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Quality Assessment of Genetic Markers Used for Therapy Stratification

I.M. Ambros, J. Benard, M. Boavida, N. Bown, H. Caron, V. Combaret, J. Couturier, C. Darnfors, O. Delattre, J. Freeman-Edward, C. Gambini, N. Gross, C.M. Hattinger, A. Luegmayr, J. Lunec, T. Martinsson, K. Mazzocco, S. Navarro, R. Noguera, S. O’Neill, U. Pötschger, S. Rumpler, F. Speleman, G.P. Tonini, A. Valent, N. Van Roy, G. Amann, B. De Bernardi, P. Kogner, R. Ladenstein, J. Michon, A.D.J. Pearson, P.F. Ambros

From the Children’s Cancer Research Institute, St Anna Kinderspital, and Department of Pathology, University of Vienna, Vienna, Austria; Unité des Marqueurs Génétiques des Cancers, Institut Gustave Roussy, Villejuif; Unité d’Oncologie Moléculaire, Centre Léon-Bérard, Lyon; Service de Génétique and Département de Pédiatrie, Section Médicale; and Institut Nationale de la Santé et de la Recherche Médicale U509 Pathologie Moléculaire des Cancers, Section de Recherche, Institut Curie, Paris, France; Centro de Genética Humana, Instituto Nacional de Saude, Lisboa, Portugal; Division of Human Genetics and Cancer Research Unit, University School, University of Newcastle upon Tyne; Sir James Spence Institute of Child Health, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom; Institute of Human Genetics, University of Amsterdam, Academic Medical Centre, Amsterdam, the Netherlands; Onco-Hematology Laboratory, Pediatrics Department, University Hospital Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; Department of Clinical Genetics, Sahlgrenska University Hospital East, Gothenburg; Woman and Child Health, Childhood Cancer Research Unit, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden; Laboratory of Tumor Genetics, National Institute for Cancer Research; Hematology-Oncology Department and Laboratory of Anatomic Pathology, Giannina Gaslini Children’s Hospital, Genoa, Italy; Department of Pathology, University of Valencia, Valencia, Spain; and Center for Medical Genetics-OK5, University of Ghent, Ghent, Belgium.

Address reprint requests to Inge M. Ambros, MD, and Peter F. Ambros, PhD, Children’s Cancer Research Institute, Kinderspitalgasse 6, A-1090 Vienna, Austria; email: ambros{at}ccri.univie.ac.at.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group.

Materials and Methods: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated.

Results: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable.

Conclusion: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
FUTURE CANCER therapies will be increasingly based on individually tailoring therapeutic tools to the malignant potential of the individual tumors and, with it, to their genetic/biologic make-up. This development implies a marked responsibility for biologists and geneticists. Every mistake may lead to wrong therapeutic decisions, with potentially fatal consequences for the patient. This fact will necessitate quality control, standardization of techniques, and a common terminology wherever genetic/biologic markers will be used for therapy stratification.

In the case of neuroblastic tumors (neuroblastoma, ganglioneuroblastoma intermixed, ganglioneuroma, and ganglioneuroblastoma nodular),^1,2 new therapeutic approaches were initiated in recent years in Europe (Localized Neuroblastoma European Study Group, Unresectable Neuroblastoma Study, European Infant Neuroblastoma Study, and High-Risk Neuroblastoma Study 1 of SIOP Europe). The clinical behavior of neuroblastic tumors, which occur in infancy and childhood, ranges from spontaneous regression and maturation to extreme aggressiveness. Therefore, some patients require a high number of currently established therapeutic modalities, whereas others can be cured by surgery alone, even if the tumor resection is incomplete.^3,4 Relapses, both localized and metastatic, can, however, also develop from localized disease, including totally resected stage I tumors.⁵ Also, the genetic composition of the tumors is diverse.^5–9 Amplification of the oncogene MYCN was one of the first known markers indicating aggressiveness irrespective of the clinical stage.^10–12 The effect of other genetic aberrations, such as the deletion at the short arm of chromosome 1, del(1)(p36.3), have been discussed more controversially in the past. ^5,6,13–20 The same holds true for the biologic significance of the DNA content of the tumor cells.^17,21–23

In the Localized Neuroblastoma European Study Group trial, the therapy stratification involved stage 2A and 2B patients (according to the International Neuroblastoma Staging System)²⁴ who did not receive cytotoxic agents after surgery if no MYCN amplification was detected in the tumor.²⁵ Other parameters, such as chromosome 1p status, tumor cell ploidy, and histologic features,^1,2,26,27 were evaluated prospectively. To guarantee a high quality and reliability of the results in all involved laboratories, the European Neuroblastoma Quality Assessment (ENQUA) Group, consisting of participants from nine European countries (11 laboratories), was launched and a quality-control study was initiated to assess the standard and the difficulties in individual laboratories and as an attempt to survey and control the quality of molecular-genetic results used for therapy stratification and prospective evaluation (MYCN status, chromosome 1p36.3, and ploidy analyses). The work resulted in recommendations for the processing of tumor material, the methods and DNA probes to be used, and the interpretation of DNA blots and fluorescent in situ hybridization (FISH) results. Moreover, specified definitions for MYCN results, chromosome 1p aberrations, and loss of heterozygosity (LOH) were formulated.²⁸

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In total, 137 tumor specimens from 17 neuroblastic tumor pieces derived from four European countries (Austria, Italy, Portugal, and Sweden) were investigated. Ten laboratories received 17 specimens each, and one participant analyzed specimens from all 17 tumors. Thirteen tumor pieces had Schwann cell stroma–poor areas, and four pieces had Schwann cell stroma–rich areas (according to Shimada et al²). No information about tumor cell content or histology was given. Each laboratory received 12 frozen tumor specimens for MYCN and chromosome 1p analyses and determination of the tumor cell content. Each series contained at least one MYCN-amplified tumor, one tumor with a chromosome 1p aberration, and one Schwann cell stroma–rich specimen. Back-up touch slides were made from all specimens and stored at -70°C for re-evaluation. The participants were asked to process the samples using the same methods as they routinely do and to report the results within 2 weeks.

MYCN Analysis
Six of 11 participants investigated the MYCN status using FISH. Four participants also used Southern blot (SB) with the probe pNb1 (M. Schwab, Heidelberg, Germany). Further single-technique approaches included SB (one participant) and polymerase chain reaction (PCR) according to the standard protocols (three participants). For FISH investigations, the probes D2Z, specific to the centromere of chromosome 2, and P5115 (both Qbiogene, Heidelberg, Germany) or LSI N-myc SpectrumOrange (Vysis, Downers Grove, IL), respectively, were applied. Data from one laboratory were not entered in the database.

Chromosome 1p Analysis
Four participants analyzed the chromosome 1p status by FISH, four others used PCR, and one laboratory used SB. In two laboratories, two methods (PCR and FISH) were used. The DNA probes used for FISH were PUC 1.77, specific to the centromeric (heterochromatic) region of chromosome 1 combined with D1Z2 binding to the chromosomal region 1p36.33. For PCR, the primers for the following loci were applied (according to frequency): D1S80, D1S76, CMM12.1, MCT118, D1S554, D1S468, D1S244, D1S260, D1S160, D1S214, D1S60, D1S200, and D1S243. The DNA probe used for SB was CEB15.

DNA Measurement
Tumor cell ploidy was determined by flow cytometric measurement (FCM) in two laboratories and by image cytometric measurement (ICM) in two others. The DNA content was indicated by absolute numbers.

Divergent Findings
The terms discrepancy or divergence were used for results that deviated from each other (ie, MYCN-amplified v nonamplified; chromosome 1p36.3 deletion/imbalance v intact 1p36.3 regions; near-triploid v near-diploid DNA content). This was done without drawing any conclusion as to the results’ correctness. For MYCN, varying degrees of amplification or gain were not judged as discrepancies, and DNA content results differing less than 0.2 from each other were not assessed as divergent. All cases with divergent findings were re-evaluated by investigating the back-up touch slides of the tumor pieces by FISH or another method or by the exchange of tumor material between two laboratories performing the same method. After reviewing the respective cases, conclusions were reached about the status of the genetic markers in the individual tumors and about the correctness of the results indicated.

Intertechnique Reliability
The intertechnique reliability was tested using the Kappa test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Three hundred fifty-two investigations were performed to determine the MYCN status, chromosome 1p36.3 integrity, and tumor cell ploidy (Tables 1 through 3). Three hundred fourteen investigations were assessable (89.2%); discrepancies were registered for 23 results (7.3%; Table 4). Tumor cell contents as indicated by the participants are listed in Table 5. After check-up and review of the divergent findings, 17 results (5.4%) were judged as errors (Tables 1 through 4). Terminology and definitions of the molecular-(cyto)genetic results are listed in Table 6.

MYCN Analyses
One hundred seventy-one MYCN investigations using SB, PCR, and FISH were performed; 160 were assessable. According to the consensus data (Table 1), 13 tumors were judged as having no amplification, two cases were judged as exhibiting a high MYCN amplification, and two others, assessed by FISH, were judged as showing a focal MYCN gain/amplification. All together, six false-negative results (two for each of the three techniques) and two false-positive results (one PCR and one FISH) were encountered (Tables 1 and 4). The intertechnique reliability was highest for SB and FISH (weighted Kappa, 0.914) and relatively low for PCR and FISH and PCR and SB (weighted Kappa, 0.569 and 0.536, respectively).

False-negative results were registered for cases 5 and 9. Although the percentage of cells with MYCN gain/amplification (definitions, Table 6) differed in the individual samples, the check-up of the back-up tumor pieces by FISH showed the presence of such cells in all pieces. Case 5 brought about the majority of divergent results (five of eight). In two laboratories, the concerned population, which was approximately 70% of tumor cells with a two- to three-fold MYCN gain in the analyzed specimens, was detected neither by SB nor by FISH. A third divergent result concerning this case was obtained by PCR. In case 9, one divergent result was observed. The sample in question contained 90% of tumor cells, 15% of which, as shown by FISH, showed a gene amplification, 75% of which showed a MYCN gain, and 10% of which displayed only single-copy signals. However, re-evaluation of this sample by PCR in another laboratory also revealed a negative result.

The false-positive results concerned cases 14 and 16. In case 14, one of the tumor samples analyzed by FISH was reported to show a high MYCN amplification in 10% of the tumor cells. This discrepancy was dissolved by using DNA probes specific to the X and Y chromosomes, which revealed a contamination with amplified cells from another patient. In case 16, one PCR result was interpreted as amplification. This result, however, was not reproducible.

Chromosome 1p36.3 Analyses
One hundred thirty-seven chromosome 1p36.3 investigations were conducted; 24 were not assessable. Twelve tumors showed no 1p36.3 aberrations by FISH and no LOH by SB and/or PCR (11 cases); three tumors showed LOH, with two having chromosome 1p imbalance and one having a deletion (Table 2; definitions listed in Table 6). The agreement between PCR and FISH results did not prove satisfactory (weighted Kappa, 0.658).

DNA Content Measurement
FCM was performed by two participants on 20 samples, and ICM was performed by two other participants on 24 samples (Table 3). After comparison of all FCM and ICM data and comparison also with the number of chromosomes 1 and 2 as determined by FISH, eight tumors were classified as near-triploid and seven tumors were classified as diploid (or tetra-, octoploid). One tumor was assessed as unclear, because the result obtained by ICM was unlikely to correspond with the number of chromosomes 1 and 2. FCM resulted in one divergent DNA value, and ICM showed five variant results. In five cases, each ICM result differed from the other ICM and FCM data, ranging from 0.2 to 0.5. One of these divergent results was judged as a discrepancy but not as a false result, because the DNA value indicated was still in the near-diploid range.

Interlaboratory Comparison
Errors were made by seven of 11 participants. The percentage of errors, related to the number of investigations performed in the individual laboratories, ranged between 2% and 18% (one to five errors of seven to 43 assessable results). Three participants made mistakes for more than one parameter analyzed (four and five errors). Four participants made mistakes for one parameter (one error each).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The quality-assessment study clearly revealed that the determination of the tumor cell content is the most important but often neglected prerequisite for the interpretation of the obtained results. It should be performed before using the material for any molecular-genetic/biologic investigation to avoid false-negative results or results that only seem to be correct but are based on the investigation of normal cells (for details, see Ambros and Ambros²⁸). In this study, it was noticed that a number of investigations were performed either without knowing the percentage of tumor cells or without taking it into consideration when interpreting the results. In addition, results that were based on the investigation of normal cells (ie, Schwann cells⁷) were given. In the case of neuroblastic tumors, it is especially the Schwann cell that may represent a source of error if it is not recognized as normal cell. For other tumors, but also for neuroblastic tumors and posttherapy specimens, the presence of, for example, granulation, fibrous and lymphoid tissue in the tumor specimen, or surrounding normal tissue can give rise to false results as well. Another problem is the investigation of the tumor cell content by means of immunologic methods. In this case, it is especially important to use appropriate antibodies.

Only a few multicenter quality-control studies have been published; for example, for metaphase FISH, resulting in 1% of errors²⁹; for the quality of PCR for detection of HIV-1 DNA, showing 14% of false-positive results owing to contamination³⁰; for the quality of genetic testing for cystic fibrosis³¹; and for ploidy measurement.^32–34 However, no published reports on quality assessment of gene amplifications exist, although they are accepted as potent prognostic markers. In this study, we noticed that the gain of a gene or its low or heterogeneous amplification cannot be reliably detected by DNA-extracting methods. Moreover, in two laboratories, gene amplification was detected neither with SB nor with FISH, although the specimens contained amplified cells. This fact may lead to the speculation that negative SB results may influence the interpretation of ambiguous FISH results or vice versa. False-negative results were also observed with PCR. False-positive results were encountered for two methods (ie, for PCR and FISH).

For LOH studies, SB and PCR are considered to be reliable techniques, as long as result interpretation is performed together with the consideration of the tumor cell content. However, if deletions or imbalances are found by FISH in a subpopulation of cells (case 13), SB/PCR results must be judged as inconclusive. With regard to chromosome 1p investigations by FISH, false-positive findings (four results) were the main problem and were attributed to insufficient hybridization. In particular, tumor specimens with either a large amount of normal cells or differentiating tumor cells or both caused difficulties because of the different conditions such cells need for optimum hybridization. Although the advantages of FISH are apparent (eg, its possibility to make analyses on the cellular level and morphologic preservation), a number of problems, such as failure to check the hybridization efficiency, have become evident and accounted for an error rate of 6.5%. Dewald et al,²⁹ who reported on a quality control study for metaphase FISH, attributed most errors to inexperience with the FISH technique.

FCM showed a lower percentage of errors than ICM. The observed deviations were mostly sufficient to misjudge tumor cell ploidy, changing it from triploid to diploid or vice versa. However, most of the ICM errors were made in one laboratory. Therefore, it cannot be concluded that ICM in general is less appropriate for measurement of the DNA content than FCM. D’Hautcourt et al,³³ who tested the quality of FCM, showed that only those laboratories that fulfilled consensus recommendations produced homogeneous results.

The common terminology (Table 6) developed by the ENQUA Group²⁸ was designed for the MYCN and chromosome 1p36.3 results but can be applied for gene amplification and chromosomal deletions/imbalances and LOH studies in general. The most important changes concern the specification of gene amplification and gain with exclusive relation to the chromosome of origin and the interpretation of the loss of chromosomal sequences dependent on the method used, which is reflected in a specified terminology. Advantages and limitations of the different techniques that were used for the determination of the MYCN status and of chromosome 1p36 changes are listed in Table 7.

	ACKNOWLEDGMENTS

 
We thank M. Zavadil and I. Walters for critical reading of the manuscript and H. Tüchler for providing us with valuable and helpful suggestions and comments.

	NOTES

 
Supported by the Directorate-General V, European Comission (Luxembourg, Belgium, no. SOC 98 201284 05F02), the Austrian Federal Ministry of Education, Science and Culture (Vienna, GZ 70.041/2-Pr/4/99), the Forschungsintitut für krebskranke Kinder im St Anna Kinderspital (Vienna, Austria), and the Associazione Italiana per la Lotta al Neuroblastoma (Genoa, Italy).

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Submitted March 5, 2001; accepted March 18, 2003.

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