., 2003).

Use of Y chromosome sequences

Male patients with female donors can be monitored using Y chromosome specific assays (Fehse et al., 2001), these are highly sensitive (0.001%) and there may always be a threshold of positive cells, therefore serial analysis is more useful, with any increase in the level of male cells being regarded as suspicious. Investigation of mixed chimerism should always be done in tandem with MRD analysis where suitable disease markers are available. Our laboratory uses an in-house assay based on detection of the TSPY repeats on Y chromosome. The use of a reiterated target makes this assay very sensitive; due to the variation in number of TSPY repeats in individual males, the AACt method is used. Although this is not an expression assay, the values obtained are sufficiently accurate to reflect fluctuation in the level of male cells against a female background. The control gene used is HBB, but other genomic sequences may be used. Pre-trans-plant DNA or buccal swab DNA from the recipient is used as the calibrator sample.

Protocol 16.2: Use of TSPY repeats on Y-chromosome in chimerism studies

REAGENTS AND CONSUMABLES_

Optical Adhesive Cover (Applied Biosystems) 96-well real-time PCR plate (Applied Biosystems) 1.5 mL amber tubes (Axygen) Aerosol-resistant pipette tips (Axygen) Universal TaqMan® Master Mix (Applied Biosystems) Molecular Biology Grade Sterile H2O

Oligonucleotide Primers (10 ^M in molecular biology grade H2O) TaqMan® probes (5 ^M in molecular biology grade H2O, in amber tubes)

Primers and probes for TSPY PCR

TSPY 1139-F TSPY 1204-R TSPY 1162-T TaqMan® Probe Beta globin 509-F Beta globin 587-R Beta globin 541-T TaqMan® Probe TAMRA

5' TGT CCT GCA TGT TGG CAG AGA 5' TCA AAA AGA TGC CCC AAA CG

5' JOE-CCT TGG TGA TGC CGA GCC GC-3'TAMRA 5' CTG GCT CAC CTG GAC AAC CT 5' CAG GAT CCA CGT GCA GCT T

5' FAM-TGC CAC ACT GAG TGA GCT GCA CTG TG-3'

EQUIPMENT_

7500 real-time PCR instrument (Applied Biosystems) Genespec 1 micro spectrophotometer (Hitachi) Centrifuge with microplate adapters Micropipettes

METHOD_

1. Purified DNA is the preferred template material; however this assay can be performed using white blood cells isolated by red cell lysis techniques or ficoll gradient separation

2. Prepare master mixes for TSPY and HBB (or preferred control gene). Set up triplicate reactions for both. Remember that each male patient requires his own individual calibrator specimen. If possible, include a known female DNA isolated at the same time as the test specimens (NAC) and NTC

Constituent

Vol./reaction TSPY

Vol./reaction HBB

TaqMan® Universal Master Mix

12.5 pL

12.5 pL

H2O

3.5 pl

5.0 pl

Forward primer (10 pM)

0.75 pl

0.75 pl

Reverse primer (10 pM)

2.25 pl

0.75 pl

Probe (5 pM)

1.0 pl

1.0 pl

DNA (100 ng/pL)

5 pL

5 pL

3. Dispense 20 pL of mix to appropriate wells of a 96-well plate (use adjacent sets of columns)

4. Add 5 pL of the test specimens, NAC and NTC controls, before adding the pre-transplant calibrator specimens

5. Seal the plate carefully with an optical adhesive cover

6. Spin briefly

7. Set up the real-time instrument as per manufacturer's instructions. Use the default Applied Biosystems thermal cycling protocol of 95°C for 10 min followed by 50 cycles of 15 sec at 95°C and 60 sec at 60°C

8. Save the data and export the raw Ct data to Microsoft Excel

9. Open the data in Excel. Sort by specimen and detector and compare the replicate values. Discard outliers and calculate the mean Ctfor each set of triplicates

10. Calculate the ACt for each specimen thus: ACt = Mean Ct (TSPY) - Mean Ct (HBB)

11. Calculate the AACt for each test specimen thus:

AACt = ACt Test - ACt Calibrator

12. Calculate the relative TSPY copy number using the formula 2-AACt

13. The sensitivity of this assay is such that low level TSPY sequences will be detected post stem cell transplant (assuming sufficient DNA is analyzed) in patients with complete donor chimerism. This is probably due to contamination with male cells during sampling.

Use of TSPY to monitor chimerism by real-time PCR

Use of TSPY to monitor chimerism by real-time PCR

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Weeks post-transplant

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Weeks post-transplant

Figure 16.5

Use of TSPY to monitor chimerism by real-time PCR.

However, it is more important to establish a threshold for each patient. Increasing levels should be investigated where possible with MRD specific assays, the underlying disease should be considered; acute leukemia patients may have at least a tenfold higher level in bone marrow compared to peripheral blood if the TSPY level is an indicator of MRD

Serial analysis of peripheral blood from a young male patient transplanted for high risk ALL is shown in Figure 16.5; he became MRD positive at fifteen weeks post-transplant and relapsed clinically at week 48.

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