Mastocytose systémique

I. Mastocytose systémique

La mastocytose systémique se caractérise par une accumulation anormale de mastocytes au

sein de différents organes, en particulier la moelle osseuse avec une atteinte osseuse dans 75%

des cas. La présentation clinique principale en est l’ostéoporose à prédominance trabéculaire

pouvant se compliquer de fractures notamment vertébrales. Ces manifestations osseuses

peuvent être les seules manifestations cliniques de la maladie et en être un mode de

révélation. L’ostéosclérose est une manifestation clinique osseuse plus rare et serait associée

aux formes les plus graves de la mastocytose systémique.

Nous avons souhaité dans cette étude étudier les caractéristiques histomorphométriques

associées à la mastocytose systémique. Pour se faire nous avons repris l’ensemble des

biopsies osseuses transiliaques réalisées dans le service d’histologie du CHU d’Angers de

1995 à 2010 pour en extraire celles dont le résultat était en faveur d’une mastocytose

systémique. Nous avons gardé pour l’analyse celles qui répondaient aux critères diagnostiques

de l’OMS à savoir le critère majeur et le critère mineur histologique correspondant à des

infiltrats denses multifocaux de mastocytes (> 15 mastocytes par aggrégat) contenant > 25%

de cellules allongées ou de morphologie atypique (Tableau 3).1845 biopsies osseuses

transiliaques ont été réalisées en 25 ans, parmi lesquelles 60 répondaient aux critères

d’inclusion.

Pour chacun des patients les éléments anamnestiques suivants ont été recueillis dans les

dossiers :

‐ les antécédents personnels de fractures vertébrales et périphériques,

‐ les facteurs de risque d’ostéoporose : âge de la ménopause, intoxication

éthylo-tabagique, indice de masse corporelle, glucocorticoïdes au long cours, pathologies

endocriniennes associées,

‐ les antécédents de traitement anti-ostéoporotique.

Pour chacun des patients les paramètres suivants ont été analysés :

- les paramètres histomorphométriques 2D des biopsies osseuses obtenus après

coloration au trichrome de Goldner, avec le bleu de toluidine et avec la réaction

TRAcP,

‐ les résultats histologiques de la biopsie ostéomédullaire réalisée le plus souvent au

décours de la biopsie osseuse ainsi que les résultats immunohistochimiques utilisant

les anticorps anti tryptase, anti-CD117 (c-kit) et anti-CD20,

‐ les radiographies du rachis vertébral dorsal et lombaire à la recherche de fractures

vertébrales,

‐ les résultats densitométriques au rachis lombaire, au col fémoral et à la hanche totale,

‐ le taux de tryptase sérique au diagnostic de la mastocytose systémique.

Résultats

Les résultats de ce travail sont détaillés dans l’article 3. Bièvement, les 60 patients ont été

adressés par les services de rhumatologie, de médecine interne, d’allergologie et des maladies

du sang. Parmi les 60 patients, 42 avaient un diagnostic de mastocytose systémique présumé

avant la réalisation de la biopsie osseuse demandée pour avoir une preuve diagnostique

histologique. Ces patients avaient des manifestations cutanées et allergiques de mastocytose.

Deux patients avaient une hémopathie associée : un myélome multiple et une

hyperéosinophilie, les autres avaient tous une mastocytose systémique indolente. Les 18

autres patients ont eu une biopsie osseuse transiliaque à la recherche d’une étiologie

expliquant la survenue de fractures vertébrales ou d’une ostéoporose densitométrique sans

étiologie évidente. Ces patients n’avaient a priori pas d’autres signes cliniques de

mastocytose systémique dont le diagnostic a donc été réalisé sur la biopsie osseuse.

Les résultats montrent une large distribution des paramètres histomorphométriques. Quel que

soit le paramètre histomorphométrique étudié il n’a pas été retrouvé de différence entre les

femmes fracturées et non fracturées et en fonction de l’indication initiale de la biopsie

osseuse. Les hommes fracturés avaient un volume trabéculaire plus bas et des surfaces

érodées (figure 25) plus importantes que les hommes non fracturés conduisant à une

désorganisation de la microarchitecture trabéculaire comme en témoigne l’augmentation de

l’index d’interconnectivité (ICI). Il n’y avait par contre aucune différence

histomorphométrique en fonction de l’indication initiale de la biopsie osseuse

Figure 25 : Augmentation des surfaces érodées (flèches), grossissement x100

Il existe également une large distribution des résultats densitométriques. Il n’a pas été observé

de différence de densité osseuse chez les femmes en fonction de leur état fracturé ou non

fracturé, ni différence selon l’indication initiale de la biopsie osseuse. Les hommes fracturés

avaient une densité osseuse significativement abaissée aux 3 sites comparé aux non-fracturés.

Les hommes dont l’indication initiale de biopsie osseuse était le bilan d’une ostéoporose

avaient également des densités osseuses aux 3 sites plus basses que ceux dont l’indication

initiale était la confirmation d’une mastocytose systémique. Hormis les patients avec

ostéosclérose, qui avaient une importante infiltration médullaire de mastocytes avec fibrose, il

n’y avait pas de différence histologique entre les patients fracturés et les patients non fracturés

et entre les patients dont le diagnostic était inconnu avant la biopsie osseuse et les autres.

Trente-trois patients ont eu une biopsie ostéo-médullaire, les nodules définis selon les critères

OMS n’étaient retrouvés que dans 21 cas (figure 26). Le taux de tryptase ne corrélait avec

aucun paramètre histomorphométrique ni résultat densitométrique.

Figure 26 : Nodule mastocytaire, coloration bleu de toluidine, grossissement x100

En plus des nodules intramédullaires, la disposition des mastocytes le long des travées est

constante. Cette disposition a été décrite en cas d’hyperparathyroïdie où l’élévation chronique

du taux de PTH augmenterait la production de C-kit par les cellules osseuses bordantes

attirant les mastocytes près des surfaces osseuses. L’autre possibilité serait l’interaction des

deux cellules via leurs intégrines comme cela a été décrit entre les précurseurs des mastocytes

et les cellules épithéliales dans le poumon et les intestins ([222] et figure 27).

Figure 27 : Interaction des mastocytes et ostéoblastes ; en haut, hypothèse moléculaire, en

bas, aspect histologique grossissement x400

ARTICLE 3

Systemic mastocytosis:

bone biopsy and histomorphometry for the diagnosis

Béatrice Bouvard, Erick Legrand, Maurice Audran, Emmanuel Hoppé, Charles

Masson, Régis Levasseur, Christian Lavigne, Anne-Bérangère Beucher, Martine

Drouet, Aline Schmidt-Tanguy, and Daniel Chappard

Systemic mastocytosis:

bone biopsy and histomorphometry for the diagnosis

Béatrice Bouvard, Erick Legrand, Maurice Audran, Emmanuel Hoppé, Charles

Masson, Régis Levasseur, Christian Lavigne, Anne-Bérangère Beucher, Martine

Drouet, Aline Schmidt-Tanguy, and Daniel Chappard

INSERM, U 922 – LHEA – IRIS-IBS Institut de Biologie en Santé (B.B., E.L., M.A.,

C.M., D.C.), Service de Rhumatologie (B.B., E.L., M.A., E.H., C.M., R.L),Service de

Médecine Interne (C.L., A.B.B.),Service d’Allergologie (M.D.), Service des Maladies

du Sang (N.I.) CHU d’Angers, 49933 ANGERS Cedex – France

please send all correspondence to:

* Béatrice BOUVARD, M.D. Tel: (33) 244 68 83 49

INSERM, U 922-LHEA

IRIS – IBS Institut de Biologie en Santé

Fax: (33) 244 68 83 50

CHU Angers e-mail:beatrice.bouvard@univ-angers.fr

49933 ANGERS Cedex - FRANCE

Short running title: bone biopsy in systemic mastocytosis

ABSTRACT

Systemic mastocytosis (SM) has frequent bone involvement leading to osteoporosis,

osteosclerosis and fractures. The aim of the study was to describe bone status of

patients with SM proved by pathological examination of transiliac bone biopsy from

1995 to 2010. We performed a retrospective analysis of histomorphometric and

histologic parameters of patients with SM. Sixty patients (37 males and 23 females)

who underwent a bone biopsy in the assessment of SM or in the assessment of

unexplained bone fragility were included. All of them had also a bone densitometry as

well as pelvic and vertebral X-rays; 33 had a bone marrow biopsy. In 18 cases bone

biopsy was done in the assessment of unexplained bone fragility and surprisingly

revealed a SM. Increase in eroded surfaces as well as a marked disorganization of

trabecular microarchitecture was observed in almost all patients. In addition to nodules

of mast cells, a high number of mast cells (MC) was directly apposed on the trabeculae,

affixed on the osteoblasts or the lining cells. One third of the bone marrow biopsies did

not exhibit MC nodules nor MC infiltration and led to a false negative diagnosis for

SM. SM can be discovered in the assessment of fracture or osteoporosis. Transiliac

bone biopsy allows to diagnose the disease more accurately than bone marrow biopsy;

it also provides a histomorphometric analysis of bone remodeling.

Abbreviations used in this paper

Aj.Ar Adjusted apposition rate

BMD Bone mineral density

BMD LS Bone mineral density at the lumbar spice

BMD TH Bone mineral density at the total hip

BMD FN Bone mineral density at the femoral neck

BMI Body mass index

BV/TV Trabecular bone volume

Ct.Th Cortical thickness

DXA Dual energy X-Ray absorptiometry

ES/BS Eroded surfaces

ICI Interconnectivity Index

ISM Indolent systemic mastocytosis

MCs Mast cells

MicroCT Microcomputed tomography

N.Oc/B.Ar Number of osteoclasts per mm2 of bone area

OS/BS Osteoid surface per surface volume

OV/BV Osteoid volume per bone volume

SM Systemic mastocytosis

SMI Structural model index

Tb.N Trabecular number

Tb.Pf Trabecular pattern factor

Tb.Sp Trabecular separation

Tb.Th Trabecular thickness

TRAcP Tartrate-resistant acid phosphatase

V*m.space Star volume of marrow spaces

Introduction

Systemic mastocytosis (SM) is a clonal hematologic disease involving mast cells (MC)

[1]. Clinical features and courses of SM vary greatly and range from completely

indolent with normal life expectancy to highly aggressive with short survival times [2].

The symptoms of SM are secondary to MC mediator release or MC infiltration into

affected tissues (histamine, serotonin, heparin, tryptase…) [3] The disease is

characterized by abnormal growth and accumulation of MCs in one or more visceral

organs such as skeleton or gastro-intestinal tract. Osteoporosis is considered to be the

most frequent skeletal manifestation and the disease can be revealed by vertebral

fractures; on the other hand, osteosclerosis or mixted pattern can also be observed but

are less frequent [4]. Histologic diagnosis of SM is usually done on a bone marrow

biopsy performed with a Jamshidi’s needle but the diagnosis cannot be ascertained in

25-30% of cases [5]. Transiliac bone biopsy is an interesting tool in the diagnosis of

idiopathic osteoporosis and other metabolic bone diseases [6-8]. Histomorphometric

analysis on 2D sections allows the evaluation of bone mass and microarchitecture

parameters; in addition it provides an accurate evaluation of bone cell activities

(osteoclasts and osteoblasts). Microtomography (microCT), a newly developed tool in

the analysis of 3D micro-architectural parameters, dœs not provide information on the

cellular content of the bone marrow but is rapid and can be used as a pre-diagnosis in

bone metastatic malignancies [9]. Bone marrow can also be studied after cytological

staining methods.

The objectives of this study were to describe histomorphometric characteristics and

bone involvement in a series of 60 patients affected by SM. The interest of bone biopsy

(with histomorphometry) and bone marrow biopsy (with immunohistochemical

staining) for the histopathological diagnosis has also been discussed.

Patients and methods

Study design

This retrospective study was conducted in a single university hospital to assess the bone

characteristics of patients in whom the diagnosis of SM was established by means of

transiliac bone biopsy. Results of 1845 transiliac bone biopsies realized from 1995 to

2010 were reviewed to select patients in whom SM has been diagnosed according the

WHO criteria [10].

Patients had two different initial indications of bone biopsy 1) to confirm the diagnosis

of clinically suspected SM, 2) in search of a secondary aetiology in unexplained

osteoporosis (associated with a low bone mineral density) or in patient with

unexplained fragility fractures (Figure 1).

Clinical characteristics

Current and former clinical manifestations of SM were searched, including cutaneous

manifestations (urticaria pigmentosa or pruritic rash), allergic manifestations to

hymenoptera venom or drugs, gastro-intestinal manifestations, hepatosplenomegaly or

adenopathy. History of non-traumatic fractures and risk factors of osteoporosis (early

menopause, tobacco and /or alcohol abuse, hypogonadism, hyperthyroidism, past or

current use of corticosteroids, familial history of fracture, BMI < 18 kg/m²) were

collected for each patient.

X-rays assessment

Pelvic, antero-posterior, lateral thoracic and lumbar spine X-rays done at the time of the

bone biopsy were reviewed by two trained investigators unaware of the patient status. A

vertebral fracture was defined as a reduction of at least 20% in the anterior, middle, or

posterior vertebral heights according to Genant’s definition [11]. Osteosclerosis was

defined as a diffuse or localized increase bone density observed on X-rays.

Bone Mineral Density assessment

Results of Bone mineral density (BMD) done at the time of the bone biopsy were also

collected. BMD was measured by dual energy X-ray absorptiometry (DXA). Lumbar

BMD was assessed from L2 to L4 in the posteroanterior view, and fractured vertebrae

were excluded from analysis. At the upper femur extremity, measurements of total hip

and femoral neck BMD were also performed. Osteoporosis was defined as a lumbar

spine or hip bone T-score of -2.5 or less [12].

Biologic parameters

Blood serum was collected at the time of biopsy and tryptase level was measured by a

commercial fluorœnzyme immunoassay (UniCAP Phadia, Uppsala, Sweden):

concentrations ≥ 20 ng/mL were considered as abnormally high and were confirmed on

another serum sample when possible [13].

Bone biopsies

All transiliac bone biopsies were performed after an informed consent was obtained

from every patient. The standardized biopsy procedure used a 7-mm internal diameter

trephine made with martensitic steel (Commeca SA, Beaucouzé, France); biopsy was

performed 2-cm below the iliac crest and 2-cm behind the anterosuperior iliac spine.

Biopsies were done after an analgesic premedication and under a bicortical anesthesia

as reported elsewhere [14]. All specimens were fixed in an alcohol-formalin solution

and embedded in poly (methylmethacrylate) without decalcification in a cold

environment to preserve bone enzyme activities. Sections, 7 μm in thickness, were

cut-dry using a Leica Polycut-S microtome. Staining for routine histomorphometry was

performed with a Goldner’s trichrome in six nonserial sections according to classical

histomorphometric procedures (one section every 100 μm). Six additional sections were

stained for the osteoclast tartrate-resistant acid phosphatase (TRAcP) [15]. Toluidine

blue staining was performed at pH 4.0 to identify MCs on 4 additional sections, 4

sections were left unstained for histodynamic analysis under UV light microscopy.

Histomorphometric analysis was performed on a Leica Quantimet Q550 image analyzer

(Leica France, Rueil Malmaison, France) and on semiautomatic systems with lab-made

software. Classical histomorphometric parameters were considered for diagnosis

according to the American Society for Bone and Mineral Research recommendations:

cortical thickness (Ct.Th, in mm), trabecular bone volume (BV/TV, in %) trabecular

characteristics including |thickness (Tb.Th, in mm), number (Tb.N, in 1/mm) and

separation (Tb.Sp, in mm). The spatial distribution of trabeculae was described by

Interconnectivity Index (ICI) and star volume of marrow spaces (_V

*_{m space.}

, in mm

³

)

[16]. Osteoid parameters (osteoid volume and surfaces, expressed in percentage) were

determined. Bone formation rate was appreciated by calculating adjusted apposition

rate (Aj.AR) derived from the measurement of mineral appositional rate and

mineralized surfaces. A special attention was paid with parameters of bone resorption:

eroded surfaces (ES/BS, in %) represent the amount of trabecular surfaces occupied by

scalloped areas reflecting the osteoclast activity. The number of osteoclasts (NOc/B.Ar,

in ¢/mm

²

) was obtained by counting the number of TRAcP positive cells. Control

values for the French population were used according to age and gender [17].

Microcomputed tomography (microCT)

Bone biopsies, still in the fixative, were scanned on a computerized microtomograph

(Skyscan 1172, Kontich, Belgium). Projections images of the whole bone biopsy height

were obtained at 80kV and 100µA with a 0.5mm aluminum filter according to

previously published protocol applied to human bone biopsies [9, 15]. The

magnification used for each biopsy was x32; a pixel corresponding to 8.29 µm and a

0.20° rotation was used at each step. The following parameters were determined with

the Ct-An 1.11 software: bone volume fraction (BV/TV

3D

,in %), trabecular thickness

(Tb.Th

3D

, in mm), number (Tb.N

3D

, in /mm) and separation (Tb.Sp

3D

, in mm),

Trabecular Bone Pattern factor (Tb.P

f

, an index of trabecular connectivity) and structure

model index (SMI, that quantifies the plate-versus-rod characteristic of trabecular bone.

Bone marrow biopsies

Bone marrow biopsies were performed with a Jamshidi’s needle (Sterylab, Milano,

Italy) at the immediate vicinity of the bone biopsy. Cores, 2 mm in diameter, were

immediately fixed in formalin, pH 7.4 and kept for 24 h at ambient temperature. Bone

marrow biopsies were then decalcified, embedded in paraffin and sectioned at 5 μm.

Standard HPS, Giemsa and toluidine blue staining were done. Immunohistochemistry

using monoclonal antibodies for tryptase, c-kit (CD117) and CD20 was performed. All

antibodies were obtained from Dako (Dako, Glostrup, Denmark).

Statistical analysis was performed using the Systat

^®

statistical software release 13

(Systat, San José, CA). For BV/TV values, the Z-score was determined to avoid

influence of gender and age. Values are expressed as mean ± SEM. We used student t

-Test for continuous variables and the Wilcoxon test for the non-parametric values. P

values less than 0.05 were considered statistically significant.

Results

Characteristics of the patients

Patients were recruited in four clinical departments: Rheumatology, Internal Medicine,

Allergy and Hematology units. From 1995 to 2010, 60 patients (37 males ranging from

30-74 y.o., 23 females ranging from 18-72 y.o.) whose characteristics are detailed in

Table 1 had a SM proved on the bone biopsy. In 42 cases, bone biopsy was realized in

the assessment of SM to provide a histologic proof of the disease; diagnosis of SM was

highly suspected because of cutaneous or allergic manifestations, with or without a high

level of serum tryptase. One patient had liver manifestations with ascites and portal

hypertension. Two patients had a SM with an associated clonal hematologic non-mast

cell lineage disease; others had an indolent systemic mastocytosis (ISM). Two patients

had already taken bisphosphonates for osteoporosis before the bone biopsy. In 18 cases,

diagnosis of SM was unknown before performing bone biopsy. These patients were

referred to the rheumatology unit for unexplained osteoporosis or unexplained fragility

fractures. Bone biopsies were done in search for a secondary osteoporosis and,

surprisingly, revealed MC nodules typical of SM.

In the whole series of 60 patients, 17 patients (6 women, 11 men) were considered as

osteoporotic according to the WHO definition of osteoporosis (with a T-score < -2.5 in

at least one site by DXA exam). None of the women had other risk factors of

osteoporosis (mean age 55.3 ± 1.9 yrs, 4 in postmenopause since 6 ± 2.7 yrs). Among

the 11 men (53.5 ± 3.5 years), 4 had other risk factors of osteoporosis. Among all SM

patients, 19 had at least one traumatic fracture: 16 patients had one or more

non-traumatic vertebral fracture (up to 8 fractures) and 4 patients had a peripheral

osteoporotic fracture (2 patients with ankle fracture, one with wrist fracture, and one

with hip fracture). Men with fractures had a significant lower BMD at lumbar spine,

total hip and femoral neck than non-fractured men. Altogether, 25 patients had an

established osteoporosis (identified by DXA) or a non-traumatic fracture, or both; 6

patients had a vertebral osteosclerosis on X-rays (Fig. 2). Figure 3 represents the

T-score distribution for lumbar spine, total hip and femoral neck.

Bone biopsies

MicroCT results

MicroCT parameters are detailed in Table 2 and Figure 5A to 5C. Data were available

for 50 patients (20 females and 30 males). There was no statistically significant

difference between females with and without fractures. Bone volume was highly

correlated between the 2D and 3D methods although microCT tended to slightly

overestimate BV/TV as previously reported [18]. The 3D models evidenced the

different types of findings ranging from osteoporosis with increased erosion areas,

normal microarchitecture or osteosclerosis with marked disorganization in the center of

the trabecular network. The most interesting finding was that men with fracture had

significant higher SMI and Tb.P

f

, confirming the marked disorganization of trabecular

microarchitecture.

Histomorphometric parameters

One biopsy was broken; it contained large nodules of MCs attesting to the diagnosis of

SM but did not permit quantitative analysis. Histomorphometric parameters are detailed

in Table 2 and main parameter distributions appear on Figure 4 showing a wide range

of distribution. High values for BV/TV and bone formation parameters were observed

in patients with osteosclerosis. The most striking finding was that 88 % of the patients

had an increase in eroded surfaces but no relationship could be found with any

microarchitectural descriptor. A marked disorganization of trabecular microarchitecture

was obvious (as evidenced by ICI, _V

*_{m space.}

). Whatever the histomorphometric

parameter considered, no significant difference was observed between women with and

without fractures and between women with a suspected diagnosis of SM and the others.

By contrast, men with fractures had a significant lower BV/TV, a significant increase in

eroded surfaces leading to a marked disorganization of the trabecular microarchitecture

as shown by an increased ICI.

Histologic analysis

In all biopsies, nodules with more than 15 cells were detected in the marrow spaces on

toluidine-blue stained sections. MCs were characterized by the presence of

metachromatic granules; they always appeared atypical with an elongated shape and in

some cases, they contained few granules scantily distributed in the cytoplasm (Figure

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