Arne Kristian Schwarz

Martin Stanulla

Gunnar Cario

Thomas Flohr

Rosemary Sutton

Anja Möricke

Philippe Anker

Maurice Stroun

Karl Welte

Claus R Bartram

Martin Schrappe

André Schrauder
email: a.schrauder@pediatrics.uni-kiel.de

: A Möricke

: M Schrappe

: M Stroun

Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute lymphoblastic leukemia

Keywords: Acute lymphoblastic leukemia, Childhood, Minimal residual disease, Ig/TCR gene rearrangements, Plasma DNA

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Over the past 40 years, a number of studies have been published on the presence of free circulating nucleic acids in the blood plasma of humans. In this context, various malignancies have been associated with increased concentrations of free plasma DNA as compared to levels in healthy controls [START_REF] Leon | Free DNA in the serum of cancer patients and the effect of therapy[END_REF][START_REF] Shapiro | Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease[END_REF][START_REF] Stroun | Isolation and characterization of DNA from the plasma of cancer patients[END_REF]. It has therefore been proposed that analyses of free plasma DNA could be a useful tool for prognosis or the early diagnosis to detect subclinical disease recurrence [START_REF] Anker | Tumor-related alterations in circulating DNA, potential for diagnosis, prognosis and detection of minimal residual disease[END_REF]. Since the introduction of PCR-based techniques in the late 1980s, a large number of tumorspecific alterations have been identified in free plasma DNA, such as K-ras mutations in patients with colorectal cancer [START_REF] Anker | K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer[END_REF], microsatellite alterations in patients with smallcell lung cancer [START_REF] Chen | Microsatellite alterations in plasma DNA of small cell lung cancer patients[END_REF], head and neck cancer [START_REF] Nawroz | Microsatellite alterations in serum DNA of head and neck cancer patients[END_REF], clear cell renal carcinoma [START_REF] Goessl | Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma[END_REF] and melanoma [START_REF] Fujiwara | Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients[END_REF], and finally aberrant promotor hypermethylation of tumor suppressor genes in patients with non-small-cell lung cancer [START_REF] Esteller | Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients[END_REF], liver [START_REF] Wong | Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients[END_REF], and breast cancer [START_REF] Silva | Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients[END_REF]. Whereas the majority of these studies examined patients with solid tumors, it has also been suggested that free plasma DNA can be used for the detection of molecular abnormalities in hematological malignancies [START_REF] Vasioukhin | Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia[END_REF][START_REF] Frickhofen | Rearranged Ig heavy chain DNA is detectable in cell-free blood samples of patients with B-cell neoplasia[END_REF][START_REF] Rogers | Relative increase in leukemia-specific DNA in peripheral blood plasma from patients with acute myeloid leukemia and myelodysplasia[END_REF].

In acute lymphoblastic leukemia (ALL), the detection of minimal residual disease (MRD) using leukemic clonespecific immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements as PCR targets provides important prognostic information on the in vivo effectiveness of early treatment [START_REF] Stanulla | Treatment of childhood acute lymphoblastic leukemia[END_REF][START_REF] Cario | Distinct gene expression profiles determine molecular treatment response in childhood acute lymphoblastic leukemia[END_REF][START_REF] Cave | Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer-Childhood Leukemia Cooperative Group[END_REF][START_REF] Van Dongen | Prognostic value of minimal residual disease in childhood acute lymphoblastic leukemia: a prospective study of the International BFM Study Group[END_REF][START_REF] Flohr | Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia[END_REF]. Up to now, most large-scale MRD studies have been performed using bone marrow (BM) aspirates. Due to a lack of study, there is no information available on both the DNA concentration in the plasma of healthy children as well as the abundance of tumorspecific plasma DNA in children with hematological malignancies (e.g., ALL) or solid tumors. As such information could potentially provide a basis for the use of plasma DNA for MRD measurements in children with malignant neoplastic diseases, we aimed to establish reference ranges for the total concentration of free plasma DNA in children and to evaluate the feasibility of MRD measurement in the early course of childhood ALL by means of plasma DNA.

Materials and methods

Patients

Peripheral blood (PB) samples were obtained from 21 children with ALL at diagnosis and during the 12-week induction therapy, 13 children with other malignancies during therapy and 12 healthy children (Table 1). All samples were collected under protocols approved by the Institutional Review Board of the Medizinische Hochschule Hannover and after informed consent was given by the parents and/or patients.

Sample processing

As previous studies have demonstrated that EDTA prevents DNA degradation [START_REF] Frickhofen | Rearranged Ig heavy chain DNA is detectable in cell-free blood samples of patients with B-cell neoplasia[END_REF] and that coagulation-associated factors may influence the concentration of cell-free DNA in serum [START_REF] Lee | Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma[END_REF], we systematically used EDTA tubes to collect PB samples and exclusively worked with PB plasma.

To determine the possible influence of the storage period on the total concentration of DNA being measured in the plasma, we performed pilot experiments in which the plasma was separated from blood cells at 6, 12, 24, 36, and 48 h after collection. The plasma DNA concentrations were stable for up to 24 h of storage, but increased after longer time intervals. These results were in accordance with previous experiments performed by Anker et al. [22]. As a consequence, all plasma samples analyzed in the present study were separated from PB within 24 h by centrifugation (2×10 min at 3,000×g). Leukocytes were isolated by Ficoll-Paque™ Plus density centrifugation (Amersham Biosciences, Piscataway, NJ). The obtained plasma samples Since the concentration of DNA in plasma was very low compared to the 500 ng used in all routine bone marrow analyses, all markers were re-evaluated for their effectiveness in detecting MRD at the low levels of DNA found in plasma. Each patient's BM mononuclear cell DNA from day 0 (150 pg/μl; diluted with AE buffer (Qiagen, Hilden, Germany) was used to prepare a tenfold dilution series in DNA isolated from the buffy coat fraction from normal individuals. The dilution series was used to test all the RQ-PCR assays for markers available for each patient with hybridization temperatures adapted according to the primerannealing temperatures. The assays used LightCycler™ FastStart DNA Master SYBR Green I on LightCycler™ equipment and all included two negative controls (H 2 O) and three buffy coat samples for defining the non-specific background signals.

We established criteria to distinguish between an informative and a non-informative marker, with the melting curve analysis being the determining factor. A marker that showed a clear distinction between buffy coat and negative control on the one hand and the patient-specific DNA (BM, day 0) on the other hand, was assessed as informative (Fig. 1).

MRD assessment in plasma and peripheral leukocytes MRD detection in PB leukocytes was conducted as published by others [START_REF] Nakao | Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by LightCycler technology[END_REF][START_REF] Van Der Velden | Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects[END_REF][START_REF] Van Der Velden | Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data[END_REF][START_REF] Van Der Velden | Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting[END_REF]. Briefly, MRD PCR targets were tested for specificity and sensitivity to enable on-time monitoring of remission samples. RQ-PCR analysis was performed either by using single PCR amplification with sequence-specific TaqMan hydrolysis probes or by a two-round nested RQ-PCR including SYBR Green I detection. The nested RQ-PCR used a first-round consensus touchdown PCR (starting at 66°C) with a decreasing annealing temperature profile (-0.5°C per cycle) over 28 cycles and 12 additional cycles at 52°C, followed by the second-round RQ-PCR with a patientspecific ASO primer and an inner consensus primer. Tenfold serial dilutions of the diagnostic leukemic DNA sample were made in normal mononuclear cell DNA (pooled DNAs of six healthy donors) and used for RQ-PCR amplification in duplicate to generate a standard curve and to determine the sensitivity of the clonespecific Ig/TCR target. After receiving the follow-up samples, a freshly prepared dilution series was used to quantify the tumor load in triplicate in the patient's remission samples.

Since the assays for MRD detection in plasma were performed at very low concentrations of DNA (about 1 ng DNA per PCR), we were not able to perform quantitative measurements and defined positivity or negativity on the basis of melting curve analysis. Each sample was analyzed in duplicate and the results for the sample assessed qualitatively and reported as double negative, single, or double positive. Thus, the specificity of MRD assays on plasma DNA was based on the combination of allelespecific oligonucleotides (ASO) and target-specific melting curve analysis. 1A) should be disregarded for further analysis of this specific MRD-target as it is an unspecific amplification, shown by the melting curve analysis (Figure 1B)

Results

Median plasma DNA concentration

The concentration of total free plasma DNA was determined in three sets of samples (I, II, and III). Set I included 150 PB plasma samples from 21 children with ALL at diagnosis and under treatment; Set II included 20 PB plasma samples from 13 children with other malignancies receiving treatment and Set III were 12 PB plasma samples from 12 healthy children (Table 1). The results revealed a wide spectrum of DNA concentrations, ranging from 2.85 to 12,260 ng/ml. Interestingly, the median concentrations between the three groups were highly comparable (I: 71 ng/ml, II: 76 ng/ml, and III: 57 ng/ml). When the ALL group was analyzed according to treatment day, it became apparent that initially, plasma DNA was elevated and upon treatment initiation rapidly returned to normal levels (Fig. 2a). Plasma DNA concentrations on days 0 and 3 were significantly higher in comparison to levels thereafter (P Mann-Whitney Test=0.001). This decrease paralleled the decline in PB leukocyte numbers (Fig. 2b) and we observed a previously unreported correlation between the concentration of plasma DNA from children with ALL and the number of PB leukocytes (Spearman coefficient of correlation=0.21, P=.001; data not shown). Two smaller peaks in DNA concentration were observed on day 33 and week 12.

Detection of MRD

MRD measurements were performed whenever less than or equal to five serial plasma samples were available which was the case for 15 of the 21 patients with ALL. Characteristics of these 21 patients are shown in Table 2 kinetics could be described for all of the 15 patients. Figure 3a shows a steady decrease in the percentage of plasma MRD-positive patients during early therapy with samples for the majority of patients becoming MRD negative by day 22. All 15 patients were below the detection limits of MRD after day 52. The earliest MRD negative results were observed at day 8 in two out of ten patients tested. When we compared the MRD measurements in plasma and leukocytes, we discovered a high concordance (86.7%) between the results. In 32 pairs, MRD was detected in both PB leukocytes and plasma, in eight pairs, MRD was solely detected in PB leukocytes, in two pairs, only the plasma sample was MRD-positive, and in 33 pairs, MRD was negative both in plasma and PB leukocytes. Similar to our observations in plasma analysis, there was a change from predominantly positive MRD signals to predominantly negative findings between day 21 and 22 in the leukocytes.

Discussion

The present study is the first approach to quantify the concentration of total plasma DNA in healthy children and children with malignant diseases by RQ-PCR. It is widely known that a substantial, if not predominant (depending on the course of therapy) portion of plasma DNA in cancer patients derives from tumor cells [START_REF] Stroun | The origin and mechanism of circulating DNA[END_REF]. Higher plasma concentrations have been described in patients with large tumors or at an advanced stage of the disease [START_REF] Shapiro | Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease[END_REF][START_REF] Stroun | Isolation and characterization of DNA from the plasma of cancer patients[END_REF][START_REF] Fujiwara | Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients[END_REF][START_REF] Stroun | Neoplastic characteristics of the DNA found in the plasma of cancer patients[END_REF][START_REF] Fournie | Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours[END_REF] and it has been postulated that circulating plasma DNA in these disease states originates largely from tumor lysis/ [START_REF] Li | Cellfree DNA is released from tumor cells upon cell death: a study of tissue cultures of tumor cell lines[END_REF].

Our results reveal that DNA is abundant at high levels in the plasma of children with ALL at initial diagnosis and decreased rapidly in the first few days after the initiation of therapy to reach a level that is comparable to that of either children with other malignancies or to healthy children. Interestingly, small increases in plasma DNA concentrations were also observed during therapy intermission periods (day 33 and week 12) when active bone marrow regeneration occurs (Fig. 2b).

We were able to demonstrate higher plasma DNA levels in patients newly diagnosed with ALL. Since the amount of total PB plasma DNA in all analyzed patients decreased rapidly and reached levels comparable to those observed in healthy subjects by the fourth treatment day, the method does not seem to be broadly applicable for prognostic purposes in childhood ALL. Thus, we explored instead the applicability of MRD techniques in plasma. For this purpose, we successfully adapted the techniques used for MRD studies in bone marrow for the use with plasma DNA. The observed high initial concentration of both total DNA as well as leukemia-specific DNA in plasma before the induction of chemotherapy was predictable. It can most likely be explained by the higher turnover rate of leukemic cells in comparison to normal cells, which, combined with incomplete processing and degradation of cellular contents would lead to higher abundance of DNA in plasma [START_REF] Rogers | Relative increase in leukemia-specific DNA in peripheral blood plasma from patients with acute myeloid leukemia and myelodysplasia[END_REF]. In this context, Frickhofen et al. speculated that, normally, there is a steady state between DNA release from cells and degradation by plasma nucleases [START_REF] Frickhofen | Rearranged Ig heavy chain DNA is detectable in cell-free blood samples of patients with B-cell neoplasia[END_REF]. On treatment day 3, after significant tumor lysis, the levels of both total DNA and MRD are still very high in plasma so that we assume that the plasma nucleases are not able to cope with the amount of circulating DNA being released from leukemic cells in these first days. Subsequently, plasma concentrations were restored rapidly which is consistent with the view that the interference with DNA replication may shift the balance towards degradation of DNA. It is interesting that the rapid decline in number of leukocytes did not lead to a transient increase in the concentration of circulating plasma DNA (Fig. 2b) suggesting mechanisms other than tumor lysis may play a role here. Unfortunately, we can not make any interferences on the plasma DNA levels of the children with other malignancies (group II) at diagnosis as in this group we only had access to plasma samples under treatment.

Nevertheless, the origin of the baseline level of plasma DNA which is also present in normal individuals remains unanswered. One hypothesis is that DNA is spontaneously and actively released from intact cells, given the evidence that there are marked differences between released DNA and cellular DNA which suggests that the extracellular DNA is not simply derived from dying cells [START_REF] Stroun | The origin and mechanism of circulating DNA[END_REF][START_REF] Jachertz | Information carried by the DNA released by antigen-stimulated lymphocytes[END_REF][START_REF] Stroun | Alu repeat sequences are present in increased proportions compared to a unique gene in plasma/serum DNA: evidence for a preferential release from viable cells?[END_REF]. This theory is supported by the correlation between the concentration of plasma DNA from children with ALL and the number of leukocytes shown in our study.

In summary, we were able to quantify plasma DNA in children using RQ-PCR and demonstrate that it is feasible to use plasma for the evaluation of DNA-based MRD kinetics during the early course of childhood ALL treatment. However, we were neither able to demonstrate a superior role of plasma DNA-based MRD analysis in comparison to conventional MRD analyses relying on DNA from BM or PB cells nor were we able to prove an added clinical value of this method in association with the already-established MRD techniques [START_REF] Coustan-Smith | Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia[END_REF][START_REF] Van Der Velden | Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL[END_REF].

Fig. 1

 1 Fig.1Allele-specific RQ-PCR for testing patients markers. a Detection of amplification products by SYBR Green. V0-V3 dilution series with day 0 BM, H 2 O: negative control. bc buffy coat to define non-specific background signals (slope -2.47; r=-0.98). b Melting curve analysis. Showing a specific melting point for V0, V1, and V2 at 88°C, identifying the marker as informative. The amplification curve of V3 which arises between V1 and V2 (Figure1A) should be disregarded for further analysis of this specific MRD-target as it is an unspecific amplification, shown by the melting curve analysis (Figure1B)

Fig. 3

 3 Fig. 3 Detection of MRD in childhood ALL. a Synopsis of MRD in plasma over the course of therapy showing a steady decrease of percentage of MRD. We performed MRD measurements when greater than or equal to five serial plasma samples had been obtained. b Comparison of MRD in PB plasma vs. MRD in peripheral leukocytes as detected in 75 paired samples indicating a high concordance of 86.7%

Table 1

 1 Characteristics of individuals included in plasma DNA analysis the respective BIOMED-2 multiplex PCR primer sets. PCR products obtained from Ig and TCR gene rearrangements were further examined by heteroduplex or gene scanning analysis to discriminate between amplifications derived from monoclonal or polyclonal lymphoid cell populations. Biclonal or biallelic PCR products were separated either by cutting out amplicons from the polyacrylamide gel or by DNA cloning. Junctional regions of monoclonal PCR products were sequenced. Thereafter, patient-specific junctional region sequences of potential MRD PCR targets were identified using the IMGT (http://www.imgt.cines.fr), Vbase (http://www.vbase.mrc-cpe.cam.ac.uk), or IgBlast (www.ncbi.nlm.nih.gov/igblast/) databases and commonly distributed hardcopy sequences. At least one allelespecific oligonucleotide (ASO) primer was designed complementary to the junctional region sequence of each target either manually or using the Primer Express software (Applied Biosystems, Darmstadt, Germany).

		No. of patients	No. of samples	Gender		Age, year (range)
				Male	Female
	ALL	21	150	8	13	5 (2-16)
	Other malignancies
	AML	2	2	1	1	13
	Anaplastic astrocytoma 1	1	1		1	11
	Hepatoblastoma	1	2	1		7
	Medulloblastoma	1	1	1		8
	Nephroblastoma	3	5	3		9 (6-12)
	Neuroblastoma	1	1	1		11
	NHL	3	4	1	1	9 (6-12)
	Rhabdomyosarcoma	1	3	1		8
	Total	13	20	10	3	9 (6-13)
	Healthy controls	12	12	10	2	8.5 (1-17)
	AML Acute myelogenous leukemia, NHL non-Hodgkin lymphoma

Identification and sequencing of patient-specific gene rearrangements was carried out as part of routine MRD analysis for the clinical trial ALL-Berlin-Frankfurt-Münster 2000

(BFM 2000)

, at the Institute of Human Genetics, University of Heidelberg, Germany. Briefly, genomic DNA from ALL samples at diagnosis was screened by PCR amplification using the BIOMED-1 primer sets for immunoglobulin kappa, IGK, deleting element (Kde) gene rearrangements (V-Kde, intron-Kde), complete and incomplete TCR delta, TCRD (V-(D)-J1, D2-J1, V2-D3, D2-D3), and TCR gamma, TCRG (V-J1.3/2.3, V-J1.1/2.1) rearrangements. Complete and incomplete IGH rearrangements (V H -(D H )-J H , D H -J H ) were identified using five V H and six D H family primers in combination with one J H consensus primer. As an extra PCR target for T-ALL patients, primer sets to detect the SIL-TAL1/2 deletion were included in the PCR screening protocol utilizing breakpoint-spanning SIL gene forward and TAL gene reverse oligonucleotides. If no DNA rearrangements were identified for IGH, IGK-Kde, TCRG, TCRD, and SIL-TAL deletion, in a subset of patients alternative targets were added such as complete and incomplete TCRB (V-(D)-J, D-J), IGK (V-J), and IGL (V-J) using

  . MRD was consistently detected until day 7 and MRD

	Fig. 2 Plasma DNA concentra-	a		100000
	tion in children with ALL, other malignancies and healthy con-trols. a Synopsis of the plasma DNA concentration in group I, patients with childhood ALL at day 0, day 3, and ≥day 4 of treatment; group II, patients with other malignancies (non-ALL); and group III, healthy controls (controls). Each box represents the values between the 1st and 3rd quartile. The bar indicates		Log10 [ng/ml] DNA concentration	100 1000 10000 10		277.00	247.60	62.13		76.34	56.55
	the median DNA concentration
	with the absolute value being
	given next to each box. b The			1
	median plasma DNA concentra-				ALL, day 0	ALL, day 3	ALL, > day 4	Non-ALL	Control
	tion and median number of leukocytes in children with ALL	b	300									14000
	over the course of therapy,
	reflecting a correlation between the plasma concentration and the number of leukocytes. For the correlation between median plasma DNA concentration and time points during ALL therapy, we only analyzed time points where greater than or equal to five samples had been collected	DNA concentration [ng/ml]	100 150 200 250									12000 4000 6000 8000 10000	number of leukocytes/µl
			50									2000
			0									0
				day 0	day 3	day 4	day 5	day 8	day 15	day 21	day 22	day 33	day 52	day 64 week
				(n=11)	(n=10)	(n=6)	(n=9)	(n=10)	(n=12)	(n=8)		(n=7)	(n=10)	(n=7)	(n=6) 12
												(n=8)
									time points
						median DNA concentration	median number of leukocytes

Table 2

 2 Characteristics of patients with acute lymphoblastic leukemia included in the present study and evaluated for plasma DNA (all patients) and plasma DNA MRD monitoring (patients #7 to #15) Male, f female, good less than 1,000 leukemic blood blasts on treatment day 8, poor more than 1,000 leukemic blood blasts, SR negative at days 33 and 78, HR still 10 -3 or more at day 78, MR all others, TEL/AML1 fusion transcript positivity, none of the patients was positive for the BCR/ ABL fusion transcript or an MLL aberration necrosis and apoptosis. In 2001, Jahr et al. described a significant increase of plasma DNA after induction of apoptosis and necrosis in both, a cell culture and a murine model system, with the increase being more impressive in the necrosis model[START_REF] Jahr | DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells[END_REF]. Similar findings were reported by[START_REF] Li | Cellfree DNA is released from tumor cells upon cell death: a study of tissue cultures of tumor cell lines[END_REF] in tumor cell lines and cell tissue cultures

	Patients Age at diagnosis	Gender WBC at	Immuno-	TEL/	Prednisone	MRD risk	Number of MRD	MRD		Relapse
	(years)		diagnosis	phenotype	AML1	response	group	markers
										Day	Day
										33	78
	ALL patients
	#1	14.9	m	31,500 Common	Neg	Good	MR	4	10 -2 Neg Bone
												marrow
	#2	15.7	m	2,500 Common	Neg	Good	HR	2	10 -2 10 -2/	-
											-3
	#3	4.3	f	3,300 common	Neg	Good	MR	2	10 -4 Neg -
	#4	4.3	f	15,000 Pre-B	Pos	Good	No markers	0	-	-	-
	#5	3.2	f	41,000 Unknown	Neg	Good	SR	2	Neg Neg -
	#6	15.5	f	38,600 T	Neg	Good	No markers	0	-	-	-
	Patients with MRD monitoring in plasma DNA
	#7	12.7	f	5,400 Common	Neg	Good	HR	3	10 -3 10 -3 -
	#8	8.6	f	7,100 T	Neg	Good	HR	2	10 -2 10 -3 -
	#9	4.6	f	2,800 Common	Neg	Good	MR	3	10 -5 10 -5 -
	#10	9.4	f	17,800 Common	Neg	Good	SR	4	Neg Neg -
	#11	2.6	m	7,200 Common	Neg	Good	MR	2	10 -4 10 -4 -
	#12	18.0	m	52,800 Pre-B	Neg	Good	SR	3	Neg Neg -
	#13	5.2	m	76,800 Common	Pos	Poor	MR	2	10 -3 10 -4 -
	#14	4.1	f	90,200 Common	Neg	Good	MR	3	10 -3 Neg Bone
												marrow
	#15	8.3	m	2,300 Common	Neg	Good	SR	3	Neg Neg -
	#16	7.8	m	33,600 Common	Neg	Good	MR	4	10 -4 Neg -
	#17	4.1	m	18,300 Common	Pos	Good	SR	2	Neg Neg -
	#18	13.4	f	125,000 Common	Neg	Poor	MR	3	10 -4 Neg Bone
												marrow
	#19	5.2	f	5,700 Common	Neg	Good	SR	3	Neg Neg Bone
												marrow
	#20	15.2	F	3,000 Common	Neg	Good	MR	2	10 -4 Neg -
	#21	3.4	F	17,100 Common	Neg	Good	MR	2	10 -3 Neg Bone
												marrow
	m

Acknowledgments We acknowledge the cooperation of all patients and their parents, nurses, and physicians who contributed to the collection of samples for this study. We thank Nicole Wittner for expert technical assistance and Dr. Martin Zimmermann for statistical tests and discussion. This work was supported by the Deutsche Krebshilfe (Bonn, Germany) and the Leukemia Research Foundation (Chicago, IL, USA).

This work was supported by the Deutsche Krebshilfe (Bonn, Foundation (Chicago, IL, USA).


Francesca Olcese

Marino Clavio
email: claviom@unige.it

Edoardo Rossi

Mauro Spriano

Filippo Ballerini

Letizia Canepa

Ivana Pierri

Sara Aquino

Riccardo Varaldo

Annunziata Manna

Vincenzo Secondo

Omar Racchi

Enrico Balleari

Fraternali Giulio

Orcioni

Michele Angelo

Riccardo Carella

Marco Ghio

Gobbi

: R Varaldo

A M Carella

: R Ghio

: M Gobbi

G F Orcioni

The addition of radiotherapy to chemotherapy does not improve outcome of early stage Hodgkin's lymphoma patients: a retrospective long-term follow-up analysis of a regional Italian experience

Keywords: Hodgkin's lymphoma, ABVD, Radiotherapy, Late toxicity, Early stage

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The addition of radiotherapy to chemotherapy does not improve outcome of early stage Hodgkin's lymphoma patients: a retrospective long-term follow-up analysis of a regional Italian experience

Francesca Olcese, Marino Clavio, Edoardo Rossi, Mauro Spriano, Filippo Ballerini, Letizia Canepa, Ivana Pierri, Sara Aquino, Riccardo Varaldo, Annunziata Manna, et al.

To cite this version:

Francesca Olcese, Marino Clavio, Edoardo Rossi, Mauro Spriano, Filippo Ballerini, et al.. The addition of radiotherapy to chemotherapy does not improve outcome of early stage Hodgkin's lymphoma patients: a retrospective long-term follow-up analysis of a regional Italian experience. Annals of Hematology, 2009, 88 [START_REF] Koontz | Combined-modality therapy versus radiotherapy alone for treatment of early-stage Hodgkin's disease: cure balanced against complications[END_REF], pp.855-861. 10.1007/s00277-009-0699-5. hal-00535030

Introduction

The successful treatment of Hodgkin's lymphoma (HL) has been one of the most significant accomplishments in cancer therapy over the last century. Since the introduc-tion of extended field radiotherapy and MOPP (mustargen, oncovin, procarbazine, and prednisone) combination chemotherapy, more than 60% of patients can be cured [START_REF] Diehl | Hodgkin's disease, from pathology specimen to cure[END_REF]. Further progress in prognostic definition has been made over the last decade [START_REF] Hasenclever | A prognostic score for advanced Hodgkin's disease[END_REF], and several randomized trials compared innovative therapies with an ABVD-based approach [START_REF] Canellos | Chemotherapy of advanced Hodgkin's disease with MOPP, ABVD, or MOPP alternating with ABVD[END_REF][START_REF] Duggan | Randomized comparison of ABVD and MOPP/ABV hybrid for the treatment of advanced Hodgkin's disease: report of an intergroup trial[END_REF][START_REF] Gobbi | ABVD versus modified Stanford V versus MOPPEBVCAD with optional and limited radiotherapy in intermediate-and advanced-stage Hodgkin's lymphoma: final results of a multicenter randomized trial by the Intergruppo Italiano Linfomi[END_REF][START_REF] Diehl | Standard and increased-dose BEA-COPP chemotherapy compared with COPP-ABVD for advanced Hodgkin's disease. Standard and increased-dose BEACOPP[END_REF]. Six to eight courses of ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) are now considered as the standard treatment for patients with advanced stage HL. On the contrary, the optimal treatment strategy for early stage HL is still the subject of intense debate [START_REF] Diehl | Hodgkin's disease, from pathology specimen to cure[END_REF][START_REF] Diehl | Early, intermediate and advanced Hodgkin's lymphoma: modern treatment strategies[END_REF]. Between 1950 and 1980, radiotherapy (RT) was preferentially employed since it was considered a less toxic curative approach than MOPP. Later trials revealed that the risk of relapse in non-irradiated sites was approximately 20-30% and that many of these relapsed patients were rescued by chemotherapy. It therefore became interesting to determine whether combined modality therapy (chemotherapy + radiotherapy, CT + RT) might improve the results as compared to radiotherapy [START_REF] Bonadonna | ABVD plus subtotal nodal versus involvedfield radiotherapy in early-stage Hodgkin's disease: long-term results[END_REF][START_REF] Koontz | Combined-modality therapy versus radiotherapy alone for treatment of early-stage Hodgkin's disease: cure balanced against complications[END_REF][START_REF] Engert | Two cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine plus extended-field radiotherapy is superior to radiotherapy alone in early favorable Hodgkin's lymphoma: final results of the GHSG HD7 trial[END_REF][START_REF] Fermè | Chemotherapy plus involved-field radiation in earlystage Hodgkin's disease. EORTC-GELA H8 Trial[END_REF][START_REF] Olweny | Chemotherapy plus involved-field radiation in early-stage Hodgkin's disease[END_REF]. Moreover, the late risk of developing second tumors and coronary heart disease after chest radiotherapy led to reconsider the application of involved or extended field radiotherapy in front line treatment [START_REF] Hoppe | Hodgkin's disease: complications of therapy and excess mortality[END_REF][START_REF] Ghalibafian | Heart and coronary artery protection in patients with mediastinal Hodgkin lymphoma treated with intensity-modulated radiotherapy: dose constraints to virtual volumes or to organs at risk[END_REF][START_REF] Travis | Evaluation of the risk of therapy-associated complications in survivors of Hodgkin lymphoma[END_REF][START_REF] Kwong | Mammographic screening in women at increased risk of breast cancer after treatment of Hodgkin's disease[END_REF][START_REF] Hodgson | Individualized estimates of second cancer risks after contemporary radiation therapy for Hodgkin lymphoma[END_REF]. Over the last decade, some trials have explored the possibility of treating HL patients with CT alone [START_REF] Laskar | Consolidation radiation after complete remission in Hodgkin's disease following six cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine chemotherapy: is there a need[END_REF][START_REF] Straus | Results of a prospective randomized clinical trial of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by radiation therapy (RT) versus ABVD alone for stages I, II, and IIIA nonbulky Hodgkin disease[END_REF][START_REF] Meyer | Randomized comparison of ABVD chemotherapy with a strategy that includes radiation therapy in patients with limited-stage Hodgkin's lymphoma: National Cancer Institute of Canada Clinical Trials Group and the Eastern Cooperative Oncology Group[END_REF]. There is now increasing evidence that early stage HL patients are likely to be cured by four to six courses of ABVD alone, thus avoiding RT altogether [START_REF] Diehl | Hodgkin's disease, from pathology specimen to cure[END_REF][START_REF] Diehl | Early, intermediate and advanced Hodgkin's lymphoma: modern treatment strategies[END_REF][START_REF] Gospodarowicz | The management of patients with limited-stage classical Hodgkin lymphoma. Hematology[END_REF].

In order to document the application of these new therapeutic concepts and to verify the outcome of early stage HL patients, we retrospectively reviewed the clinical features, therapy, and long-term outcome of 139 stage I-II HL patients who were diagnosed and followed up in an Italian northern region (Liguria) from 1995 to 2007, and who received either chemotherapy (ABVD) alone or a combined modality treatment (CT+RT).

Patients and methods

Clinical data were retrospectively collected through the analysis of records of HL patients diagnosed and treated in eight oncology-hematology divisions from 1995 to 2007. Physicians in charge in different hospitals were interviewed to review the charts. The staging procedures included physical exam, total body CT scan, and bone marrow biopsy.

Only stage I and II HL with available follow-up information on response to therapy, long-term outcome, and toxicity were included in the study. Histological type was reassessed according to the current World Health Organization Classification [START_REF] Jaffe | World Health Organization Classification of tumors. Pathology and genetics of tumors of haematopoietic and lymphoid tissues[END_REF]. Bulk disease was defined as a nodal tumor mass >10 cm. Response to therapy was assessed with physical and radiological imaging (ultrasonography and CT). Only in the last 5 years, 18-FDG PETs have been employed in defining initial stage and response as well.

The median follow-up is 60 months (range 12-175).

Overall survival (OS) was calculated from the beginning of treatment to death by any cause, or to the last follow-up visit. Disease-free survival (DFS) was calculated from the date of the first complete remission to the first relapse. Overall DFS and overall survival (OS) curves were calculated according to the Kaplan and Meier method. Univariate logistic-regression methods were used to evaluate the prognostic importance of various characteristics with respect to complete remission (CR) achievement and overall survival. We considered a two-tailed p value of 0.05 or less as statistically significant.

Patients' features and therapy

Sixty-three patients received chemotherapy alone (CT group) and 76 received an association of CT and radiotherapy (CT+RT group). Radiotherapy was started within 1 month of the completion of chemotherapy.

Target volumes for involved-field radiotherapy initially included involved nodal regions, while target volumes for extended field included the mantle field, spleen, and para-aortic nodes. Patients received a median of 30 Gy.

Main clinical, laboratory, and histological features of the whole cohort of patients are reported in Table 1, whereas Table 2 reports relevant prognostic data according to the treatment they received.

As shown in Table 2, the two therapeutic groups were comparable for clinical and histologic features. Mediastinal involvement (not bulky) was more frequent in patients receiving combined therapy.

ABVD was the regimen of choice in the CT group (95%). A median of six courses were given. In the CT+RT group, 53% of patients received ABVD and 42% were given the Stanford V regimen.

Results

Response to therapy

Details about type of treatment and response to therapy are reported in Table 3.

Briefly, CR rate after CT+RT was higher than what was achieved with CT alone (96% vs. 86%, respectively, p = 0.03). Although early application of salvage 4 reports the CR rate according to number of ABVD courses, stage, mediastinal involvement, or bulky disease. Statistical analysis revealed no clinical factors affecting the CR rate in either of the therapeutic groups, or in the cohort as a whole. In stage II patients and in those with mediastinal involvement or mediastinal bulky disease, the combined modality of therapy increased the probability of achieving CR.

Outcome, long-term toxicity

Relapse rate (12%) was the same in both groups (Table 5) and DFS curves (Fig. 1) were comparable (83% and 80% in CT and CT+RT groups, respectively, p=0.47). All relapses occurred within 38 months. Neither stage (I or II) nor mediastinal bulky disease affected the incidence of relapse (Table 6). Treatments at first relapse are reported in Table 5. There was no statistically significant difference with regards to the complete response rate to salvage therapy between the CT group (71%) and the CT+RT group (43%) (p=0.27).

Sixty-one (97%) patients in the first cohort (CT alone) and 73 patients (96%) among those who were initially treated with CT+RT are alive after a median follow-up of 50 and 90 months, respectively. Causes of death, mostly disease related, are reported in Table 7.

Figure 2 shows that overall survival of the two therapeutic groups is comparable. No second tumors have been reported among patients receiving chemotherapy alone, whereas a second neoplasia has been diagnosed in four patients in the CT+RT group (5%, p=0.09). The development of a tumor in two cases (breast and lung cancer) is possibly radiotherapy related.

Figure 3 reports the second neoplasia-free survival rates of the CT and CT+RT groups (100% and 92%, respectively, p=0.2).

One patient treated with CT+RT developed coronary heart disease.

Discussion

The aim of this retrospective study was to review long-term results and toxicity in the management of early stage HL in a regional Italian area (Liguria). We found that the front line therapeutic approach always included chemotherapy, which was followed by radiotherapy in 55% of patients (see Table 7). However, our analysis was not aimed at

A months

Fig. 1 Stage I-II Hodgkin's lymphoma disease-free survival comparing these therapeutic strategies. This retrospective review of the Ligurian experience confirms the previously reported clinical features and histologic distribution of early stage HL, including the rarity of presentation below the diaphragm (5%) [23]. The only relevant exception to reported data is that females are more frequently affected than males. The longer median follow-up in the CT+RT group is due to the fact that, for a long period of time, CT+ RT was considered standard therapy for limited stage HL patients.

Long-term outcome data show that a high cure rate can be achieved with limited side effects in the vast majority of early stage HL patients using either of the therapeutic strategies.

Considering the retrospective, non-randomized nature of this survey, a comparison between the two treatment modalities regarding the efficacy at inducing CR is beyond its aims. Moreover, as many as 36 (42%) patients in the combined modality group did not receive ABVD (Stanford V). Despite these limits, we show that the upfront combination of CT and involved or extended field RT led to an increased CR rate among stage II patients, in those with mediastinal involvement and mediastinal bulky disease. However, long-term observation showed that omitting radiotherapy did not influence relapse rate or duration of first CR, even in patients with mediastinal involvement or bulky disease. As a matter of fact, both groups showed relapse rates of about 12% and response to salvage treatment was similar.

The administration of ABVD alone as first line therapy is associated with a more frequent and earlier use of HDT compared to the combined modality approach (8 vs 2 patients, respectively).

Our study shows that both CT and CT+RT allow us to achieve optimal long-term control of early stage HL, with no statistically significant differences in DFS or in overall survival, as confirmed by a number of recent randomized trials [START_REF] Laskar | Consolidation radiation after complete remission in Hodgkin's disease following six cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine chemotherapy: is there a need[END_REF][START_REF] Straus | Results of a prospective randomized clinical trial of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by radiation therapy (RT) versus ABVD alone for stages I, II, and IIIA nonbulky Hodgkin disease[END_REF][START_REF] Meyer | Randomized comparison of ABVD chemotherapy with a strategy that includes radiation therapy in patients with limited-stage Hodgkin's lymphoma: National Cancer Institute of Canada Clinical Trials Group and the Eastern Cooperative Oncology Group[END_REF].

Nonetheless, radiotherapy, especially when administered in extended field has been associated with an increased risk of second neoplasia [START_REF] Hoppe | Hodgkin's disease: complications of therapy and excess mortality[END_REF][START_REF] Travis | Evaluation of the risk of therapy-associated complications in survivors of Hodgkin lymphoma[END_REF][START_REF] Hodgson | Individualized estimates of second cancer risks after contemporary radiation therapy for Hodgkin lymphoma[END_REF]. In our series, a second tumor was diagnosed in four patients who had been exposed to RT, even though a clear relationship with RT was only found in two of the cases. No second tumors occurred among the patients who had not been exposed to RT, and despite the increased use of further salvage chemotherapy and HDT, no secondary myelodysplastic syndromes or sAML were observed either. However, there is no statistically significant difference in the second neoplasia-free survival rate in the two therapeutic groups, and the second neoplasia was the cause of death in only one patient. Only one patient who received mediastinal radiotherapy developed coronary disease. However, an increased incidence of delayed heart complications is expected in the future in patients submitted to mediastinal radiotherapy, as previously reported [START_REF] Ghalibafian | Heart and coronary artery protection in patients with mediastinal Hodgkin lymphoma treated with intensity-modulated radiotherapy: dose constraints to virtual volumes or to organs at risk[END_REF][START_REF] Travis | Evaluation of the risk of therapy-associated complications in survivors of Hodgkin lymphoma[END_REF]. A longer period of observation is needed to reveal small differences in toxicity and non-lymphoma-related mortality. Hoppe showed in fact that the risk of death from Hodgkin's disease is 17% at 15 years of follow-up and increases only slightly thereafter, whereas the risk of death from other causes is also 17% at 15 years, but increases sharply thereafter for at least 25 years [START_REF] Hoppe | Hodgkin's disease: complications of therapy and excess mortality[END_REF].

Although quality of life has not been specifically addressed, both initial therapeutic strategies were well tolerated and did not strongly affect quality of life. This may be related to the improved radiotherapy techniques as well as to the use of advanced supportive therapy.

In conclusion, our retrospective study shows that an upfront combination of CT and limited RT is an effective and well-tolerated option for early stage HL because it achieves a very high percentage of long lasting CR, even if the use of RT is associated with a certain risk of developing a second tumor.

Administering four to six courses of ABVD as the first line approach allows us to obtain similar, optimal, long-term disease control, as indicated by the DFS and overall survival curves without exposing patients to the risk of a second neoplasia. There were no differences in the overall survival and second neoplasia-free survival of CT and CT+RT patients in our study; however, the small number of patients in our cohort may have prevented us from identifying a statistically significant difference. Innovative techniques, such as 3-D radiotherapy or radiotherapy with modulated intensity, may reduce the early and late toxic effects [START_REF] Ghalibafian | Heart and coronary artery protection in patients with mediastinal Hodgkin lymphoma treated with intensity-modulated radiotherapy: dose constraints to virtual volumes or to organs at risk[END_REF][START_REF] Eich | Current role and future developments of radiotherapy in early-stage favourable Hodgkin's lymphoma[END_REF][START_REF] Hoppe | Hodgkin's lymphoma: the role of radiation in the modern combined strategies of treatment[END_REF][START_REF] Macdonald | New strategies for the treatment of early stages of Hodgkin's lymphoma[END_REF], although results must be confirmed by controlled studies. Chemotherapy alone proved to be effective and can be used in early stage HL patients when RT is not available or when patients or physicians decide to exclude it from the first line approach.

Fig. 3 Fig. 2

 32 Fig. 3 Stage I-II Hodgkin's lymphoma second neoplasia-free survival

Table 1

 1 Patients' clinical and histological features (all patients)

	Patients		139
	Male/female		58 (42%)/81 (58%)
	Median age (range)	31 (14-79)
	Histology	Nodular sclerosis (NS)	115 (83%)
		Type I	24/115 (21%)
		Type II	30/115 (26%)
		Not specified	61/115 (53%)
		Lymphocyte prevalence (PL)	14/139 (10%)
		Mixed cellularity (MC)	10/139 (7%)
	Presentation	Above the diaphragm	132 (95%)
		Below the diaphragm	7 (5%)
		Mediastinal involvement	83 (60%)
		Mediastinal bulky	17/83 (20%)
	Stage	I A	13 (9%)
		I B	5 (4%)
		I E	1 (1%)
		II A	71 (50%)
		II B	49 (35%)
	Hematological	Median leukocytes x 10 9 /L	9.150 (4.2-2.7)
	parameters	(125 pts)
		Median Hb g/dl (125 pts)	13 (8.4-17)
		Median plt x 10 9 /L (121 pts)	309 (86-574)
		Median albumin g/dl (93 pts) 4 (2.1-5.1)
		Median LDH U/l (121 pts)	408 (59-1100)
		Above normal range	35/121(29%)
		Median ESR (in 80 pts)	28 mm/h (2-187)
		Median PCR (in 54 pts)	3.4 (0-139)
		Median β2 microglobulin mg/	1.8 (0.2-15)
		l (in 49 pts)

Table 2

 2 Patients' clinical and histological features according to first line therapy

			First line therapy without radiotherapy (RT)	First line therapy including RT	p value
			N=63 (45%)	N=76 (55%)
	Male/female		25 (40%)/38 (60%)	33 (43%)/43 (57%)	0.55
	Median age		31	30
	Histology	LP	7 (11%)	7 (9%)	0.88
		NS	52 (83%)	63 (83%)
		MC	4 (6%)	6 (8%)
	Mediastinal involvement	31 (49%)	52 (68%)	0.01
	Mediastinal bulky disease	8/31 (26%)	11/52(21%)	0.77
	Stage	I A	6 (10%)	7 (9%)	0.91
		I B	2 (3%)	3 (5%)
		I E	-	1 (1%)
		II A	32 (51%)	39 (51%)
		II B	23 (36%)	26 (34%)
	LDH above normal range	11/50 (22%)	24/71 (34%)	0.11
	LP lymphocyte predominance, NS nodular sclerosing, MC mixed cellularity

Table 4

 4 Factors affecting CR rate

		CT	CT+RT	All patients
		CR (%)	CR (%)	CR (%)
	ABVD 6 courses	A: 33/41 (80%)
	ABVD <6 courses	B: 18/19 (95%)
	Stage I	C: 7/8 (87%)	I: 11/11 (100%)	Q: 18/19 (95%)
	Stage II	D: 51/55 (93%)	L: 65/65 (100%)	R: 116/120 (97%)
	Mediastinal involvement	E: 25/28 (89%)	M: 52/52 (100%)	S: 73/82 (89%)
	No mediastinal involvement	F: 30/32 (94%)	N: 24/24 (100%)	T: 53/57 (93%)
	Mediastinal bulky	G: 5/9 (56%)	O: 12/12 (100%)	U: 17/22 (77%)
	No mediastinal bulky	H: 16/19 (84%)	P: 40/40 (100%)	V: 109/117 (93%)
	Statistical comparison between groups of data: A vs. B p=0.14; C vs. D p=0.88; E vs. F p=0.43; G vs. H p=0.12; C vs. I p=0.42; D vs. L p=
	0.04; E vs. M p=0.04; F vs. N p=0.32; G vs. O p=0.02; H vs. P p=0.03; Q vs. R p=0.86; S vs. T p=0.31; U vs. V p=0.99

Table 5

 5 Relapse, therapy at relapse Statistical comparison between groups of data: C vs. D p=0.80; E vs. F p=0.13; G vs. H p=0.95; I vs. L p=0.61; M vs. N p=0.02; O vs. P p= 0.87; C vs. I p=0.64; D vs. L p=0.58; E vs. M p=0.08; F vs. N p=0.06; G vs. O p=0.34; H vs. P p=0.32; Q vs. R p=0.63; S vs. T p=0.83; U vs. V p=0.87

		First line therapy without radiotherapy	First line therapy including	p value
		(RT) N=58 complete responses	RT N=76 complete responses
	Relapses	7 (12%)	9 (12%)	0.62
	CR length in relapsed patients months	15 (5-38)	14 (4-31)
	Therapy of relapse
	Chemotherapy (CT)+RT	2/7 (28.6%)	2/9 (22%)	0.48
	CT	1/7 (14.2%)	3/9 (34%)
	CT+HDT	2/7 (28.6%)	1/9 (11%)
	RT alone	2/7 (28.6%)	1/9 (11%)
	No therapy	-	2 (22%)
	Response to therapy at relapse
	CR	5/7 (71%)	3/7 (43%)	0.28
	PR	-	2/7 (28.5%)
	Not evaluated	2/7 (29%)	2/7 (28.5%)

Table 7

 7 Long-term outcome and late toxicities

		First line therapy	First line therapy	p
		without radiotherapy	including RT N=	value
		(RT) N=63 pts	76 pts
	First CR	33 (7-134)	73 (7-144)
	length,
	months
	Patients alive	61 (97%)	73 (96%)	0.59
	Patients dead	2 (3%)	3 (4%)	0.59
	Overall	40 (12-144)	87 (12-175)
	survival,
	months
	Median	55	98
	follow-up
	Patients with	-	4 (5%)	0.09
	second
	neoplasia
	Rectum	-	1
	Lung	-	1
	Breast	-	1
	Prostate	-	1
	Coronary	-	1
	heart disease
	Causes of
	death
	HL	2	2
	Second	-	1
	neoplasia
	Patients
	undergoing
	salvage
	HDT

0.05


Palle Pedersen

Nils Milman
email: nils.mil@dadlnet.dk

Extrinsic factors modifying expressivity of the HFE variant C282Y, H63D, S65C phenotypes in 1,294 Danish men

Keywords: Expressivity, Genotypes, Hemochromatosis, Mutations, Penetrance, Scandinavia

Abstract This study analysed the influence of extrinsic factors on the phenotypic expression of HFE gene variants in ethnic Danish men. A cohort of 6,020 men aged 30-53 years was screened for HFE C282Y, H63D and S65C variants. Serum iron, serum transferrin, transferrin saturation, and serum ferritin were analysed in 1,452 men and 1,294 men completed a questionnaire on factors, which could influence iron balance. The C282Y allele was present in 5.6%, H63D in 12.8% and S65C in 1.8% of the men. In the entire series, 3% had elevated iron status markers (transferrin saturation ≥50%, ferritin ≥300μg/L). Selfreported liver disease had an elevating effect and peptic ulcer a lowering effect on iron status markers. Age increased the fraction of men with elevated ferritin from 8.3% at 32-38 years to 16.2% at 46-53 years of age (p=0.002). Blood donation had a lowering effect on iron status markers (p=0.0001). Alcohol consumption elevated serum iron and serum ferritin (p=0.001). Meat consumption had an elevating effect (p=0.02) and milk consumption a lowering effect (p=0.03) on serum ferritin. There was no influence of vitamin-mineral tablets on iron status markers. In adjusted logistic regression analysis, the HFE genotype had the highest impact on iron status markers; high alcohol consumption was significantly associated with elevated transferrin saturation. High age and high alcohol consumption were significantly associated with elevated ferritin and high egg consumption and blood donation was significantly associated with normal ferritin levels. In conclusion, the expressivity of HFE variant phenotypes in Danish men was enhanced by alcohol and meat consumption and decreased by milk and egg consumption and blood donation.

Genetic HFE hemochromatosis is inherited as an autosomal recessive trait [START_REF] Milman | Clinically overt hereditary hemochromatosis in Denmark 1948-1985: epidemiology, factors of significance for long-term survival, and causes of death in 179 patients[END_REF][START_REF] Milman | Evidence that the Cys282Tyr mutation of the HFE gene originated from a population in Southern Scandinavia and spread with the Vikings[END_REF][START_REF] Milman | Inheritance of hemochromatosis: family studies[END_REF] and the phenotypic penetrance of the most important HFE mutation or variant, i.e. C282Y, has been assessed in various studies using phenotypic, clinical and genetic criteria [START_REF] Burt | The significance of haemochromatosis gene mutations in the general population: implications for screening[END_REF][START_REF] Olynyk | A population-based study of the clinical expression of the hemochromatosis gene[END_REF][START_REF] Mcdonnell | Population-based screening for hemochromatosis using phenotypic and DNA testing among employees of health maintenance organizations in Springfield, Missouri[END_REF][START_REF] Mclaren | for the Hemochromatosis and Iron Overload Screen (HEIRS) Study Research Investigators (2008) Clinical manifestations of hemochromatosis in HFE C282Y Homozygotes identified by screening[END_REF][START_REF] Phatak | Prevalence and penetrance of HFE mutations in 4865 unselected primary care patients[END_REF][START_REF] Jackson | HFE mutations, iron deficiency and overload in 10, 500 blood donors[END_REF][START_REF] Beutler | Penetrance of 845G A (C282Y) HFE hereditary haemochromatosis mutation in the USA[END_REF][START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF]. A longitudinal study of C282Y homozygous individuals, followed up for more than 10 years, assessed the fraction, who developed disease due to iron overload [START_REF] Allen | Iron overload related disease in HFE hereditary hemochromatosis[END_REF].

The expressivity of the HFE variant C282Y (c845 G→A), H63D (c187 C→G) and S65C (c193 A→T) phenotypes also depend on extrinsic factors or modifiers of iron balance, such as dietary iron intake, bioavailabilty of dietary iron, blood losses at menstruation and pregnancy as well as blood donation. The aim of the present study was to analyse environmental extrinsic factors, which might influence the phenotypic expression of the HFE variants on iron status markers.

Methods

Participants and study design

The study was performed in 2000-2001 and described in previous papers [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF][START_REF] Elsass | Assessment of the psychological effects of genetic screening of hereditary hemochromatosis[END_REF][START_REF] Pedersen | Frequencies of the haemochromatosis (HFE) gene variants C828Y, H63D and S65C in 6, 020 ethnic Danish men[END_REF]. It was approved by The Danish Scientific Ethical Committee and fulfilled The Declaration of Helsinki. Ethnic Danish men aged 30-53 years (n=10,993) living in Naestved and Vordingborg municipalities were drawn from the Census Registry and invited to participate.

In the first phase of the study, HFE genotypes were assessed on saliva samples from the participants. In the second phase, participants with HFE variants plus participants in whom the genotype could not be assessed on saliva, were invited to genetic retesting and analysis of iron status markers. Participants with elevated iron status markers were invited to a reanalysis.

Iron status

Blood samples were drawn in the fasting state (except in 250 men) between 0800 and 1000 hours. Analyses of iron status markers, i.e. serum iron, serum transferrin and serum ferritin were performed on Dimension® RxL Clinical Chemistry System with heterogeneous Immunoassay Module (Dade Behring Inc., Deerfield, Illinois, USA, now merged with Siemens Healthcare Diagnostics GmbH, Eschborn, Germany) [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF]. The serum transferrin saturation percent (TSAT) was calculated from serum iron and serum transferrin concentrations as: serum iron (μmol/L) × 100/serum transferrin (μmol/L) × 2. Transferrin saturation values ≥50% and serum ferritin values ≥300μg/L were considered elevated [START_REF] Milman | Iron status markers in hereditary haemochromatosis: distinction between individuals being homozygous and heterozygous for the haemochromatosis allele[END_REF][START_REF] Milman | Serum ferritin in Danes: studies of iron status from infancy to old age, during blood donation and pregnancy[END_REF].

Questionnaire

The questionnaire yielded information concerning factors, which might influence iron status, such as blood donation, iron supplements, self-reported disease, alcohol consumption and dietary habits comprising consumption of meat, milk and egg. As previously reported [START_REF] Elsass | Assessment of the psychological effects of genetic screening of hereditary hemochromatosis[END_REF], the questionnaire also assessed the psychological consequences of genetic screening. reference interval was defined as the 2.5-97.5 interpercentile range. The significance level was set at 0.05. The association between HFE genotype and the risk of having elevated transferrin saturation and/or serum ferritin was assessed by conditional logistic regression with adjusted odds ratios (OR) and 95% confidence intervals (CI). HFE genotype, age, blood donation, self-reported disease, vitamin mineral tablets containing iron, alcohol, meat, milk and egg consumption were included as covariates.

Statistics

The predicted phenotypic penetrance of the HFE genotypes, adjusted for other covariates, was calculated as previously described [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF]. The probability for a specific HFE genotype to present elevated transferrin saturation or elevated serum ferritin is an estimate of penetrance when other modifiers are in the reference state. The reference group was wt/wt homozygous men aged 32-39 years, who never had donated blood, had no self-reported disease and low alcohol, meat, milk and egg consumption.

Results

Salvia samples were obtained from 6,567/10,993 (60%) men. In 1,064/6,567 (16%) men, genotype could not be assessed. They were invited to genetic testing and analysis of iron status markers, which was obtained in 522/1,064 (49%).

HFE genotype was assessed in 6,020 men. The 2,149 men carrying a HFE variant were invited to analysis of iron status markers being obtained in 930/2,149 (43%) men. In the entire series, iron status markers were obtained in 1,452/ 6,020 (24%) men of whom 1,294/1,452 (89%) completed the questionnaire. Data from the questionnaire are summarized in Table 1.

HFE allele frequencies

The HFE genotypes C282, H63, and S65 are designated wild type (wt). In the entire series, the allele frequency of C282Y, H63D, and S65C variants was 5.6%, 12.8% and 1.8%, respectively [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF][START_REF] Pedersen | Frequencies of the haemochromatosis (HFE) gene variants C828Y, H63D and S65C in 6, 020 ethnic Danish men[END_REF].

Iron status markers

In total 250/1,452 (17%) men were non-fasting. Serum iron and transferrin saturation was significantly lower in fasting than in non-fasting men (Table 2). Consequently, we used only serum iron values in fasting men in the calculation of transferrin saturation and the statistical analyses.

Age vs iron status markers

Age had no influence on transferrin saturation values. There was no significant difference in serum ferritin levels in the various age groups (Table 3) and the lower limit of the 95% reference interval for ferritin remained unchanged from 32 to 53 years of age. However, the upper limit for ferritin and the percentage of men having elevated ferritin ≥300μg/L increased gradually with age.

Alcohol vs iron status markers

There was a significant elevation of serum ferritin and serum iron with increasing alcohol consumption (Table 4).

Blood donation vs iron status markers

In blood donors, serum ferritin, serum iron and transferrin saturation decreased and serum transferrin increased with the number of blood donations (Table 5 and Fig. 1). Blood donors who were bled more than two years ago had significantly lower median ferritin than non-donors (Mann-Whitney's test, p=0.04), but similar serum iron, serum transferrin and transferrin saturation.

Donors bled from one up to more than four times during the last two years had higher serum transferrin than donors who were bled more than two years ago and non-donors (Mann-Whitney's test, p<0.001). Donors bled more than 4 times during the last two years had lower serum iron and transferrin saturation than non-donors (p=0.01 and p= 0.0001, respectively).

Dietary factors vs iron status markers

Consumption of meat had an elevating effect and consumption of milk a lowering effect on serum ferritin levels (Table 6). There was no significant influence of egg consumption and vitamin-mineral tablets containing iron. A few took oral iron, they had low iron status due to iron deficiency, which obviously was the indication for treatment.

Self-reported disease vs iron status markers Self-reported disease had a significant influence on transferrin saturation (Table 7). Although median serum ferritin level was higher in men with liver disease and lower in men with peptic ulcer, the differences were not statistically significant, probably due to the low number of men in the groups.

Logistic regression analysis

C282Y homozygotes had an 86-fold higher risk of elevated ferritin and a 66-fold higher risk of elevated transferrin saturation than wt/wt homozygotes (Tables 8 and9). C282Y/H63D compound heterozygotes had a 7.2-fold higher risk of having elevated transferrin saturation and a 3.3-fold higher risk of having elevated ferritin than wt/wt homozygotes. Of the remaining genotypes, C282Y heterozygotes and H63D homozygotes had higher risk of having elevated transferrin saturation. High alcohol consumption was associated with elevated transferrin saturation. High age and high alcohol consumption were significantly associated with elevated ferritin and high egg consumption and blood donation was significantly associated with normal ferritin levels.

Discussion

In genetic terms, penetrance describes the proportion of individuals carrying a particular variation of a gene, i.e. the genotype that expresses an associated trait, i.e. the phenotype, while expressivity refers to variations of a phenotype in individuals carrying a particular genotype. The present study assessed the penetrance of the three most frequent HFE variants, i.e. the influence of the genotype on the phenotype, as well as the impact of age and environmental factors on the expressivity of the phenotype. Men are more predisposed to iron overload than women, i.e. the influence of the HFE variants and modifying factors are more likely to be expressed in men than in women [START_REF] Milman | Clinically overt hereditary hemochromatosis in Denmark 1948-1985: epidemiology, factors of significance for long-term survival, and causes of death in 179 patients[END_REF][START_REF] Allen | Iron overload related disease in HFE hereditary hemochromatosis[END_REF]. Due to iron losses at menstruation and pregnancy, iron overload is less frequent in fertile women [START_REF] Milman | Iron status in 268 Danish women aged 18-30 years: influence of menstruation, contraceptive method, and iron supplementation[END_REF]. Allen et al. [START_REF] Allen | Iron overload related disease in HFE hereditary hemochromatosis[END_REF] followed up 203 C282Y homozygous individuals over 12 years; 28% of men vs 1% of women developed iron overload-related disease.

In our series, the phenotypic penetrance in C282Y homozygous men (n=23) was close to 100% and higher than the penetrance in C282Y/H63D compound heterozygous [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF].

HFE variant homozygous and compound heterozygous men had significantly higher transferrin saturation than wt/wt homozygous men [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF]. However, even among the wt/wt homozygotes, 6% had elevated transferrin saturation, 13% elevated serum ferritin and 1% elevation of both iron status markers [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF], suggesting that other genetic factors (besides HFE) and/or environmental factors influence iron homeostasis. C282Y homozygous or C282Y/H63D compound heterozygous individuals have an excess dietary iron absorption and may over time develop iron-overload-associated organ damage. However, both the phenotypic penetrance and the expressivity of HFE hemochromatosis are incomplete and several homozygotes and compound heterozygotes do not develop clinical iron overload disease [START_REF] Allen | Iron overload related disease in HFE hereditary hemochromatosis[END_REF][START_REF] Andersen | Hemochromatosis mutations in the general population: iron overload progression rate[END_REF]. Modifier genes and/or environmental factors may cause this variability in penetrance.

We observed an age-associated increase in high serum ferritin values ≥300μg/l, although there was no significant increase in median serum ferritin levels (Table 3). In H63D heterozygotes, the percent of men with elevated serum ferritin rose significantly more with age than in wt/wt homozygotes (χ 2 test, p=0.004), stressing the impact of H63D on body iron status.

Age-related increase in high serum ferritin values have been reported in other Danish studies [START_REF] Milman | Serum ferritin in Danes: studies of iron status from infancy to old age, during blood donation and pregnancy[END_REF][START_REF] Milman | Iron status in Danish men 1984-94: a cohort comparison of changes in iron stores and the prevalence of iron deficiency and iron overload[END_REF] due to genetic modifiers of iron absorption and/or changes in dietary habits with increased meat [START_REF] Lyhne | Danskernes Kostvaner 2000-2002. Hovedresultater (Danish Nutrition Habits[END_REF] and alcohol consumption (www.sst.dk) A similar age dependent effect was not observed for transferrin saturation. Danish men have a dietary iron intake of median 10.9 mg/day (10-90 percentile 7.2-15.3) [START_REF] Lyhne | Danskernes Kostvaner 2000-2002. Hovedresultater (Danish Nutrition Habits[END_REF] with a bioavailability of 15-20%. Cook [START_REF] Cook | Hemochromatosis: effect of iron fortification of foods[END_REF] reported that iron fortification in foods could increase the phenotypic penetrance of hemochromatosis. In Denmark, food iron fortification was stopped in 1987.

The Danish Health Authorities recommended a maximum alcohol intake for men of 21 drinks/week corresponding to 252 g ethanol/week (www.sst.dk). High alcohol consumption (>21 drinks/week) was associated with elevated serum iron. Among Danish men 45-66 years of age, ~20% consume more than 21 drinks per week (www.sst.dk). However, the increase in serum iron was not high enough to elevate transferrin saturation. Likewise, high alcohol consumption was associated with elevated serum ferritin (Table 4). The positive association between alcohol intake and serum ferritin has been observed in previous Danish surveys [START_REF] Milman | Relationship between serum ferritin, alcohol intake, and social status in 2235 Danish men and women[END_REF]. The mechanisms by which alcohol increase serum ferritin are not completely understood, but alcohol can induce hepatocellular injury, suppress erythropoiesis, reduce erythrocyte survival [START_REF] Baynes | Interaction of alcohol, iron and hemochromatosis[END_REF] enhance iron absorption and possibly also ferritin synthesis [START_REF] Moirand | Increase in glycosylated and nonglycosylated serum ferritin in chronic alcoholism and their evolution during alcohol withdrawal[END_REF], all of which contribute to elevated ferritin. Alltogether, alcohol promotes and accelerates the phenotypic penetrance of HFE variants and the expressivity with development of hepatic damage [START_REF] Loreal | Liver fibrosis in genetic hemochromatosis. Respective roles of iron and noniron-related factors in 127 homozygous patients[END_REF][START_REF] Brissot | Clinical spectrum of hepatic disease in hemochromatosis[END_REF].

Donation of blood has a profound impact on iron status [START_REF] Milman | Serum ferritin in Danes: studies of iron status from infancy to old age, during blood donation and pregnancy[END_REF][START_REF] Milman | Iron status in Danes updated 1994. I: prevalence of iron deficiency and iron overload in 1332 men aged 40-70 years. Influence of blood donation, alcohol intake, and iron supplementation[END_REF][START_REF] Milman | Iron stores in male blood donors: evaluation by serum ferritin[END_REF][START_REF] Milman | The influence of blood donation on iron stores assessed by serum ferritin and haemoglobin in a population survey of 1433 Danish males[END_REF]. Among Danish men, ~28% are blood donors [START_REF] Milman | Iron status in Danes updated 1994. I: prevalence of iron deficiency and iron overload in 1332 men aged 40-70 years. Influence of blood donation, alcohol intake, and iron supplementation[END_REF]. With an increasing number of blood donations, serum ferritin and eventually serum iron and transferrin saturation decrease. Blood donation is clearly an effective way to reduce body iron stores and decrease the phenotypic penetrance and expressivity of the HFE variants. In patients with HFE associated iron overload, phlebotomies are usually performed until serum ferritin reaches a level of 50-100μg/L.

In our adjusted logistic regression analysis, the HFE genotype had the highest impact on iron status markers followed by blood donation, alcohol intake, age, and egg consumption.

Consumption of meat (beef, pork, poultry) has a positive effect on iron status [START_REF] Leggett | Prevalence of haemochromatosis amongst asymptomatic Australians[END_REF][START_REF] Bergström | Sex differences in iron stores of adolescents: what is normal?[END_REF] and men with a high meat intake displayed higher serum ferritin compared to those with a low intake. In addition to "the meat factor", which enhances iron absorption, meat contains heme iron, which has a higher bioavailability than non-heme iron. Therefore, high meat consumption may increase the phenotypic penetrance and expressivity of the HFE variants. Danish men consume median 132 g meat (beef, pork, poultry) per day (10-90 percentile 46-158) and ~80% consume meat on a daily basis [START_REF] Lyhne | Danskernes Kostvaner 2000-2002. Hovedresultater (Danish Nutrition Habits[END_REF].

Drinking large quantities of milk inhibits iron absorption in a dose related manner, probably due to the calcium content [START_REF] Barton | Calcium inhibition of inorganic iron absorption in rats[END_REF]. Danish men consume median 248 g milk/day and ~66% consume milk on a daily basis [START_REF] Lyhne | Danskernes Kostvaner 2000-2002. Hovedresultater (Danish Nutrition Habits[END_REF]. Consequently, devoted milk consumption may lower the phenotypic penetrance of the HFE variants. Other studies have shown that regular tea drinking causes a 30% decrease in iron absorption in hemochromatosis patients [START_REF] Kaltwasser | Clinical trial on the effect of regular tea drinking on iron accumulation in genetic haemochromatosis[END_REF].

Egg contains sulphides, which can inhibit iron absorption, but appeared to have no effect on iron status even in a quantity of more than four eggs per week. The recommended intake is 3-4 eggs/week. Danish men consume median 2 eggs/week and ~5% consume more than 7 eggs per week [START_REF] Lyhne | Danskernes Kostvaner 2000-2002. Hovedresultater (Danish Nutrition Habits[END_REF].

Multivitamin-multimineral tablets containing 14-20 mg of ferrous iron had no influence on iron status as previously reported in Danish studies [START_REF] Milman | Iron status in Danish men 1984-94: a cohort comparison of changes in iron stores and the prevalence of iron deficiency and iron overload[END_REF]. Among Danish men, ~30% use daily vitamin-mineral supplements containing median 14 mg ferrous iron [START_REF] Milman | Iron status in Danes updated 1994. I: prevalence of iron deficiency and iron overload in 1332 men aged 40-70 years. Influence of blood donation, alcohol intake, and iron supplementation[END_REF]. Obviously, iron absorption from these preparations is very low, probably due to the content of other essential divalent metal ions (zinc, copper, manganese ect.), which compete with iron about the absorption capacity in the gastrointestinal tract [START_REF] Rossander-Hultén | Competitive inhibition of iron absorption by manganese and zinc in humans[END_REF][START_REF] O'brien | Prenatal iron supplements impair zinc absorption in pregnant Peruvian women[END_REF]. However, inappropriate use of iron tablets on vague indications such as fatigue ect. without an established diagnosis of iron deficiency may promote iron overload in C282Y homozygous individuals [START_REF] Grønbaek | Preclinical hereditary hemochromatosis -is there an indication for preventive screening?[END_REF][START_REF] Bell | The clinical expression of hemochromatosis in Oslo, Norway. Excessive oral iron intake may lead to secondary hemochromatosis even in HFE C282Y mutation negative subjects[END_REF][START_REF] Hennigar | Hemochromatosis caused by excessive vitamin iron intake[END_REF][START_REF] Perez | Hemochromatosis presenting as acute liver failure after iron supplementation[END_REF].

Men reporting having peptic ulcers had significantly lower iron status than men without ulcers. Helicobacter pylori infection may induce peptic ulcer, has a negative influence on iron status [START_REF] Milman | Serum ferritin, hemoglobin, and Helicobacter pylori infection: a seroepidemiological survey comprising 2754 Danish adults[END_REF] and may thereby reduce the phenotypic penetrance of HFE variants. In general, pathological blood losses tend to lower the penetrance and expressivity of the HFE variants

In conclusion, the C282Y variant have a high phenotypic penetrance in Danish men [START_REF] Pedersen | Genetic screening for HFE hemochromatosis in 6,020 Danish men: penetrance of C282Y, H63D, and S65C variants[END_REF]. The expressivity of the C282Y and H63D variants may be promoted by high alcohol and meat consumption and decreased by high milk consumption and blood donation.

Fig. 1

 1 Fig.1Influence of blood donation on iron status markers

Table 3

 3 Influence of age on iron status markers, median (95% interval)

	Age (y) n a	Ferritin	Ferritin	n b	TSAT (%)
			(μg/L)	≥300 μg/L (%)
	32-39	363 149 (25-417) 8.3	327 30 (13-68)
	40-46	433 153 (25-519) 13.9	301 29 (12-59)
	47-53	656 153 (23-566) 16.2	574 28 (12-61)
	P-value		0.09*	0.002**		0.07*

a) The 3 groups had similar HFE genotype distribution, χ 2 test, p=0.3 b) Fasting men *) Kruskal Wallis' test **) χ 2 test

Table 4

 4 Influence of alcohol consumption on iron status markers, median values

	Drinks a /	n b Ferritin	Transferrin	n c Iron	TSAT
	week	(μg/L)	(μmol/L)	(μmol/L)	(%)
	<5	586 145	29	500 17	29
	5-15	481 149	30	404 18	30
	16-21	144 156	30	109 17	28
	>21	70 225	31	45 20	32
	P-value*	0.0001	0.2	0.01	0.3
	a) One drink = 12 g ethanol
	b) The 4 groups had similar HFE genotype distribution, χ 2 test p=0.2
	c) Fasting men
	*) Kruskal-Wallis' test

*) Kruskal-Wallis' test

Table 7

 7 Self-reported disease and iron status markers in Danish men,

	median values
	Disease	n	Ferritin	Transferrin	n a	Iron	TSAT
			(μg/L)	(μmol/L)		(μmol/L)	(%)
	Liver	no 1268 151	30	1048 17	29
	disease	yes 15	202	28	12	21	36*
	Peptic	no 1226 153	29	1011 18	29
	ulcer	yes 57	125	30	49	16*	26*
	a) Fasting men
	Mann-Whitney's test: * p<0.05 ** p<0.001 compared with the "no"
	groups

**) Kruskal-Wallis' test

  and may be

	Table 8 Logistic regression analysis of risk factors associated	Independent variable	β	OR Exp (β)	95% CI	P-value
	with elevated transferrin
	saturation ≥50%	HFE genotype				0.0001
		C282Y/C282Y	4.456	86.1	21.6-343.5	0.0001
		C282Y/H63D	1.977	7.2	2.6-19.9	0.0001
		H63D/H63D	1.700	5.5	2.0-14.9	0.001
		H63D/S65C	0.498	1.6	0.2-14.6	0.7
		C282Y/wt	1.186	3.3	1.4-7.9	0.01
		H63D/wt	0.968	2.6	1.2-6.0	0.02
		S65C/wt	-0.177	0.8	0.2-4.2	0.8
		Alcohol (drinks/week)				0.07
		6-15	0.095	1.1	0.7-1.8	0.7
		16-21	0.578	1.8	0.94-3.4	0.07
		>21	0.862	2.4	1.1-5.1	0.03
		Egg (no./week)
	Logit-value (α) in this model is	>4	-0.772	0.5	0.2-1.04	0.06
	-3.586.
	Table 9 Logistic regression analysis of risk factors associated	Independent variable	β	OR Exp (β)	95% CI	P-value
	with elevated serum ferritin
	≥300 μg/L	HFE genotype				0.0001
		C282Y/C282Y	4.189	66.0	10.7-404.9	0.0001
		C282Y/H63D	1.209	3.3	1.6-7.1	0.002
		H63D/H63D	0.682	2.0	0.9-4.2	0.08
		H63D/S65C	-0.825	0.4	0.05-3.7	0.5
		C282Y/wt	-0.308	0.7	0.4-1.4	0.4
		H63D/wt	0.084	1.1	0.7-1.8	0.8
		S65C/wt	0.231	1.3	0.5-2.9	0.6
		Age (years)				0.03
		39-45	0.585	1.8	1.03-3.1	0.04
		46-53	0.680	2.0	1.2-3.3	0.01
		Blood donation last 2 years				0.0001
		0	-0.0605	0.5	0.3-0.96	0.04
		1-4	-2.032	0.1	0.04-0.4	0.001
		>4	-3.450	0.03	0.004-0.3	0.001
		Alcohol (drinks/week)				0.0001
		6-15	0.271	1.3	0.9-2.0	0.20
		16-21	0.777	2.2	1.2-3.8	0.006
		>21	1.453	4.3	2.2-8.2	0.0001
		Egg (no./week)
	Logit-value (α) in this model is	>4	-0.716	0.5	0.3-0.95	0.04
	-2.66

Ann Hematol (2009) 88:957-965

Acknowledgements The authors express their gratitude to the staff at the Department of Clinical Biochemistry at Naestved Hospital for their entusiasm at the performance of this study, especially to Chief Physician Arne Bremmelgaard for devoted support and guidance and to Laboratory Technician Gitte Vedel Melsen for excellent technical assistance.


Hermann Heimpel
email: hermann.heimpel@uniklinik-ulm.de

Ulrich Dührsen

P Hofbauer

V Rigamonti-Wermlinger

Ernst-Dietrich Kreuser

Klaus Schwarz

Max Solenthaler

S Pauls

E.-D Kreuser

Bulky extramedullary hematopoiesis is not a rare complication of congenital dyserythropoietic anemia

Keywords: Congenital dyserythropoietic anemia, Extramedullary hematopoiesis, Imaging techniques

Bulky extramedullary hematopoiesis, usually detected in the thorax by imaging techniques, is a wellknown complication in many types of congenital anemias. Here, we describe 12 cases of congenital dyserythropoietic anemia with extramedullary hematopoiesis which was always located in the paravertebral space of the thoracic spine and in other paraspinal regions in a few cases. All bulks were originally detected in chest radiographs and confirmed by imaging techniques such as computed tomography and/or magnetic resonance imaging. In some cases, thoracotomy was performed for suspected malignancy. Although the true prevalence is not known, paravertebral masses in patients with CDA of any type are not uncommon and should be the first differential diagnosis considered when masses adjacent to the spine are detected in this disorder.

Introduction

Bulky extramedullary hematopoiesis, usually detected in the thorax by imaging techniques, is a well-known complication in many types of congenital anemias. It has been reported in up to 15% of patients with thalassemia intermedia [START_REF] Taher | Thalassemia intermedia: revisited[END_REF] and also in hereditary hemolytic anemias, such as sickle-cell-disease [START_REF] Gumbs | Thoracic extramedullary hematopoiesis in sickle-cell disease[END_REF], hereditary spherocytosis [START_REF] Bastion | Massive mediastinal extramedullary hematopoiesis in hereditary spherocytosis: a case report[END_REF][START_REF] Pulsoni | Mediastinal extramedullary hematopoiesis as first manifestation of hereditary spherocytosis[END_REF] or pyruvate kinase deficiency [START_REF] Plensa | Paravertebral extramedullary hematopoiesis due to kinase deficiency[END_REF]. In the majority of cases it is detected accidentally on routine X-ray of the thorax [START_REF] Bowling | Pulmonary extramedullary hematopoiesis[END_REF]. Usually it is asymptomatic, but may present a problem of differential diagnosis and result in extensive diagnostic procedures, which may prompt thoracotomy if a neoplastic lesion is suspected.

Here, we report six cases of bulky extramedullary erythropoiesis in a cohort of patients with congenital dyserythropoietic anemias (CDAs) observed since 1971 and review six further cases published as case reports in the literature.

Materials and methods

All cases were extracted from the data bank of the German registry on CDAs. The registry collates all cases of CDA seen in the departments of pediatric and adult hematology of the university of Ulm, as well as cases treated by other institutions known to the registry by correspondence and subsequent regular follow-up ("Own cases"). Collection and analysis of data was approved by the ethical committee of the University of Ulm, Germany. For data privacy protection, all cases were coded using a unique patient's number (CDA-UPN), thereby preventing recurrence of individual data. Also, patients reported in the literature also received a CDA_UPN. Due to the interest on this very rare disorder, many cases were published more than once; utmost care was taken to exclude double notifications. Diagnosis including classification of the type of CDA followed the definition criteria published before [START_REF] Heimpel | Congenital dyserythropoietic anemia type II: epidemiology, clinical appearance, and prognosis based on long-term observation[END_REF] and by detection of mutations in the CDAN1 gene [START_REF] Heimpel | Congenital dyserythropoietic anemia type I (CDA I): molecular genetics, clinical appearance and prognosis based on long-term observation[END_REF] in cases 026/ 01 and 398/01.

In four of our six patients, computed tomography (CT) and magnetic resonance imaging (MRI) were available as print outs or digital data. The CT scans were performed after contrast medium application (portalvenous phase, slice thickness of 5 mm). In case 252/01, only a chest CT was performed, in patient 065/01, the CT scan included the chest as well as the upper abdomen.

In patient no. 203/01, MRI of the cervical spine, the thorax, and the whole abdomen was acquired with the following axial sequences: native T1-weighted (FLASH 2 D) and T2-weighted sequences (HASTE) as well as contrast-enhanced T1-weighted sequences (VIBE) with fat saturation.

In patient no. 065/01, MRI of the thorax was performed with the following sequences: T2-weighted turbo spin echo (TSE) sequence (native) and T1-weighted spin echo sequence native and after contrast medium application in transversal and coronal slices.

Results

Pertinent data of 12 cases from 11 families are shown in Table 1. Eight and two out of 12 cases are of the less uncommon types II and I, respectively. In case 203/01 with additional thrombocytopenia, a GATA1 mutation was detected. In all cases masses were originally detected by chest X-ray and confirmed by computed tomography or magnetic resonance imaging. In case 252/01, a contrastenhanced CT was performed for confirmation of pulmonary embolism after splenectomy at the age of 42 years (Fig. 1); however, when previous radiographs films where retro- spectively analyzed, the mass was already seen at the age of 35 years. Age at which the thoracic mass was first detected varied between 11 and 52 years.

In three cases, the lesions caused symptoms. In case 065/ 01, the large bulks (Fig. 2) probably contributed to the respiratory problems in addition to anemia and heart failure. The masses were removed by thoracotomy at the age of 48 years but had regrown, and at the age of 63 years they reached the same size as before. Bilateral pleural effusions with respiratory distress were reported in case 023/01; the patient's condition improved after thoracocentesis and additional treatment with low-dose hydroxyurea and transfusions. In patient 398/01, the left-sided lesions caused neuropathic pain, which disappeared after local irradiation. In three cases, the detection of intrathoracic masses prompted thoracotomy before CT became available.

Histology and/or fine needle aspiration cytology was obtained in three cases The material showed mixed hematopoiesis, with preponderance of erythroblasts show-ing abnormalities on morphology as seen in the bone marrow. In case 026/01, a touch imprint made from the tumor bulk showed identical alterations as seen in the bone marrow (Fig. 3).

On computed tomography and magnetic resonance imaging, the paravertebral masses were located paraspinal and bilateral. On CT, the masses were isointense to the muscle tissue, relatively homogeneous and show no significant contrast enhancement. On MRI, the masses showed a homogenous contrast enhancement. No direct connection to the bone marrow of the vertebrae body could be found. The bone marrow in the tumor´s area showed no alteration in the contrast enhancement compared to the bone marrow in other spine regions. On T2-weighted images, the larger masses showed a hyperintense signal in the central area of the mass (Fig. 4) as a sign of edema.

Discussion

Extramedullary hematopoiesis beyond liver and spleen has been observed in both neoplastic and benign blood disorders. Within the latter, masses of extramedullary hematopoiesis may present in a variety of hereditary anemias with expansion of the erythroid bone marrow, such as thalassemia, sickle cell disease, and hemolytic anemias, particularly in hereditary spherocytosis (HS) [START_REF] Bowling | Pulmonary extramedullary hematopoiesis[END_REF][START_REF] Koch | Nonhepatosplenic extramedullary hematopoiesis: associated diseases, pathology, clinical course, and treatment[END_REF]. No figures on the prevalence within the different types of hemolytic anemia are available; the fact that most case reports describe bulky extramedullary hematopoiesis in HS may be just due to the higher prevalence of HS as compared with other types of hereditary hemolytic anemias. Altogether the occurrence in 12 cases with CDAs is known (Table 1). In a 32-year-old female with CDA I (026/ 01), a right-sided large paravertebral tumor was removed by thoracotomy. The same was true in a female with CDA II (065/01). Here, a thoracic mass was removed at the age of 48 years. Eighteen years later the bulk had regrown, as shown in Fig. 2. Considering the rareness of the CDAs, with an estimated prevalence of one in 0.5 to one million families (publication pendent), the number of observations of bulky extramedullary disease seems to be rather high. Again, we cannot provide sound data on the prevalence. Data to be obtained in cases of the German registry does not include routine CT scans of the thorax. As shown in case 252/01, chest X-ray alone may fail to detect paravertebral bulks and such masses may have remained undetected in other cases.

In CDAs, bulky extramedullary hematopoesis is usually confined to the paravertebral space of the thoracic spine, extending to the pleural space and compressing the lung in single cases. In one case, foci extended to the upper lumbar spine and in another case a presacral mass was detected. This observation favors the hypothesis that this pattern is rather due to the extrusion of the hyperplastic hemopoietic (erythroid) marrow rather than deposition of circulating hemopoietic stem cells, as assumed to occur in non-bulky hemopoietic foci observed in liver and spleen. However, the alternative hypothesis that the bulks observed develop from heterotopic hemopoietic cells is not excluded and is supported by autopsy findings failing any continuity between the ribs and the paravertebral masses [START_REF] Verani | Intrathoracic extramedullary hematopoiesis: report of a case in a patient with sickle-cell disease-beta-thalassemia[END_REF]. We did not observe compression of the spinal cord by a paravertebral mass, as described in thalassemia [START_REF] Hassoun | Spinal cord compression secondary to extramedullary hematopoiesis: a noninvasive management based on MRI[END_REF][START_REF] Saghafi | Extramedullary hematopoiesis with spinal cord compression in beta-thalassemia intermedia[END_REF]. However, this may also occur in other forms of ineffective erythropoiesis and therefore this possibility should be included in the differential diagnosis of neurological deficits of the lower limbs in patients with CDA. Extramedullary hematopoiesis is often considered as a compensatory mechanism in certain benign (congenital hemolysis) as well as malignant (myeloid metaplasia, myelopthisis) forms of chronic anemia. However, at least for the case of CDA, this is not a satisfying explanation. In our own cases, hemoglobin concentration at the time the bulk first was detected varied between 7.6 and 11.6 g/dl. In a report on three siblings of Moroccan origin with CDA type II, thoracic masses were detected in two of them, including one female who even had a normal hemoglobin [START_REF] Lugassy | Paravertebral extramedullary hematopoiesis associated with improvement of anemia in congenital dyserythropoietic anemia type II[END_REF]. A lifelong erythroid hyperplasia is the most plausible explanation.

On computed tomography, the paravertebral masses are typically bilateral, isointense to the muscle tissue, homogeneous, and show no significant contrast enhancement [START_REF] Gumbs | Thoracic extramedullary hematopoiesis in sickle-cell disease[END_REF]. During therapy, the size as well as the density of these paravertebral tumors may change. Differentiating neurogenic tumors such as neurofibromatosis type I or lymphoma has to be taken into consideration based on chest X-ray and also CT imaging alone. Additionally, imaging methods can be necessary to differentiate these masses. Single photon emission tomography (SPECT) after application of 99-TClabeled antineutrophilic antibodies [START_REF] Denzlinger | 54jährige Patientin mit Anämie, Ikterus, Hepatosplenomegalie und paravertebralen Raumforderungen[END_REF] specifically detects hematopoietic tissue. The combined SPECT/CT technique provides functional and morphologic information about the tumors and yields a high accuracy in diagnosis of an extramedullary hematopoiesis.

In conclusion, physicians who care for patients with CDA should be aware of the possibility that any masses detected in the thorax or adjacent to the spine could be extrusions of the hyperplastic marrow. No surgical interventions should be done unless there is evidence of neoplastic features of the lesion. Diligent analysis of the tissue structure, CT-directed needle aspirates and CT scans to detect hemopoietic bulk at other sites should be performed before the decision of a surgical removal is made in order to avoid iatrogenic damage in a chronic, nonmalignant disease.

Fig. 1

 1 Fig. 1 CT-scan of a 42-year-old female with CDA II (UPN 252/01)

Fig. 2 XFig. 3

 23 Fig. 2 X-ray of a 63-year-old female with CDA II (UPN 065/01)

Fig. 4

 4 Fig. 4 MRI sequences of the thorax from patient no. 065/01. Left Smooth delined paraspinal masses with hyperintense signal in T2 w sequence (axial slice) into the right paraspinal mass which corresponds to an edema. Right Contrast-enhanced T1 w sequence (coronar) with homogenous contrast enhancement of the masses

Table 1

 1 CDA_UPN: code of the German Registry on CDAs Age

	CDA_UPN	Type	Sex	Country	Age	Method	site	Symptoms	Sx	Hemoglobin g/dl
	026/01	I	F	DE	32	X-ray	Thorax right, paravertebral	No	No	9.3
	398/01	I	F	CH	56	X-ray, CT	Thorax bi, paravertebral	Yes	No	11.6
	065/01	II	F	DE	48	X-ray, CT, SPECT	Thorax bi paravertebral,	Yes	No	7.6
							pleura bi presacral
	116/01 [13]	II	F	IL	55	X-ray	Thorax bi paravertebral	No	No	10.7
	116/02 [13]	II	F	IL	51	X-ray, CT	Thorax bi, paravertebral	No	No	13
	182/01 [15]	II	F	CZ	41	X ray, MRI, CT	Thorax bi, LS paravertebral	No	No	8.0
	245/01 [16]	II	M	BR	13	X-ray, CT	Paravertebral	No	No	9.1
	252/01	II	M	DE	42	CT	Thorax bi paravertebral	No	No	10
	405/01	II	F	DE	56	X-ray	Thorax ?paravertebral	No	Yes	9.9
	322/01 [17]	II	M	GB	63	CT	Thorax left, paravertebral	No	Yes	11.5
	023/01 [18]	III	F	NL	33	X-ray, CT, SPECT	Thorax bi, paravertebral	Yes	No	7.6
	203/01	var	M	DE	39	X-ray, CT	Thorax bi, paravertebral	Yes	Yes	7.4

age (years) when extramedullary bulk was first detected, Country country of residence at time of diagnosis, Bi, bilateral, Sx splenectomy