PAX7 Expression in Rhabdomyosarcoma, Related Soft...

PAX7 Expression in Rhabdomyosarcoma, Related SoftTissue Tumors, and Small Round Blue Cell Neoplasms

Gregory W. Charville, MD, PhD,* Sushama Varma, BSc,* Erna Forgo, MD,*Sarah N. Dumont, MD, PhD,w Eduardo Zambrano, MD, MS,*z Jonathan C. Trent, MD, PhD,y

Alexander J. Lazar, MD, PhD,8 and Matt van de Rijn, MD, PhD*

Abstract: Rhabdomyosarcoma, the most common soft tissue

malignancy of childhood, is a morphologically variable tumor

defined by its phenotype of skeletal muscle differentiation. The

diagnosis of rhabdomyosarcoma often relies in part on the

identification of myogenic gene expression using immuno-

histochemical or molecular techniques. However, these tech-

niques show imperfect sensitivity and specificity, particularly in

scant tissue biopsies. Here, we expand the toolkit for rhabdo-

myosarcoma diagnosis by studying the expression of PAX7, a

transcriptional regulator of mammalian muscle progenitor cells

implicated in the pathogenesis of rhabdomyosarcoma. Im-

munohistochemical analysis of tissue microarrays using a

monoclonal anti-PAX7 antibody was used to characterize PAX7

expression in 25 non-neoplastic tissues, 109 rhabdomyosarco-

mas, and 697 small round blue cell or other soft tissue tumors.

Among non-neoplastic tissues, PAX7 was specifically expressed

in adult muscle progenitor cells (satellite cells). In embryonal

rhabdomyosarcoma, PAX7 expression was positive in 52 of 63

cases (83%), negative in 9 of 63 cases (14%), and focal in 2 of 63

cases (3%). PAX7-positive embryonal rhabdomyosarcoma cases

included several showing focal or negative myogenin expression.

PAX7 expression in alveolar rhabdomyosarcoma was positive in

6 of 31 cases (19%), negative in 14 of 31 cases (45%), and focal

in 11 of 31 cases (36%). In addition, PAX7 was expressed in 5 of

7 pleomorphic rhabdomyosarcomas (71%) and 6 of 8 spindle

cell rhabdomyosarcomas (75%). Among histologic mimics, only

Ewing sarcoma showed PAX7 expression (7/7 cases, 100%). In

contrast, expression of PAX7 was not seen in the large majority

(688/690, 99.7%) of examined cases of other soft tissue tumors,

small round blue cell neoplasms, and leukemias/lymphomas. In

summary, immunohistochemical analysis of PAX7 expression

may be a useful diagnostic tool in the assessment of skeletal

muscle differentiation in human tumors.

Key Words: rhabdomyosarcoma, PAX7, immunohisto-

chemistry, cancer stem cell, Ewing sarcoma

(Am J Surg Pathol 2016;40:1305–1315)

Rhabdomyosarcomas represent a category of softtissue malignancies exhibiting features of skeletal

muscle differentiation. Two major subtypes of rhabdo-myosarcoma—alveolar rhabdomyosarcoma (ARMS) andembryonal rhabdomyosarcoma (ERMS)—have beendifferentiated on the basis of their histomorphologic ap-pearance, clinical behavior, and molecular pathogenesis.The majority of rhabdomyosarcomas occur in the young,making rhabdomyosarcoma the most common soft tissuemalignancy of childhood which accounts for an estimated40% of pediatric soft tissue tumors.1

The morphologic diagnosis of rhabdomyosarcoma isoften difficult owing to its variable “small round blue cell” or“spindle cell” histomorphology. Thus, immunohistochemicaldetection of genes expressed during myogenic differentiationrepresents an important diagnostic tool.2 In particular, theimmunohistochemical detection in tumor cell nuclei of my-ogenin (MYOG) and MYOD1, transcription factors thatfunction as regulators of myogenesis, currently serves as thebenchmark for rhabdomyosarcoma diagnosis.3 Althoughthe expression of either MYOG or MYOD1 is sufficient todiagnose rhabdomyosarcoma, the sensitivity and specificityof these 2 immunohistochemical markers, even when usedin combination, is imperfect.2,4 Negative or focal MYOGexpression, particularly in ERMS, has been noted in aconsiderable proportion of cases.5–8 Furthermore, the inter-pretation and practical utility of MYOD1 immunostaininghas been hindered by high background and cytoplasmicstaining that renders complex the assessment of nuclear ex-pression.3,6,9 These limitations are often compounded byscant diagnostic tissue in biopsy specimens.

PAX7 is a paired box transcription factor requiredfor the developmental specification of mammalianskeletal muscle stem cells, also known as satellite cells, arare population of tissue-resident adult stem cells thatreside at the periphery of the skeletal muscle fiber sand-wiched between the basal lamina and the muscle fibersarcolemma.10 PAX7 is known to play a critical role inmammalian myogenesis, albeit partially redundant with

From the Departments of *Pathology; zPediatrics, Stanford UniversitySchool of Medicine, Stanford, CA; wDepartment of Medical On-cology, Institut Gustave-Roussy, Villejuif, France; yDepartment ofInternal Medicine, and Sylvester Comprehensive Cancer Center,Division of Hematology/Oncology, University of Miami MillerSchool of Medicine, Miami, FL; and 8Department of Pathology,The University of Texas MD Anderson Cancer Center, Houston,TX.

Conflicts of Interest and Source of Funding: The studies reported hereinwere supported by departmental research funds. The authors havedisclosed that they have no significant relationships with, or financialinterest in, any commercial companies pertaining to this article.

Correspondence: Gregory W. Charville, MD, PhD, Department ofPathology, Stanford University School of Medicine, 300 PasteurDrive, Lane 235, Stanford, CA 94305-5324(e-mail: [email protected]).

Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

ORIGINAL ARTICLE

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mailto:[email protected]

its paralog PAX3.11 In the hierarchy of transcriptionalregulation of mammalian myogenesis (Fig. 1A), PAX7controls early lineage specification, whereas MYOD1 andMYOG regulate subsequent lineage commitment andterminal differentiation.12,13 Translocations involvingPAX7 or PAX3 and FKHR underlie the large majority ofARMS, indicating a role for PAX7/3 in the pathogenesisof myogenic tumors. Intriguingly, recent elegant geneticstudies in mice have shown that inducing oncogenic mu-tations in skeletal muscle progenitors, including thoseexpressing PAX7, gives rise to rhabdomyosarcomaamong other tumors.13 Although PAX7 expression hasbeen observed at the mRNA level in a subset of rhab-domyosarcomas,14 its expression at the protein level andin particular its immunohistochemical evaluation as adiagnostic tool have yet to be studied. Here, we explorethe diagnostic utility of immunohistochemical analysesof PAX7 expression in rhabdomyosarcoma by charac-terizing its expression in a series of alveolar and ERMSs,related soft tissue tumors, histologic mimics, and non-neoplastic tissues.

MATERIALS AND METHODS

Tissue Microarrays and CasesThe tissue microarrays (TMAs) were constructed

using a tissue arrayer (Beecher Instruments, Silver Spring,MD) as previously described.15 Tissues were evaluated as0.6-mm diameter cores taken from representative areas of

each formalin-fixed paraffin-embedded block. Cores werenot considered if targeted tissue was not included on thearray, as assessed morphologically for each core. TwoTMAs (Stanford Tissue Microarray Database #412 and#414) consisted of 50 and 95 cores, respectively, of non-neoplastic tissues with each tissue represented by a vari-able number (2 to 13) of cores. An additional TMA(Stanford Tissue Microarray Database #23) included ar-chival material from 63 ERMS cases, 16 ARMS cases,and 8 spindle cell rhabdomyosarcoma cases, with 62represented in triplicate cores, 7 represented in duplicatecores, and 18 represented in single cores. These rhabdo-myosarcoma cases were used in 2 prior studies.6,16 Of theERMS cases, 14 represented recurrences. Of the ARMScases, 5 represented recurrences. Another TMA (StanfordTissue Microarray Database #166) contained themajority of soft tissue tumors and histologic mimicsof rhabdomyosarcoma, cases of which were eachrepresented as single tissue cores. Cases of synovial sar-coma, tenosynovial giant cell tumor, granular cell tumor,and schwannoma were represented as single cores on aseparate TMA (Stanford Tissue Microarray Database#170). Hematologic malignancies were retrieved from thesurgical pathology and consultation files of the StanfordHealth Care Department of Pathology and were eval-uated as single cores on 2 TMAs (Stanford TissueMicroarray Database #99-102).

Cases of mesenchymal chondrosarcoma, pleomor-phic rhabdomyosarcoma, and CIC-DUX4 round cell

A

B

Lineagespecification

Lineagecommitment

Terminaldifferentiation

PAX7 MYOD1 MYOG

Myoblasts Muscle fibersSatellite cellsEmbryonic

precursor cells

C

FIGURE 1. PAX7 expression in skeletal muscle satellite cells. A, Schematic showing transcriptional regulation of mammalianmyogenesis by PAX7, MYOD1, and MYOG. B, Representative image showing PAX7 expression localized to satellite cells in adultskeletal muscle (tongue) by immunohistochemistry. C, Magnified image of area highlighted by black dashed line in (B), showingPAX7 expression localized to satellite cells in adult skeletal muscle.

Charville et al Am J Surg Pathol � Volume 40, Number 10, October 2016

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sarcoma were retrieved from the surgical pathology andconsultation files of the Stanford Health Care Depart-ment of Pathology. Genetic confirmation by reversetranscription with polymerase chain reaction was pre-viously performed for the case of CIC-DUX4 round cellsarcoma.

Histologic and Molecular ClassificationHistologic classification of rhabdomyosarcomas

was performed as previously described.6 The originalhematoxylin and eosin (H&E)-stained slides were re-viewed by 3 pathologists. For cases without a definitehistomorphologic diagnosis, the diagnosis of rhabdo-myosarcoma was confirmed with immunohistochemistryfor desmin, MYOG, and MYOD1. The minimum criteriafor classification as rhabdomyosarcoma were: (1) im-munohistochemical detection of nuclear-localized MY-OD1 or MYOG expression or (2) morphologic evidenceof rhabdomyoblasts and immunohistochemical evidenceof desmin expression. All cases were classified into his-tologic subtypes of rhabdomyosarcoma using H&E-stained slides and International RhabdomyosarcomaStudy Group/Children’s Oncology Group criteria.

For correlation of translocation status with PAX7expression in ARMS, cases of ARMS from The Uni-versity of Texas MD Anderson Cancer Center, archivedbetween 1957 and 2001, were assembled into a TMA(n=39). All cases on this array were represented as 2separate cores. Gene translocation status was determinedby fluorescence in situ hybridization using PAX3, PAX7,and FOXO1 loci-specific probes, as previously de-scribed.17 Only ARMS cases with molecularly definedPAX3/7-FOXO1 translocations were used for this portionof the study (n=15).

ImmunohistochemistryAfter antigen retrieval, microarray sections were

stained with antibodies according to standard protocols forformalin-fixed paraffin-embedded material. A monoclonalantibody against PAX7 raised in mouse was purchasedfrom the Developmental Studies Hybridoma Bank (IowaCity, IA).18 Anti-PAX7 antibody from hybridoma super-natant was used at a dilution of 1:200 (by volume) fol-lowing antigen retrieval using citrate buffer. A monoclonalantibody against MYOG (clone F5D) raised in mouse waspurchased from Dako (Carpinteria, CA). Anti-MYOGantibody was used at a dilution of 1:80 (by volume) fol-lowing antigen retrieval using heat. For both PAX7 andMYOG, immunoreactivity was considered positive if>20% of tumor cells showed staining with appropriatenuclear chromogen localization. If 1% to 20% of tumorcells showed appropriate staining, the immunoreactivitywas considered focal. If <1% of tumor cells showed ap-propriate staining, the immunoreactivity was considerednegative. A monoclonal antibody against MYOD1 (clone5.8A) raised in mouse was purchased from Leica (BuffaloGrove, IL). Anti-MYOD1 antibody was used at a dilutionof 1:10 (by volume) following heat-induced antigen

retrieval using the Leica Epitope Retrieval Solution 2(EDTA-based solution at pH 9.0).

RESULTSPAX7 expression has been used in the past as a

marker of mammalian skeletal muscle stem cells, or“satellite cells.”10,19,20 We, therefore, first sought to vali-date the specificity of the immunohistochemical detectionof PAX7 by analyzing its expression in skeletal muscleand other non-neoplastic tissues. Anti-PAX7 antibodyshowed strong, specific nuclear localization in rare cellslocated at the periphery of the muscle fiber (Figs. 1B andC), consistent with expression in muscle satellite cells.Constituent myonuclei of skeletal muscle fibers, in addi-tion to other muscle-resident cell types, showed no iden-tifiable PAX7 expression.

Having confirmed the specificity of the anti-PAX7antibody for satellite cell nuclei in skeletal muscle, we nextanalyzed its expression in a broad range of nonpathologictissue specimens (Fig. 2). These tissues included thyroid,tonsil, liver, skin, adrenal gland, large and small bowel,placenta, breast, prostate, seminal vesicle, cervix, peripheralnerve, salivary gland, bladder, heart, parotid gland, endo-metrium, hematopoietic marrow, kidney, pancreas, ovary,parathyroid, and salivary gland. Although no nuclearstaining was seen in the evaluated tissues, nonspecific cy-toplasmic or extracellular staining was present in thyroid,oxyntic stomach, and submandibular gland (Fig. 2). Ofnote, no significant nuclear immunoreactivity was observedin tissues known to express the other diagnostically relevantpaired box transcription factors PAX2 (seminal vesicle andendometrium), PAX5 (tonsil), and PAX8 (kidney, thyroid,and endometrium) (Fig. 2). Taken together, these findingsindicate that PAX7 is a specific marker of skeletal muscleprogenitors in non-neoplastic human tissue.

Given that rhabdomyosarcoma is thought to rep-resent a tumor with characteristics of skeletal muscledifferentiation, we hypothesized that detection of PAX7protein by immunohistochemistry may be diagnosticallyuseful in the identification of rhabdomyosarcoma. Weanalyzed PAX7 expression in 63 cases of ERMS. Amongthese cases, 52 (83%) showed positive staining, 9 (14%)showed negative staining, and 2 (3%) showed focalstaining (defined as nuclear immunoreactivity in 5% to20% of lesional cells). Several PAX7-expressing ERMScases showed negative or scant MYOG expression(Figs. 3A–D). Although the majority of ERMS casesshowed only nuclear localized PAX7 immunoreactivity,rare cases showed strong nuclear and cytoplasmic re-activity (Fig. 3B). Cytoplasmic immunoreactivity withoutnuclear immunoreactivity was not observed in any cases.

We identified 16 cases of ERMS that were negativefor nuclear anti-MYOG immunoreactivity (Fig. 3D). Ofthese cases, 12 (75%) were positive for PAX7 expression.Among the additional 15 cases showing MYOG ex-pression in <20% of the lesional cell population, PAX7expression was present in all cases (15/15, 100%). Therewere 31 MYOG-positive cases (expression in >20% of

Am J Surg Pathol � Volume 40, Number 10, October 2016 PAX7 Expression in Rhabdomyosarcoma

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cells), 27 of which were PAX7 positive (86%). The 2 casesof focal PAX7 expression both showed focal MYOGexpression in <20% of cells. The PAX7-expressing casesof ERMS were derived from various anatomic locations,including retroperitoneal, paratesticular, orbital, vaginal,and thoracic sites. All 6 cases of botryoid ERMS showedpositive PAX7 expression (6/6, 100%). In addition, themajority of examined recurrent ERMS cases (9/14, 64%)were immunoreactive for PAX7.

The finding of heterogenous PAX7 expression inERMS cases led us to further characterize the PAX7-negative subset. There was no clear trend in histologicsubtype or anatomic site among the 9 PAX7-negativetumors. Although a subset (5/9, 56%) of PAX7-negativeERMS showed MYOG expression, the remainder (4/9,44%) was MYOG negative (Fig. 4). Four of 9 PAX7-negative cases were recurrences (44%).

Nuclear anti-MYOD1 reactivity was present in 36.5%of ERMS cases (23/63). Interestingly, all 23 MYOD1-positive cases were also MYOG positive (23/23, 100%).Of 54 PAX7-positive ERMS cases, 22 (41%) wereMYOD1 positive. A single PAX7-negative case showedMYOD1 expression (in addition to MYOG expression).

Spindle cell rhabdomyosarcoma is a histologic var-iant of rhabdomyosarcoma that is distinguished from othersubtypes by its clinical presentation, morphologic appear-ance, and prognosis.21 Earlier studies have suggested thatspindle cell rhabdomyosarcomas exhibit a greater degree ofmyogenic differentiation relative to other variants, based onthe expression of late myogenesis markers, such as titin and

myoglobin.22 We, therefore, analyzed PAX7 expression in aseparate cohort of spindle cell rhabdomyosarcomas. Of 8cases, 6 (6/8, 75%) showed nuclear anti-PAX7 immunor-eactivity. These 6 PAX7-positive spindle cell rhabdomyo-sarcomas were also MYOG positive. The 2 PAX7-negativecases (2/8, 25%) were also MYOG negative. Therefore,although PAX7 is an early marker of myogenic differ-entiation in comparison with MYOG, the 2 markersshowed equal sensitivity and specificity in the examinedspindle cell rhabdomyosarcomas.

In contrast to the spindle cell variant, pleomorphicrhabdomyosarcoma represents a subtype with higher-grade histologic features and a more aggressive clinicalbehavior. Previous studies have shown MYOG andMYOD1 expression in 71% and 53% of pleomorphicrhabdomyosarcomas, respectively.23,24 In a cohort of 7cases of pleomorphic rhabdomyosarcoma, we identifiedPAX7 expression in 5 (5/7, 71%). Thus, in this set ofcases, the frequency of PAX7 expression in pleomorphicrhabdomyosarcoma is similar to that of MYOG.

The evaluated ARMSs were PAX7 positive in 6 of31 (19%) cases (Fig. 5). Compared with ERMS, a greaterproportion of ARMS cases showed negative (14/31, 45%)or focal (11/31, 36%) staining. As above, focal stainingwas identified as <20% of cells showing nuclear anti-PAX7 immunoreactivity (Fig. 5B). MYOG and MYOD1expression were additionally analyzed in a 16-case cohortof ARMS cases. As previously reported,5,7 the large ma-jority of ARMS cases were MYOG positive (13/16, 81%).One of 3 MYOG-negative cases of ARMS showed PAX7

FIGURE 2. Limited PAX7 expression in non-neoplastic tissues. Representative immunohistochemical detection of PAX7 ex-pression in skin (A), colon (B), seminal vesicle (C), kidney (D), tonsil (E), and stomach (F).




expression. Two of 3 PAX7-positive cases showed focalanti-MYOG staining in <20% of lesional cells, whereas 1case showed staining inB70% of cells. No case of PAX7-

positive ARMS was a recurrence. Five of 7 ARMS caseswith focal PAX7 expression showed MYOG positivity in>50% of cells. One of 4 ARMS cases with focal PAX7

PAX7MYOGH&E

A

B

C

D

FIGURE 3. PAX7 expression in ERMS. Representative H&E and immunohistochemical analysis of PAX7-positive ERMS casesshowing examples of focal (A, B) or negative (C) MYOG expression by immunohistochemistry. D, Venn diagram summary ofPAX7, MYOD1, and MYOG expression in cases of embryonal and ARMS for which all 3 markers were analyzed in this study.Percentage of cases showing expression of each combination of markers is in parentheses.




expression was a recurrence. Among the cases of ARMS,7 were classified histologically as the solid variant, in-cluding 2 cases negative for PAX7 expression, 2 casesshowing positive PAX7 staining, and 3 cases showingfocal PAX7 staining. Nuclear anti-MYOD1 immunor-eactivity was identified in 4 ARMS cases (4/16, 25%).Three of 4 MYOD1-positive cases also expressed MYOGand PAX7, whereas 1 case was positive for MYOG, butnot PAX7, expression.

Given the observation of PAX7 expression in asubset of ARMS cases, we hypothesized that its ex-pression would correlate with underlying PAX3/PAX7-FOXO1 translocation. To test this possibility, we ana-lyzed PAX7 expression in a cohort of 15 molecularlydefined ARMS cases, which included 10 cases with aPAX3-FOXO1 gene fusion and 5 cases with a PAX7-FOXO1 gene fusion. Among the 15 cases, 7 (7/15, 47%)showed PAX7 expression, which was focal in 4 of 7PAX7-positive cases. Remarkably, all 7 cases with PAX7expression were defined by a PAX3-FOXO1 trans-location, whereas none of the 5 ARMS cases with aPAX7-FOXO1 gene fusion showed PAX7 expression.Thus, PAX7 expression correlates with the presence ofPAX3-FOXO1 gene fusion in ARMS.

To examine the specificity of anti-PAX7 immunor-eactivity for diagnostic evaluation of rhabdomyosarcomaand other soft tissue tumors, we studied PAX7 expressionin TMA samples of 697 additional nonrhabdomyosarcomaspecimens, which focused on soft tissue tumors, smallround blue cell tumors, and leukemia/lymphoma (Fig. 6and Table 1). Among these tumors, only Ewing sarcomashowed consistent PAX7 expression (7/7, 100%). In all 7

cases of Ewing sarcoma, nuclear anti-PAX7 immunor-eactivity was present in >50% of lesional cells (Fig. 6A).Among the 690 remaining specimens, 688 (99.7%) werenegative for PAX7 expression—2 of 22 examined cases(9%) of synovial sarcoma showed PAX7 expression. Ofnote, these synovial sarcoma cases did not have molecularconfirmation of the t(X;18) translocation. Both PAX7-expressing cases of synovial sarcoma were of the mono-phasic spindle cell subtype.

DISCUSSIONThe assessment of soft tissue sarcomas represents a

diagnostically challenging area owing to morphologicoverlap and incomplete molecular characterization ofmany of the involved entities. This is particularly true forrhabdomyosarcoma, which has a number of histologicmimics and distinguishing molecular features that can bechallenging to detect. Accurate distinction betweenrhabdomyosarcoma and its mimics is critical as sub-sequent treatment and overall prognosis is based on thespecific type of sarcoma. Here, we explore the moleculardiagnostics of rhabdomyosarcoma with a particular focuson the expression of PAX7 in alveolar and embryonalsubtypes, non-neoplastic tissues, and histologically sim-ilar soft tissue or small round blue cell tumors.

In our studies, PAX7 showed particular utility inthe evaluation of ERMS, where we found that PAX7expression was present in 86% of cases. This finding is ofparticular significance in the diagnosis of ERMS because,although MYOG is a relatively specific marker of rhab-

PAX7MYOGH&E

A

B

FIGURE 4. Spectrum of PAX7-negative ERMS. Representative H&E and immunohistochemical analysis of PAX7-negative ERMScases showing examples of positive (A) or negative (B) MYOG expression by immunohistochemistry.




domyosarcoma, a number of prior studies have notedthat MYOG expression is frequently negative or scant incases of ERMS. Hostein et al7 found that 37 of 64 ERMScases (58%) showed MYOG expression in <50% of tu-mor cells. Twenty-two cases (34%) showed MYOG ex-pression in <29% of cells. Dias et al5 likewise found that15 of 17 ERMS cases (88%) showed MYOG expression in<50% of tumor cells with 59% of cases (10/17) showingMYOG expression in <10% of cells. We find a similarfrequency of ERMS cases with limited MYOG expressionin the present study, with 31 of 63 cases (49%) beingMYOG-negative or MYOG-focal (expression in <20% oftumor cells). Importantly, 75% of MYOG-negative casesand 100% of MYOG-focal cases showed PAX7 expressionin >20% of cells. In contrast, only 5 of 63 ERMS cases(8%) showed MYOG staining in the absence of PAX7staining. These findings highlight the potential diagnosticvalue of PAX7 immunohistochemistry in the evaluation ofrhabdomyosarcoma, especially those of the embryonalsubtype.

Of particular interest, we observe that, among alarge and diverse array of neoplasms, PAX7 expression islimited to rhabdomyosarcoma and Ewing sarcoma. Thisfinding corroborates similar observations of PAX7 ex-pression in 3 cases of Ewing sarcoma in a prior study,25

which did not examine PAX7 expression in rhabdomyo-sarcoma itself. On the basis of these findings, we concludethat Ewing sarcoma should be considered in the differentialdiagnosis of a PAX7-expressing small round blue cell ne-oplasm. Helpful clues to differentiating rhabdomyosarcomaand Ewing sarcoma include immunohistochemical analysisof desmin expression, which is seen in 83% of rhabdo-myosarcomas,26 but only 2% of Ewing sarcomas,27 andmolecular genetic evidence of Ewing sarcoma–relatedtranslocations. Biologically, the identification of PAX7expression in the majority of studied Ewing sarcoma casesis consistent with the molecular features of primitive mes-oderm characteristic of this entity and accounts for theobserved myogenic differentiation seen in rare cas-es.23,24,27,28 Future studies aimed at inducing the terminal

PAX7MYOGH&E

A

B

C

FIGURE 5. Heterogenous PAX7 expression in ARMS. Representative H&E and immunohistochemical analysis of ARMS casesshowing examples of positive (A), focal (B), and negative (C) PAX7 expression with associated MYOG expression.




differentiation of myogenic tumors may have additionalutility in Ewing sarcoma.

Taken together, the studies presented herein suggestseveral opportunities for—and limitations of—imple-mentation of analyses of PAX7 expression in clinicalpractice. Currently, the standard for demonstrating skel-etal muscle differentiation in rhabdomyosarcomas isidentification of nuclear immunoreactivity of anti-MYOG or anti-MYOD1 antibodies. In our studies,PAX7 expression, in comparison with analysis of eitherMYOD1 or MYOG, was a more sensitive marker ofskeletal muscle differentiation in ERMSs: the sensitivityof PAX7, MYOG, and MYOD1 expression in ERMSwas 86% (54/63), 75% (47/63), and 36.5% (23/63), re-spectively. Among ARMS cases, MYOG was a moresensitive marker, with the sensitivity of PAX7, MYOG,and MYOD1 expression being 63% (10/16), 81% (13/16),and 25% (4/16), respectively. It is important to note,however, that there were several cases of both ERMS andARMS that showed expression of only one of eitherPAX7 or MYOG. Notably, we found that PAX7 is notentirely specific for rhabdomyosarcoma, as it is expressedin all 7 analyzed cases of Ewing sarcoma. Excluding casesof Ewing sarcoma, PAX7 expression was seen in only0.3% of nonrhabdomyosarcomas. Although MYOD1 andMYOG are regarded as quite specific markers of myogenicdifferentiation, prior studies have noted nuclear immunor-eactivity for these antibodies in rare nonrhabdomyosarcomacases. In 1 such study,9 7 of 107 nonrhabdomyosarcomas(6.5%) showed MYOG expression, whereas “cytoplasmic

and nonspecific background staining and reactivity of non-myoid tissues” by anti-MYOD1 antibody “hindered itspractical utility in paraffin-embedded samples,” indicatingthat, like PAX7, there are caveats to the interpretation ofpositive MYOD1/MYOG immunostaining alone as evi-dence of skeletal muscle differentiation.

One can, therefore, imagine multiple approachesto the utilization of PAX7 analyses in the immuno-histochemical evaluation of candidate rhabdomyosarco-mas. There is no certain advantage to using PAX7expression in lieu of MYOG expression in a primaryscreen for skeletal muscle differentiation. Although PAX7was a somewhat more sensitive marker in ERMS, MYOGwas somewhat more sensitive in ARMS. If PAX7 analy-ses are used as a primary screening marker, lack ofexpression would require additional studies with MYOGor MYOD1 to completely rule-out skeletal muscle dif-ferentiation, given that PAX7 does not identify all tumorsshowing myogenic gene expression. In this scenario,identification of PAX7 expression would be diagnostic ofa rhabdomyosarcoma, unless a small round blue cellmorphology was identified, in which case additional im-munohistochemical or molecular studies would be re-quired to exclude a Ewing sarcoma, as discussed above.Alternatively, PAX7 studies may be used secondarily incases showing desmin expression, but a lack of MYOG orMYOD1 expression in an initial screening assessment, toaccount for the MYOG/MYOD1-negative or MYOG/MYOD1-focal rhabdomyosarcomas identified in thisstudy. In another scenario, the practicing pathologist may

FIGURE 6. PAX7 expression in soft tissue tumors and histologic mimics of rhabdomyosarcoma. Representative im-munohistochemical detection of PAX7 expression in Ewing sarcoma (A), gastrointestinal stromal tumor (B), uterine leiomyoma(C), dedifferentiated liposarcoma (D), leiomyosarcoma (E), and osteosarcoma (F).




find utility for PAX7 expression analyses in evaluating asmall round blue cell tumor concerning for Ewing sar-coma. In this setting, our studies suggest that identi-fication of a PAX7-positive, desmin-negative tumorwould suggest the diagnosis of Ewing sarcoma and wouldwarrant follow-up molecular analyses for Ewing-relatedtranslocations.

PAX7 is one of 9 members of the paired box (Pax)family of genes, each of which has a unique function andpattern of expression. PAX2, PAX5, and PAX8 are Paxgene lineage markers that are routinely assessed in currentclinical practice. Intriguingly, recent work has shown thatan antibody against PAX5, a marker of the B-cell lineage,shows immunoreactivity toward a subset of ARMS, butnot ERMS.29 Indeed, this finding can be a diagnosticpitfall in morphologically challenging cases of ARMS.30

Because anti-PAX5 immunoreactivity correlated with thepresence of PAX3/PAX7 translocations in a small subsetof studied cases, the anti-PAX5 immunoreactivity seenin ARMS was attributed to cross-reactivity with PAX3/PAX7.29 These studies did not correlate anti-PAX5immunoreactivity with specific translocations (ie, PAX3vs. PAX7). In the present study, we observe that a majorityof ERMS cases show robust PAX7 expression, whereasPAX7 expression is relatively limited in ARMS. Given thatSullivan and colleagues see PAX5 immunore-activity primarily in ARMS, our findings suggest that theobserved PAX5 cross-reactivity is most likely attributableto recognition of PAX3 and not PAX7. In addition, we findno evidence of significant immunoreactivity of the anti-PAX7 antibody with non-neoplastic cells other than theexpected localization within skeletal muscle satellite cells.This finding suggests that there is limited cross-reactivity ofanti-PAX7 antibody for nonmyogenic Pax genes.

PAX7 has been used extensively in the past in basicscience studies as a genetic marker of the adult mouseskeletal muscle satellite cell population. Indeed, recentstudies in which the entire population of PAX7-expressing cells was conditionally ablated from adultmice showed that PAX7-expressing satellite cells are re-sponsible for the robust skeletal muscle regenerative ca-pacity seen in adult animals.31 Subsequent studies showedthat the level of PAX7 expression correlates with “stemcell” attributes in satellite cells.20 These findings raise thehypothesis that PAX7 expression also identifies cells withtumorigenic stem cell properties in human rhabdomyo-sarcoma. The heterogeneity of PAX7 expression observedin our studies suggests that rhabdomyosarcomas varyconsiderably in terms of the frequency of PAX7-expressing cells, mirroring the clinical variability oftumor recurrence and patient survival in rhabdomyo-sarcoma. If PAX7 expression correlates with a cancerstem cell phenotype, evaluation of PAX7 may haveprognostic significance in rhabdomyosarcoma. Addi-tional studies designed to correlate features of PAX7 ex-pression with clinical behavior of rhabdomyosarcomasamples will be critical in evaluating this possibility.

Fusion of PAX3/7 with FOXO1 represents a mo-lecular lesion associated with the majority of cases ofARMS. Our observation of PAX7 expression in a subsetof ARMS cases raised the intriguing question of whetherPAX7 expression in ARMS relates to the underlying ge-netic translocation. Interestingly, in a set of molecularlydefined ARMS cases, we find that, although PAX7expression is seen in 70% of cases (7/10) bearing thePAX3-FOXO1 translocation, no cases bearing thePAX7-FOXO1 translocation express PAX7. What accountsfor this surprising correlation? The anti-PAX7 antibodyused in this study was originally raised against an immu-nogen consisting of amino acids 352-523 of chicken Pax7.18

This immunogen represents a distal segment of PAX7, in-cluding the C-terminus. Therefore, although the antibody’sepitope has not been mapped, it falls within this distalregion against which the antibody was raised. Conversely,the segment of human PAX7 that is involved in the PAX7-

TABLE 1. Summary of PAX7 Expression inRhabdomyosarcomas, Small Round Blue Cell Neoplasms, andOther Soft Tissue Tumors

Tumor Type

Total

Cases

PAX7

Expressing

(n [%])

ERMS 63 54 (86)ARMS* 31 17 (55)Spindle cell rhabdomyosarcoma 8 6 (75)Pleomorphic rhabdomyosarcoma 7 5 (71)Leiomyosarcoma 62 0 (0)Ewing sarcoma 7 7 (100)Gastrointestinal stromal tumor 51 0 (0)Neuroblastoma 4 0 (0)Atypical lipomatous tumor 10 0 (0)Glomus tumor 11 0 (0)Angiosarcoma 10 0 (0)Osteosarcoma 13 0 (0)Myxoid liposarcoma 10 0 (0)Hemangioendothelioma 8 0 (0)Leiomyoma 20 0 (0)Dedifferentiated liposarcoma 10 0 (0)Desmoplastic small round cell tumor 6 0 (0)Extraskeletal myxoid chondrosarcoma 11 0 (0)Solitary fibrous tumor 7 0 (0)Dermatofibrosarcoma protuberans 10 0 (0)Desmoid-type fibromatosis 19 0 (0)Synovial sarcomaw 22 2 (9)Ovarian fibroma 9 0 (0)Nodular fasciitis 9 0 (0)Granular cell tumor 20 0 (0)Schwannoma 22 0 (0)Sarcoma with CIC-DUX4translocation

1 0 (0)

Mesenchymal chondrosarcoma 5 0 (0)Leukemia/lymphomaz 311 0 (0)Tenosynovial giant cell tumor 29 0 (0)

*Including cases from both ARMS cohorts used in this study.wCases of synovial sarcoma used in this study have not been molecularly de-

fined by the presence of t(X;18).zIncluding 89 diffuse large B-cell lymphomas, 35 grade 1 follicular lympho-

mas, 44 grade 2 follicular lymphomas, 54 grade 3 follicular lymphomas, 19 mar-ginal zone lymphomas, 11 mantle cell lymphomas, 26 chronic lymphocyticleukemias, 12 lymphoblastic lymphomas (7 T cell and 5 B cell), 8 peripheral T-celllymphomas, 3 angioimmunoblastic lymphomas, 5 anaplastic large cell lymphomas,and 5 lymphoplasmacytic lymphomas.




FOXO1 translocation consists of an N-terminal region(amino acids 1 to 383) that has little overlap with the im-munogen sequence: the portion of PAX7 bearing the epit-ope targeted by the antibody used in this study includesonly approximately 30 amino acids retained in the fusionprotein.24 Although a second, intact PAX7 allele encodinga version of PAX7 including the antibody-targeted epitopeis retained, previous elegant studies have shown sig-nificantly reduced expression of the intact PAX7 allelerelative to the translocation allele in PAX7-FOXO1ARMS.With this information in mind, our observation of limitedPAX7 expression in ARMS cases bearing the PAX7-FOXO1 translocation can be accounted for by loss of ex-pression of the antibody epitope in the fusion protein anddecreased or loss of expression of the normal PAX7 allele.

In summary, PAX7 expression is a useful markerof skeletal muscle differentiation in rhabdomyosarcoma,particularly of the embryonal subtype, with specificityfor rhabdomyosarcoma and Ewing sarcoma. PAX7may be especially helpful in cases of ERMS in whichMYOG immunoreactivity is limited or undetectable. Ad-ditional molecular or immunohistochemical studies arewarranted if PAX7 expression is seen in a case for whichEwing sarcoma is on the clinical or histopathologic differ-ential diagnosis. The development of new antibodiesand techniques for analysis of gene expression arelikely to enhance our capacity for diagnostic assessment ofmyogenic differentiation in human cancers. Future studiesof PAX7 expression in rhabdomyosarcoma will enhanceour understanding of its significance as a prognostic markeror surrogate marker of underlying molecular lesions.

ACKNOWLEDGMENTSThe authors thank Dr Yaso Natkunam (Stanford

University School of Medicine, Stanford, CA) for sharingTMA material. The authors also thank Dr Cristina Anto-nescu (Memorial Sloan-Kettering Cancer Center, NewYork, NY) for performing reverse transcription with pol-ymerase chain reaction studies during the diagnosis of theCIC-DUX4 round cell sarcoma included in this study. Theauthors acknowledge Ivy Mangonon of the Stanford HealthCare clinical laboratory (Stanford, CA) and Eileen Maisenof the Stanford Health Care Department of Pathology(Stanford, CA) for technical advice and support. The au-thors thank Norman Cyr of Stanford Health Care De-partment of Pathology (Stanford, CA) for assistance withimages.

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