Protocol for the Examination of Specimens From Patients (Children and Young Adults) With Rhabdomyosarcoma Open Access

This protocol applies to rhabdomyosarcoma in children and young adults only. It excludes nonrhabdomyosarcomatous soft tissue sarcomas. There is no American Joint Committee on Cancer/International Union Against Cancer TNM classification system for the staging of rhabdomyosarcoma. The Intergroup Rhabdomyosarcoma Study Postsurgical Clinical Grouping System is recommended.

First priority should always be given to formalin-fixed tissue for morphologic evaluation. Special studies (eg, reverse transcriptase–polymerase chain reaction) are critical to the molecular workup of rhabdomyosarcoma and require at least 100 mg of viable snap-frozen tissue as the second priority for workup (note A).

For more information, contact The Children's Oncology Group Biopathology Center, Columbus, Ohio; telephone: (614) 722-2890 or (800) 347-2486.

• I. Cytologic Material (note B)

  •  A. Clinical Information

    • 1. Patient identification

      •  a. Name

      •  b. Identification number

      •  c. Age (birth date)

      •  d. Sex

    • 2. Responsible physician(s)

    • 3. Date of procedure

    • 4. Other clinical information

      •  a. Relevant history (eg, previous diagnoses, treatment, family history) (note C)

      •  b. Relevant findings (eg, imaging studies)

      •  c. Clinical diagnosis

      •  d. Procedure (eg, fine-needle aspiration)

      •  e. Anatomic site(s) of specimen

  • B. Macroscopic Examination

    • 1 Specimen

      •  a. Unfixed/fixed (specify fixative)

      •  b. Number of slides received

      •  c. Quantity and appearance of fluid specimen

      •  d. Other materials received

      •  e. Results of intraprocedural consultation

    • 2. Material submitted for microscopic examination (eg, smear, cytocentrifuge, touch or filter preparation, cell block)

    • 3. Special studies (specify) (eg, immunohistochemistry, molecular analysis, cytogenetic analysis) (note A)

  • C. Microscopic Evaluation (note D)

    • 1. Adequacy of specimen (if unsatisfactory for evaluation, specify reason)

    • 2. Tumor, if present

      •  a. Histologic type, if possible (subtype may also be suggested)

      •  b. Other features (eg, anaplasia)

    • 3. Other pathologic findings (eg, necrosis)

    • 4. Results/status of special studies (specify)

    • 5. Comments

      •  a. Correlation with intraprocedural consultation, as appropriate

      •  b. Correlation with other specimens, as appropriate

      •  c. Correlation with clinical information, as appropriate

• II. Incisional Biopsy (Needle or Wedge) (note E)

  • A. Clinical Information

    • 1. Patient identification

      •  a. Name

      •  b. Identification number

      •  c. Age (birth date)

      •  d. Sex

    • 2. Responsible physician(s)

    • 3. Date of procedure

    • 4. Other clinical information

      •  a. Relevant history (eg, previous diagnoses, treatment, family history) (note C)

      •  b. Relevant findings (eg, imaging studies)

      •  c. Clinical diagnosis

      •  d. Procedure (eg, core needle biopsy, wedge biopsy)

      •  e. Anatomic sites(s) of specimen

  • B. Macroscopic Examination

    • 1. Specimen

      •  a. Unfixed/fixed (specify fixative)

      •  b. Number of pieces

      •  c. Dimensions

      •  d. Descriptive features

      •  e. Orientation, if designated by surgeon

      •  f. Results of intraoperative consultation

    • 2. Tissue submitted for microscopic examination, as appropriate

      •  a. Entire specimen

      •  b. Selected sample

      •  c. Frozen section tissue fragment(s) (unless saved for special studies)

    • 3. Special studies (specify) (eg, immunohistochemistry, electron microscopy, fluorescence in situ hybridization, reverse transcriptase–polymerase chain reaction, cytogenetic analysis) (note A)

  • C. Microscopic Evaluation (note D)

    • 1. Tumor

      •  a. Histologic type

      •  b. Histologic subtype, if possible

      •  c. Venous/lymphatic vessel invasion, if possible to determine

      •  d. Evaluate for anaplasia, cambium layer

    • 2. Additional pathologic findings, if present

    • 3. Results/status of special studies (specify)

    • 4. Comments

      •  a. Correlation with intraoperative consultation, as appropriate

      •  b. Correlation with other specimens, as appropriate

      •  c. Correlation with clinical information, as appropriate

• III. Resection (note F)

  • A. Clinical Information

    • 1. Patient identification

      •  a. Name

      •  b. Identification number

      •  c. Age (birth date)

      •  d. Sex

    • 2. Responsible physician(s)/clinic(s)

    • 3. Date of procedure

    • 4. Other clinical information

      •  a. Relevant history

        •  (1) previous diagnoses

        •  (2) surgery and date(s)

        •  (3) radiation and date(s)

        •  (4) chemotherapy and date(s)

        •  (5) others (note C)

      •  b. Relevant findings

      •  c. Clinical diagnosis

      •  d. Procedure (specify anatomic site[s])

        •  (1) type of excision or resection (eg, cystectomy)

        •  (2) list all anatomical structures removed

        •  (3) lymph node dissection

      •  e. Operative findings (documentation of areas of concern marked by surgeon)

      •  f. Anatomic site(s) of specimen(s)

  • B. Macroscopic Examination

    • 1. Specimen

      •  a. Organ/tissues included

      •  b. Unfixed/fixed (specify fixative)

      •  c. Size (3 dimensions)

      •  d. Descriptive features

      •  e. Orientation, if indicated by surgeon

      •  f. Results of intraoperative consultation (frozen sections or touch preparations)

    • 2. Tumor

      •  a. Anatomical site(s) involved by tumor

      •  b. Size (3 dimensions)

      •  c. Descriptive characteristics (eg, firm/soft, color, consistency, necrosis, biopsy scars)

      •  d. Anatomic extent (structures involved by tumor and depth of invasion)

      •  e. Relation to margins (note G)

      •  f. Additional tumors

    • 3. Additional pathologic findings, if present

    • 4. Lymph nodes submitted

    • 5. Margins (note G)

    • 6. Tissues submitted for microscopic examination (1 section for each centimeter of maximal tumor diameter and/or different gross appearances)1 

    • 7. Special studies (specify) (eg, immunohistochemistry, electron microscopy, fluorescence in situ hybridization, reverse transcriptase–polymerase chain reaction, cytogenetic analysis) (note A)

  • C. Microscopic Examination (note D)

    • 1. Tumor

      •  a. Histologic type

      •  b. Histologic subtype

      •  c. Venous/lymphatic vessel invasion, if possible to determine

      •  d. Evaluate for anaplasia, cambium layer

      •  e. Closest distance to margin (note G)

      •  f. Evaluate posttherapy tumors for differentiation, necrosis, and fibrosis

    • 2. Additional pathologic findings, if present

    • 3. Results/status of special studies (specify) (note A)

    • 4. Comments

      •  a. Correlation with intraoperative consultation, as appropriate

      •  b. Correlation with other specimens, as appropriate

      •  c. Correlation with clinical information, as appropriate (notes A and H)

A minimum of 100 mg of viable tumor should be snap-frozen for potential molecular studies.2 If the reservoir of tissue is limited, the pathologist can keep the frozen tissue aliquot used for frozen section (usually done to determine sample adequacy and viability) in a frozen state (−80°C or lower) for potential molecular studies. Translocations may be detected using reverse transcriptase–polymerase chain reaction or fluorescence in situ hybridization on touch preparations made from frozen tissue.

In cases in which histologic diagnosis of rhabdomyosarcoma (RMS) is difficult, immunostaining with monoclonal antibodies against the intranuclear myogenic transcription factors MyoD1 and myogenin, and a polyclonal antibody preparation against desmin (P-DES) is suggested. Nearly all RMS tumors are positive for P-DES, myogenin, and MyoD1.3,4 Polyclonal desmin is 35% more sensitive in the detection of RMS as compared with monoclonal desmin.4 

MIC-2 staining has also been used to rule out extraosseous Ewing sarcoma (EOE)/primitive neuroectodermal tumor (PNET).5 Although some RMSs demonstrate immunopositivity to MIC-2,4 it is often weakly granular and intracytoplasmic, as opposed to the distinct plasma membrane staining seen in EOE/PNET. Since some RMSs can demonstrate focal membranous MIC-2 staining, the MIC-2 immunostain should always be done in a panel that includes more specific myogenic stains, as defined above. MyoD1 and myogenin immunostaining have not been demonstrated in EOE/PNET.

The immunopositivity of RMS to p53 antibodies has been researched because of the association of some RMSs with Li-Fraumeni syndrome.4 Although immunopositivity is low among random RMS samples, strong immunopositivity to p53 antibody (grade 3 or 4 in immunopositivity) in RMS samples is strongly correlated with the presence of any anaplasia in alveolar RMS (ARMS) and diffuse anaplasia in embryonal RMS (ERMS).4,6 

The incidence of t(1;13) (PAX7-FKHR) and t(2;13) (PAX3-FKHR) is strongly correlated with ARMS. The most common gene fusions are PAX3-FKHR and PAX7-FKHR, with the former being more prevalent. Studies suggest that patients with ARMS expressing the PAX3-FKHR gene product have a lower event-free survival than PAX7-FKHR–positive ARMS,4 but the significance of the translocations must still be elucidated. More recent data indicate that when gene fusion status is compared in patients with metastatic disease at diagnosis, a striking difference in outcome is seen between PAX3-FKHR and PAX7-FKHR (estimated 4-year overall survival of 75% for PAX7-FKHR and 8% for PAX3-FKHR; P = .002).2 These translocations may be found in up to 85% of ARMS cases.2 

A major limitation of a fine-needle aspiration biopsy is that while criteria are usually sufficient to diagnose RMS (with supportive immunostains) (note A), the RMS tumor is not readily subclassified into spindle cell, botryoid, embryonal, or alveolar subtypes.7 The presence of a monotonous, monomorphous nuclear cytology with coarse chromatin and prominent nucleoli is suggestive of a diagnosis of ARMS, with or without the presence of giant cells, whereas a heterogeneous nuclear pattern with hyperchromatic irregular nuclei suggests a diagnosis of ERMS. However, either pattern may be missed due to sampling error in a mixed RMS, and even 1 focus of ARMS predicates a biologic course that requires therapeutic management as ARMS.8 Moreover, histologic patterns, such as spindle cell RMS or botryoid RMS, are not well appreciated on fine-needle aspiration biopsy.7 Since ultimately the gold standard for therapy in RMS is histologic subtyping and not grading,4 fine-needle aspiration biopsy does not readily identify the therapeutic course that must be taken with a patient, even if a generic diagnosis of RMS is made. Another limitation of fine-needle aspiration biopsy is the inability to bank tissue for additional molecular diagnostic studies1 (note A). Fine-needle aspiration biopsy does lend itself to use of fluorescent in situ hybridization studies for pertinent translocations but is still hampered by sampling error. Overall, fine-needle aspiration biopsy can provide a specific diagnosis of a suspected soft tissue sarcoma in about 20% of all cases.9 

Relevant historical factors include any previous therapy, family history of malignancy, and the presence of congenital anomalies. If preoperative therapy has been given, assessment may be limited to the estimate of viable and necrotic RMS.1 The tumor may also show extreme cytodifferentiation and nuclear pleomorphism. These factors may preclude accurate subtyping of the RMS.

There is a specific concern for increased risk of a familial cancer when the specific diagnosis of ERMS or other soft tissue sarcoma is made within the first 2 years of life, especially in a male child.10 Such syndromes include Li-Fraumeni syndrome, basal cell nevus syndrome, neurofibromatosis, and pleuropulmonary blastoma syndrome (pleuropulmonary blastoma plus associated malignancies).1 A genetic predisposition to cancer is thought to be present in 7% to 33% of children with soft tissue sarcomas.11 

Rhabdomyosarcoma is specifically associated with a variety of congenital anomalies.12 These include congenital anomalies of the central nervous system, genitourinary tract, gastrointestinal tract, and cardiovascular system.

The International Classification of Rhabdomyosarcoma is recommended.13 Although undifferentiated sarcoma is a diagnosis of exclusion, its response to therapy is similar to ARMS and was therefore included. This classification has been further modified by the Intergroup Rhabdomyosarcoma Study Group to include the anaplastic variant of RMS (Table 1).4 

The importance of accurate subtyping of RMS is evident in the superior prognosis of the botryoid and spindle cell variants to other types of ERMS. Both types can show an extensive degree of rhabdomyoblastic differentiation. Diagnosis of the botryoid variant requires at least one microscopic field demonstrating a cambium layer (condensed layer of rhabdomyoblasts) underlying an intact epithelium. A gross demonstration of a botryoid configuration (“grapelike”) tumor is not required.

Spindle cell variants show a fascicular, spindled “leiomyomatous” pattern of growth that may also be nested or storiform. Some tumors demonstrate marked collagen deposition. Spindle cell tumors rarely occur outside the paratesticular region; association with intense chronic inflammation in the paratesticular region may be associated with improved prognosis,14 although this assessment has yet to be proven in a prospective study.

Classical ARMS presents with rhabdomyoblasts lining cleftlike spaces, but these diagnostic criteria may be obscured in the solid subtype; tumor cells may sit on or palisade about fibrovascular septae and fill these spaces. It is rare for solid ARMS to be present without evidence of cleftlike spaces somewhere in the specimen, therefore adequate sampling is imperative. Recognition of palisading tumor cells about septae necessitates greater vigilance. Although the prognosis for classic and solid variants is equally poor, improved recognition of the variation in histologies should improve the recognition of this tumor subtype.

Anaplasia is a histologic feature that may be found in any histologic subtype of RMS. Evidence of diffuse anaplasia may be significant in that retrospective studies are indicative of a poor prognosis4 with presence of this nuclear change. This finding has added importance in that anaplasia is more common in ERMS, which normally has an intermediate to superior prognosis. Anaplasia was absent from earlier classification schemes owing to its rarity (2% to 3% of RMS cases)4 and a lack of a good definition prior to 1993.15 The criteria are large, lobate hyperchromatic nuclei (at least 3 times the size of neighboring nuclei) and atypical (obvious, multipolar) mitotic figures. Anaplasia is further defined as to the distribution of the cells: focal (group I) anaplasia, which consists of a single or a few cells scattered among nonanaplastic cells, or diffuse (group II), in which clusters or sheets of anaplastic cells are evident. Anaplasia is now being studied prospectively in Children's Oncology Group Soft Tissue Sarcoma Clinical Trials to see if it warrants a separate therapeutic categorization in future studies.3 

While undifferentiated sarcoma is a diagnosis of exclusion, it has a treatment regimen similar to that of ARMS and therefore is included here. This sarcoma consists mostly of medium-sized cells with indistinct cytoplasm and oval nuclei with prominent chromocenter. The cells are packed in sheets with no structure except perhaps a delicate fibrovascular septum or spindled-storiform pattern. Necrosis or inflammation is not prominent. Approximately three fourths of tumors will stain with vimentin antisera, but no other diagnostic stains have been identified. Often a combination of immunostains, electron microscopy, and cytogenetic/molecular studies is required to exclude other sarcomas from the undifferentiated category.

Fewer than 1000 cases of pediatric soft tissue sarcomas are seen annually in the United States. One half of these are RMS, for which a consensus treatment approach combining surgery, radiation therapy, and multiagent chemotherapy has emerged.16 In contrast, optimal management for NRSTS has been less clear. Combined-modality treatment approaches have not been evaluated systematically, however, due to the diversity of tumor types. An NRSTS grading system has been devised17 (Table 2), which appears to predict clinical behavior.

A number of sarcomas with a predilection for children1 are not addressed in either the International Classification of Rhabdomyosarcoma or the NRSTS grading system. These include EOE/PNET, desmoplastic small round cell tumor, and malignant rhabdoid tumor. Currently, EOE/PNET is treated in the same manner as intraosseous Ewing sarcoma protocols; desmoplastic small round cell tumor and malignant rhabdoid tumor are consistently high-grade and aggressive lesions.

Core needle biopsies can obtain sufficient material for special studies and morphologic diagnosis, but sampling problems may limit tumor subtyping or grading.18 Open incisional biopsy is the generally preferred and most widely used technique because it consistently provides a larger sample of tissue and maximizes the opportunity for a specific pathologic diagnosis.1 Excisional biopsy may not include an adequate margin of normal tissue even with an operative impression of total gross removal.1 

Resection specimens may be intralesional, marginal, wide, or radical in extent.19 Intralesional resections extend through tumor planes, with gross or microscopic residual tumor identifiable at surgical margins. A marginal resection involves a margin formed by inflammatory tissue surrounding the tumor. A wide, radical resection has surgical margins that extend through normal tissue, usually external to the anatomic compartment containing the tumor. For all types of resections, marking (tattoo with ink followed by use of a mordant) and orientation of the specimen (prior to cutting) are mandatory for accurate pathologic evaluation.1 

The extent of resection (ie, gross residual disease vs complete resection) has the strongest influence on local control of malignancy.20,21 The definition of what constitutes a sufficiently “wide” margin of normal tissue in the management of RMS has evolved over time from resection of the whole muscle to resection with a 2- to 3-cm margin.16 For NRSTS, narrower margins (1–2 cm) may be adequate for low-grade tumors, whereas wider margins (greater than 5 cm) may be needed for higher-grade tumors.16 

The TNM staging system recommended by this protocol is a surgical, site-based, pretreatment assessment, which is used to plan therapy; the system is highly predictive of outcome1,4 and has been specifically structured to assess RMSs. The American Joint Committee on Cancer/International Union Against Cancer TNM staging systems currently do not apply to RMS. Clinical classification usually is carried out by the referring physician before treatment, during initial evaluation of the patient or when pathologic classification is not possible.

• T1  Tumor confined to anatomic site of origin

• T2  Tumor extension

• a  Tumor 5 cm or less in diameter

• b  Tumor greater than 5 cm in diameter

• N0  Regional lymph nodes not clinically involved

• N1  Regional lymph nodes clinically involved

• NX  Regional lymph node clinical status unknown

• M0  No distant metastasis

• M1  Distant metastasis present

The Intergroup Rhabdomyosarcoma Study Postsurgical Clinical Grouping System is shown below. The Clinical Grouping System is used to plan radiation therapy and relies on pathologic examination.1