Laurence de Leval, MD, PhD,* David Waltregny, MD, PhD,w Jacques Boniver, MD, PhD,*

Use of Histone Deacetylase 8 (HDAC8), a New Marker

of Smooth Muscle Differentiation, in the Classification

of Mesenchymal Tumors of the Uterus

Laurence de Leval, MD, PhD,* David Waltregny, MD, PhD,w Jacques Boniver, MD, PhD,*

Robert H. Young, MD,z Vincent Castronovo, MD, PhD,w and Esther Oliva, MDz

Am J Surg Pathol Volume 30, Number 3, March 2006

ESTs vs. SMTs

ESTs may be hypocellular secondary to fibrous or myxoid background

smooth muscle tumors.

leiomyoma variants, typically HCL, or less frequently highly cellular intravenous leiomyomatosis

ESTs.

markers of smooth muscle

1) smooth muscle alpha-actin (SMA) actin isoform mostly restricted to smooth mucle cells, myoe

pithelial cells, and myofibroblasts

2) Desmin Intermediate filament also expressed in some ESTs.

markers of smooth muscle

3) h-caldesmon a cytoplasmic protein ,interactions with actin a

nd tropomyosin more specific than desmin in the diagnosis of

uterine SMTs.

4) smooth muscle myosin heavy chain widely used in the diagnosis of smooth muscle

tumors of the uterus.

marker of ESTs

CD10 specific marker of ESTs CD10 in a substantial proportion of SMTs, espe

cially leiomyosarcomas and highly cellular leiomyomas,

SMTs that more often may be mistaken for ESTs.

Histone deacetylases (HDACs)

Histone deacetylases (HDACs)

Histone core represses transcription in vivo and in vitro.

Deacetylzed histone amino termini decreasing their affinity for DNA

allow the termini to be displaced from the nucleosomeincreasing access to transcription factors

NATURE |VOL 389 | 25 SEPTEMBER 1997

Histone deacetylases 8(HDAC8)

mainly a cytosolic distribution expressed by cells showing smooth muscle differe

ntiation(visceral and vascular smooth muscle cells, myoepithelial cells , myofibroblasts)

not been assessed in mesenchymal neoplastic tissues, and it remains to be determined

can be used as a marker of smooth muscle differentiation in neoplastic conditions

Study Materials 15 typical leiomyomas (LMs), 9 highly cellular leiomyomas (HCLs), 8 epithelioid smooth muscle tumors (epithelioid SMT

s), 13 leiomyosarcomas (LMSs), 17 endometrial stromal tumors (ESTs) (4 endometrial stromal nodules and 13 low-grade en

dometrial stromal sarcomas) (3 with sex-cord-like differentiation and 5 with smoot

h muscle differentiation)

Study Materials

10 samples including myometrium and endometrium were examined as controls.

from the Pathology Departments of the Massachusetts General Hospital and of the C.H.U. Sart-Tilman, and from the consultation files of one of us (R.H.Y.).

Immunohistochemistry

All cases were stained with specific antibodies against HDAC8, desmin, h-caldesmon, a-SMA, smooth muscle myosin heavy chain, and CD10. using the avidin-biotin detection system

Negative controls were performed Cases were considered negative when less than 5

% of the tumor cells were immunoreactive.

Immunohistochemistry Five-micrometer, formalin-fixed, paraffin-embedded t

issue sections deparaffinized in xylene rehydrated in graded alcohols. blocking of the endogenous peroxidase activity with

0.3% hydrogen peroxide in methanol for 30 minutes heat-induced antigen retrieval cool down incubation with primary antibodies.

Result- Normal Uterine Tissues

HDAC8 strongly stained myometrial and vascular smooth muscle cells (Fig. 1A).

Result- Normal Uterine TissuesHDAC8 In the endometrium, endometrial stromal and glandular cells were uniformly negativearteriolar smooth muscle cells stained positive

Result- Normal Uterine Tissues

HDAC8 The same distribution of expression was observed in foci of adenomyosis

Result- Normal Uterine Tissues

HDAC8only stains the vascular walls in an endometrial polyp

Result- Normal Uterine Tissues

Desmin, h-caldesmon, and smooth muscle myosin showed the same staining pattern than that observed for HDAC8.

SMA displayed moderate but extensive staining of the endometrial stroma.

CD10 stained strongly and diffusely the endometrial stroma, and it was negative in the myometrium.

Result-Conventional Leiomyomas

HDAC8Conventional leiomyoma

moderate to strong HDAC8 expression in most tumor cells (90%–100%)

Result-Conventional Leiomyomas

smooth muscle myosin

conventional leiomyoma

strongly positive

Result-Conventional Leiomyomas

Desmin, h-caldesmon, and SMA were also diffusely and strongly expressed by all tumors.

CD10 showed variable positivity in six cases

(weak to moderate [2] and moderate to strong [4] in up to 60% of tumor cells).

Result-Highly Cellular LeiomyomasHDAC8

8 of 9 HCLs were positive

(ranging from 40% to 90% positive tumor cells)

(intensity of staining being weak in 1 case, moderate in 4 cases, and strong in 4 cases )

Result-Highly Cellular Leiomyomas

smooth muscle myosin

9/9 strongly expressed

(in 7/9 neoplasms at least 80% of the tumor cells.)

desmin –9/9 strongest positivity (3+ in all cases).

h-caldesmon – 9/9 varied from 10% to 90%.

Result-Epithelioid Smooth Muscle Tumors

HDAC8 (8/8)

diffuse cytoplasmic and membranous staining

weak (4 cases) moderate (3 cases)moderate to strong(1) positive cells varied from 40% to 100%

Result-Epithelioid Smooth Muscle Tumors

smooth muscle myosin(3/8)

diffuse cytoplasmic and membranous staining

(20% to 70% of neoplastic cells)

h-caldesmon – 1/8 in one third of the tumor cells.

Result-LeiomyosarcomasHDAC8 11/13Strongly positive (3) 15% - 90% cellsModerate positive (6) in 20% - 100% of cellsweak to moderate positivity (2) in 35% - 50% of cells

Result-LeiomyosarcomasSmooth muscle myosin (10/13)

strongly positive(9) in 25 - 100%

moderate staining (1) of 10% tumor cells

h-caldesmon –12/13 strongly positive (11) 15% to 100% cells weak staining (1) in 45% of cells

SMA –12/13 20% to 100% of tumor cells

CD10 –5/13 >30% of tumor cells, strong immunoreactivity (4)

Result-Endometrial Stromal Tumors

HDAC8 (0/9) negative in conventional ESTs

Result-Endometrial Stromal TumorsSmooth muscle myosin (0/9) negative in a conventional EST

Result-Endometrial Stromal Tumors

HDAC8 (0/3)negative in 3 ESTs with sex-cord differentiation

Result-Endometrial Stromal TumorsEST with smooth muscle differentiation

Result-Endometrial Stromal TumorsHDAC8 (5/5) EST with smooth muscle differentiationweak to moderate positity

staining restricted to areas of smooth muscle differentiation

Result-Endometrial Stromal TumorsSmooth muscle myosin(3/5)positive in an area of smooth muscle differentiation

*Only in areas of smooth muscle differentiation.

In conventional areas of EST (1) and in areas of smooth muscle differentiation (3).

In conventional areas of EST and in areas of smooth muscle differentiation (4) and only in metaplastic areas (1).

Discussion

☆Our current study confirms the lack of specificity of CD10

☆HDAC8 in LMSs tended to be less intense ☆ESTs may be positive for actin and desmin

☆SMA also stained endometrial stromal cells surrounding endometrial glands

Discussion-about CD10

CD10 Chu and Arber first reported

16/16 ESSs versus 2/19 SMTs (2 cellular leiomyomas)

several follow-up studies CD10 is expressed to variable extent in uterine SMTs, bein

g more commonly positive in LMSs and also other sarcomas

McCluggage WG, Sumathi VP, Maxwell P.Oliva E, Young RH, Amin MB, et al.Toki T, Shimizu M, Takagi Y, et al.

☆ desmin displayed the highest intensity and extent of expression In HCLs.

☆ all uterine epithelioid SMTs were HDAC8 positive, although the degree of positivity was usually weak to moderate.

☆most epithelioid SMTs were completely negative for both desmin and h-caldesmon

Discussion

HDAC8 expression is seen in the myometrium and vascular wall

s not in the endometrial stroma, endometrial glands, or en

dothelium current findings confirm the results of the original

work. Waltregny D, Glenisson W, Tran SL, et al. Histone deacetylas

e HDAC8 associates with smooth muscle alpha-actin and is essential for smooth muscle cell contractility. FASEB J. 2005;19:966–968.

☆HDAC8 and h-caldesmon only stained areas of smooth muscle differentiation, but HDAC8 was more sensitive in detecting smooth muscle differentiation

北醫 萬方醫院

HCL 27 Pt’(95~22y/o)

1 Pt’

LMS 6 Pt’ (78~30 y/o)

2 Pt’ (44~57y/o)

Epithelioid SMT

2 Pt’ (45~53 y/o)

1 Pt’ (36 y/o)

EST 8 Pt’ (36~51 y/o)

6 Pt’ (42~55 y/o)