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, myoepithelial 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 and 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, especially leiomyosarcomas and highly cellular leiomyomas,  SMTs that more often may be mistaken for ESTs.

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 nucleosome  increasing access to transcription factors  NATURE |VOL 389 | 25 SEPTEMBER 1997

Histone deacetylases 8(HDAC8)  mainly a cytosolic distribution  expressed by cells showing smooth muscle differentiation(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 SMTs),  13 leiomyosarcomas (LMSs),  17 endometrial stromal tumors (ESTs) (4 endometrial stromal nodules and 13 low-grade endometrial stromal sarcomas) (3 with sex-cord-like differentiation and 5 with smooth 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 tissue 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 Tissues HDAC8 In the endometrium, endometrial stromal and glandular cells were uniformly negative arteriolar 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 HDAC8 only 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 HDAC8 Conventional 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 Leiomyomas HDAC8 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-Leiomyosarcomas HDAC8 11/13 Strongly positive (3) 15% - 90% cells Moderate positive (6) in 20% - 100% of cells weak to moderate positivity (2) in 35% - 50% of cells

Result-Leiomyosarcomas Smooth muscle myosin (10/13) strongly positive(9) in % 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 Tumors Smooth 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 Tumors EST with smooth muscle differentiation

Result-Endometrial Stromal Tumors HDAC8 (5/5) EST with smooth muscle differentiation weak to moderate positity staining restricted to areas of smooth muscle differentiation

Result-Endometrial Stromal Tumors Smooth 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, being 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 walls  not in the endometrial stroma, endometrial glands, or endothelium  current findings confirm the results of the original work.  Waltregny D, Glenisson W, Tran SL, et al. Histone deacetylase 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 HCL27 Pt’(95~22y/o) 1 Pt’ LMS6 Pt’ (78~30 y/o) 2 Pt’ (44~57y/o) Epithelioid SMT 2 Pt’ (45~53 y/o) 1 Pt’ (36 y/o) EST8 Pt’ (36~51 y/o) 6 Pt’ (42~55 y/o)