ALK-1 protein expression and ALK gene rearrangements aid in the diagnosis of inflammatory myofibroblastic tumors of the female genital tract.
The aim of this study was to better define the histologic features of IMT in the female genital tract and to investigate the utility of detecting ALK-1 expression by immunohistochemistry (IHC) and ALK gene rearrangements by fluorescence in-situ hybridization (FISH).
MATERIALS AND METHODS
A SNOMED search of the Mayo Clinic surgical pathology records from January 1, 2004 to January 1, 2011, was performed for IMT. Cases with primary site specified in the female genital tract were included. Thirteen cases were retrieved during that period. All cases were found to be outside referrals, seen as part of the consultation service by various members of the gynecologic pathology working group. Of these 13 cases, 8 (62%) had material available for review. The cases were rereviewed by 2 pathologists (N.E.F. and D.A.B.), including sections stained with hematoxylin-eosin from paraffin-embedded tissue as well as immunohistochemistry available from the original workup to confirm the diagnoses.
ALK-1 IHC Studies
ALK-1 immunohistochemistry was performed using 4-|im sections cut from formalin-fixed, paraffin-embedded blocks. The sections were placed on charged slides, then dried, and melted in a 62[degrees]C oven for 20 minutes. Slides were deparaffinized through xylenes and graded alcohols to tap water, and heat-induced epitope retrieval was performed by treating the slides in a Dako PT Link (Dako, Carpinteria, California), containing a solution of 1 mM ethylenediaminetetraacetic acid, pH 8.0, preheated to 97[degrees]C, for 30 minutes. All remaining steps were performed at room temperature. Endogenous peroxidase was blocked by placing the slides in a 1:1 solution of 3% hydrogen peroxide to absolute methanol. Slides were placed on the Dako Autostainer (Dako); primary antibody (mouse monoclonal ALK antibody, clone ALK1, Dako; 1:100 dilution) was applied and incubated for 30 minutes. Antigen-antibody reaction was visualized by an enhanced polymer-based detection system, ADVANCE (Dako), with 20-minute incubation for ADVANCE Link and 20-minute incubation for ADVANCE horseradish peroxidase. Diaminobenzidine (DAB+, Dako) was employed for 5 minutes as the chromogen. All slides were counterstained with hematoxylin, dehydrated, and had a coverslip applied for microscopic examination. The positive control was from a known CD30+ case of anaplastic large cell lymphoma. The negative control was a mouse immunoglobulin G1 serum substitution for the primary antibody (ALK-1). (3)
A dual-color, break-apart FISH probe strategy was used to determine ALK rearrangement (Abbott Molecular, Des Plaines, Illinois). The ALK region is flanked by 2 probes: a 250-kilobase (kb) probe labeled in spectrum orange (herein referred to as red [R]), which is telomeric to the breakpoint, and a 300-kb probe labeled in spectrum green (G), which is centromeric to the breakpoint. The close proximity of these probes results in a normal overlap of the R and G signals, referred to as a fusion (F) signal. Thus, a normal nucleus with 2 normal copies of the ALK gene will contain 2 fusion signals (2F). If a genetic rearrangement has split one copy of the ALK gene, the R and G will separate, resulting in a 1R1G1F pattern (the residual 1F corresponding to the normal ALK gene). Gains or losses of an intact gene region will result in gains or losses of a fusion signal (1F or 3F). Standard scoring criteria for interphase FISH were applied, which dictates at least one signal width must be present between 2 signals to count them as 2 separate signals. This minimizes the effect of artifactual signal splitting. The 5-[micro]m sections of formalin-fixed, paraffin-embedded tissues on glass slides were heat-treated in a 90[degrees]C oven (15 minutes), deparaffinized in xylene (2 X15 minutes), and dehydrated (2 X 100% ethanol). Air-dried, deparaffinized sections were microwave-treated (10 minutes) in 10 mM citric acid (pH 6.0), digested with Digestall 3 (Invitrogen, Carlsbad, California; 20 minutes) at 37[degrees]C, sequentially dehydrated in alcohols (70%, 85%, and 100%), and again, air-dried. The breakapart ALK probe working solution was applied to the entire slide, and co-denatured within a HYBrite unit (Abbott Molecular) at 80[degrees]C (5 minutes). The slides were then hybridized overnight in a humidified chamber at 37[degrees]C. Following hybridization, the slides were washed in 0.1% NP40/2X SSC at 72[degrees]C (2 min) and were then rinsed at room temperature in 0.1% NP40/2X SSC (1 minute). Slides were DAPI counterstained--4' ,6-diamidino-2-phenylindole dihydrochloride (1000 ng/mL in antifade mounting solution; Abbott Molecular)--and then coverslips were applied for examination.
The anatomic locations for the 8 IMTs included in this study were myometrium (n = 4) as well as endometrium, fallopian tube, cervix, and cervical polyp (n = 1 each). The age of the patients at time of diagnosis ranged from 25 to 52 years, with a mean age of 39 years. One patient was known to be 4 months postpartum. Histologic features ranged from bland spindle cells to striking cytologic atypia with pleomorphic vesicular nuclei and prominent nucleoli. All cases had low mitotic activity (ranging from 0 to 3 mitoses per 10 high-power fields), scattered mixed inflammatory infiltrates (composed of lymphocytes, histiocytes, eosinophils, and occasional neutrophils), and lacked true tumor necrosis. All 8 cases contained distinct areas of spindle cells embedded in a prominent myxoid background (Figure 1). Four tumors had striking cytologic atypia sufficient to be classified as IMT with atypical features, demonstrated by hypercellularity or fascicular growth or both and significant nuclear pleomorphism with large hyperchromatic or vesicular nuclei and prominent nucleoli (Figure 2).
Immunohistochemical staining was variable with CD10, keratin, and smooth muscle markers, including smooth muscle actin (SMA) and desmin, for both percentage of tumor cells that stained and the strength of the staining. Most cases showed at least partial SMA and desmin staining (up to 75% of tumor cells) and focal CD10 staining (<25% of tumor cells). ALK-1 staining was positive with a diffuse cytoplasmic staining pattern ([greater than or equal to] 25% of tumor cells) in 7 cases (case 1 to 7; Figure 3, a). The FISH studies detected ALK gene rearrangements in 5 cases (Figure 3, b). All 5 cases with abnormalities detected by FISH also had ALK-1 IHC staining (cases 1 to 5). Two (cases 6 and 7) were ALK-1 IHC positive only. A single tumor (case 8) was negative by both IHC and FISH, but demonstrated classical morphology for IMT. The morphology, IHC profile, and FISH results are summarized in the Table.
IMT is a predominantly benign, mesenchymal neoplasm, characterized histologically by interlacing fascicles of slender and stellate myofibroblasts, with eosinophilic cytoplasm that can have a hyalinized appearance, often intermixed with hypocellular areas of spindled to plump cells set in a myxoid stroma. A variable number of scattered and mixed inflammatory cells are usually present. The cytology is often bland, but IMT can show striking atypia, including vesicular or hyperchromatic nuclei with prominent nucleoli, which is seen relatively often in our experience. Occasionally, increased mitotic activity with atypical mitoses and focal necrosis can be present. "Atypical histology," including prominent fascicular growth, cytologic pleomorphism, atypical mitoses, and necrosis, has been reported to correlate with aggressive behavior.
IMT has been reported rarely in the uterus, cervix, or fallopian tube as individual case reports, (5-7) and one small series by Rabban and coworkers. (8) Most of these tumors occurred in the uterine corpus, and several presented as masses that prolapsed through the cervical os, as in our series. The morphologic and immunohistochemical features of these neoplasms, as demonstrated in this and prior studies, emphasize the difficulties in distinguishing these largely benign neoplasms from more common and clinically aggressive sarcomas of the female genital tract. The demonstration of ALK-1 expression, in most IMTs of the female genital tract, in this study and in prior reports, (7,8) and our demonstration of ALK gene rearrangements by FISH in IMT of the female genital tract, confirm the utility of these techniques in establishing this difficult morphologic diagnosis.
Overexpression of ALK-1 protein is variably present when considering IMT of all anatomic sites, from 10% to 100%, depending on the site studied. (4,9) ALK-1 staining is present in a significant proportion of IMTs from anatomic sites in which it is most commonly encountered, including lung (45%), gastrointestinal tract (60%), urinary bladder (62%-71%), and peritoneum (100%). (4,9,10,11) We demonstrated strong ALK-1 IHC positivity with a cytoplasmic staining pattern in 7 of 8 cases (88%), confirming the findings by Rabban and coworkers, (8) regarding ALK-1 IHC positivity in uterine IMT. This current series and the prior series both demonstrate more than 25% of tumor cells with ALK-1 staining, although the staining was heterogenous in our series, with ALK-12 areas present in all neoplasms in our study. Despite this variability, ALK-1 positivity has been shown to be relatively specific for IMTs. Rabban and coworkers (8) detected no ALK-1 staining among 7 uterine leiomyomas, 6 uterine leiomyosarcomas, 4 uterine carcinosarcomas, 4 endometrial stromal sarcomas, and 1 uterine lymphangiomyomatosis. Sukov and coworkers (11) failed to detect ALK-1 immunostaining in various spindle cell lesions of the bladder, including 16 leiomyosarcomas, 8 sarcomatoid carcinomas, 3 embryonal rhabdomyosarcomas, 3 reactive myofibroblastic proliferations, 1 malignant solitary fibrous tumor, and 1 schwannoma. However, Li and coworkers (12) showed that up to 28% of all soft tissue tumors have low-level ALK-1 expression, including leiomyosarcoma (3 of 18; 17%) and rhabdomyosarcoma (4 of 7; 57%), although the IMT was the only tumor to show consistent strong staining (4 of 4; 100%).
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This relative specificity of ALK-1 may be valuable diagnostically because IMT has a variable immunoprofile for other markers. As demonstrated in our cases (Table) and by others, IMTs can stain with smooth muscle markers (SMA and desmin) and can show focal keratin positivity as well as variable CD10 staining. Given the variety of tumors that may have a myxoid appearance in the female genital tract, immunohistochemical staining panels that include these antibodies are often used to aid in their differential diagnosis. Immunostains for smooth muscle markers, including SMA, caldesmon, and desmin, are typically strong in leiomyoma and leiomyosarcoma, but obviously play no role in differentiating those 2 neoplasms. (13) Strong CD10 immunostaining is usually present in endometrial stromal sarcoma, but can be a relatively nonspecific marker in this region. (14) Skeletal muscle markers, myoD1, and myogenin can be of value if embryonal rhabdomyosarcoma is a diagnostic consideration.
Approximately one-half of IMTs at various sites have been shown to harbor clonal cytogenetic aberrations of the anaplastic lymphoma kinase (ALK) gene on the short arm of chromosome 2 at 2p23. (15) This finding not only supports the assertion that IMT is truly a neoplastic process but also can serve as a reliable diagnostic tool. Our study, which detected ALK gene rearrangements by FISH in 5 of 8 cases (63%), confirms that this is also true for IMTs of the female genital tract. The presence of the ALK gene rearrangement in spindle cell neoplasms appears to be highly specific for IMT, thus far. Sukov and coworkers (11) found ALK gene rearrangements in 67% of tumors in a series that included 21 cases of IMTs of the urinary bladder, but found no such ALK gene rearrangement in the other tumors among 32 non-IMTs tested, including 16 leiomyosarcomas and 3 embryonal rhabdomyosarcomas. Li and coworkers (12) studied 249 soft tissue tumors and found only IMTs (2 of 3 cases; 75%) to contain ALK gene rearrangements by FISH, and whereas 4 non-IMTs, including 2 leiomyosarcomas, 1 rhabdomyosarcoma, and 1 malignant fibrous histiocytoma, demonstrated ALK gene amplification, no non-IMTs had rearrangements of the ALK gene. There was also a strong correlation between ALK-1 IHC overexpression and ALK gene rearrangements in our study and in studies by other investigators. Five of our cases had both ALK-1 IHC staining and FISH abnormalities, 2 with ALK1 IHC staining only, and most important from a diagnostic standpoint, only a single case was negative for both. Given the heterogeneity of ALK-1 immunohistochemical overexpression and reported overlap in staining with other tumors in the differential diagnosis, testing for characteristic ALK gene rearrangements in suspected cases may allow definitive diagnosis of IMT and may confirm the diagnosis in cases with only focal ALK-1 positivity.
The importance of reliable diagnostic tools to aid in differentiating IMT from myxoid sarcomas of the female genital tract is highlighted by the preliminary diagnoses of the referral pathologists of the tumors in this study, which included leiomyosarcoma in almost all of the cases. The morphologic appearance of IMTs, with bland to atypical spindle cells in a myxoid background and occasional mitotic activity and even necrosis, may be highly suggestive of leiomyosarcoma in the female genital tract. Because the prognosis between largely benign IMT and leiomyosarcoma is so discrepant and a diagnosis of leiomyosarcoma will inevitably result in hysterectomy, differentiating between these entities is of great clinical importance. Our study demonstrates that immunohistochemical overexpression of ALK-1 protein is a valuable adjunct to establishing the diagnosis of IMT in these circumstances. Although ALK-1 immunopositivity often correlates with the presence of ALK gene rearrangements, given the reported overlap with other tumors in the differential diagnosis and the often heterogenous staining pattern of ALK-1, detection of the rearrangement of the ALK gene may confirm the diagnosis of IMT in the face of morphology that is highly suggestive of malignancy.
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Neil E. Fuehrer, MD; Gary L. Keeney, MD; Rhett P. Ketterling, MD; Ryan A. Knudson, BS; Debra A. Bell, MD
Accepted for publication August 4, 2011.
From the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
The authors have no relevant financial interest in the products or companies described in this article.
Presented as a poster at the annual meeting of the United States and Canadian Academy of Pathology, March 2, 2011; San Antonio, Texas.
Reprints: Debra A. Bell, MD, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Hilton 11, Rochester, MN 55905-0001 (e-mail: Bell.Debra@mayo.edu).
Findings in 8 Cases of Inflammatory Myofibroblastic Tumor of the Female Genital Tract Case Site Age, SMA, Desmin, Keratin, No. y n = 7 n = 7 n = 6 1 Myometrium 48 Pos Pos 2 Myometrium 26 Neg Neg Neg 3 Cervix 48 Pos Pos Pos 4 Cervical polyp 42 Neg Pos Pos 5 Endometrium 32 N/A Pos Neg 6 Fallopian tube 25 Pos Pos Neg 7 Myometrium 52 Pos Neg N/A 8 Myometrium 40 Pos N/A N/A Analyzed, No. (%) 5 (71) 5 (71) 2 (33) Case Site CD10, Atypical Myxoid ALK No. n = 5 Cytology, Change, IHC, n = 8 n = 8 n = 8 1 Myometrium Pos (focal) No Yes Pos 2 Myometrium Pos Yes Yes Pos 3 Cervix N/A Yes Yes Pos 4 Cervical polyp Pos (focal) Yes Yes Pos 5 Endometrium Pos (focal) No Yes Pos 6 Fallopian tube N/A Yes Yes Pos 7 Myometrium Neg No Yes Pos 8 Myometrium N/A No Yes Neg Analyzed, No. (%) 4 (80) 4 (50) 8 (100) 7 (88) Case Site ALK No. FISH, n = 8 1 Myometrium Pos 2 Myometrium Pos 3 Cervix Pos 4 Cervical polyp Pos 5 Endometrium Pos 6 Fallopian tube Neg 7 Myometrium Neg 8 Myometrium Neg Analyzed, No. (%) 5 (64) Abbreviations: ALK, anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; N/A, not available; Neg, negative; Pos, positive; SMA, smooth muscle actin.
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|Title Annotation:||Original Article|
|Author:||Fuehrer, Neil E.; Keeney, Gary L.; Ketterling, Rhett P.; Knudson, Ryan A.; Bell, Debra A.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Jun 1, 2012|
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