Li-Fraumeni syndrome

The College of American Pathologists offers these protocols to assist pathologists in providing clinically useful and relevant information when reporting results of surgical specimen examinations. The College regards the reporting elements in the "Surgical Pathology Cancer Case Summary" section of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice.

The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons has mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with the document. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of this document.

PROTOCOL FOR THE EXAMINATION OF SPECIMENS FROM PATIENTS (CHILDREN AND YOUNG ADULTS) WITH RHABDOMYOSARCOMA

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.

Important Note.-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 snapfrozen 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.

EXPLANATORY NOTES

A: Special Studies.-Frozen Tissue.-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[degrees]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.

Immitnohistochemistry.-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

Chromosomal Translocations.-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

B: Cytologic Material.-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

C: Relevant History.-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.

D: Histologic Type.-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 histologie 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.

Nonrhabdomyosarcomatous Soft Tissue Sarcomas (NRSTSs).-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 multi-agent 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.

E: Incisional Biopsy.-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

F: Resection.-Resection specimens may be intralesional, marginal, wide, or radical in extent." 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

G: Margins.-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

H: TNM Stage and Clinical Grouping.-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.

References

1. Coffin CM, Dehner LP. Pathologic evaluation of pediatric soft tissue tumors. Am J Clin Pathol. 1998;109(suppl 1):S38-S52.

2. Qualman SJ, Morotti RA. Risk assignment in pediatric soft-tissue sarcoma: an evolving molecular classification. Curr Oncol Rep. 2002;4:123-130.

3. Parham DM. Pathologic classification of rhahdomyosarcomas and correlations with molecular studies. Mod Pathol. 2001;14:506-514.

4. Qualman SJ, Coffin CM, Newton WA, et al. Intergroup Rhabdomyosarcoma Study: update for pathologists. Pediatr Dcv Pathol. 1998;1:550-561.

5. Ambros IM, Amhros PF, Strehl S, Kovar H, Gadner H, Salzer-Kuntschik M. MIC2 is a specific marker for Ewing's sarcoma and peripheral primitive neuroectodermal tumor: evidence for a common histogenesis of Ewing's sarcoma and peripheral neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer. 1991;67:1886-1893.

6. Bridge JA, Liu J, Qualman SJ, et al. Genomic gains and losses are similar in genetic and histologic subsets of rhabdomyosarcoma, whereas amplification predominates in embryonal with anaplasia and alveolar subtypes. Genes Chromosom Cancer. 2002;33:310-321.

7. DeAlmeida M, Stastny JF, Wakely PE, et al. Fine-needle aspiration biopsy of childhood rhabdomyosarcoma: reevaluation of the cytologic criteria for diagnosis. Diagn Cytopathol. 1994;11:231-236.

8. Pappo AS. Rhabdomyosarcoma and other soft tissue sarcomas in children. Curr Opin Oncol. 1996;8:311-316.

9. Cost MJ, Campman SC, Davis RL, et al. Fine needle aspiration cytology Ot sarcoma: retrospective review of diagnostic utility and specificity. Diagn Cytopathol. 1996;15:23-32.

10. Birch JM, Hartley AL, Blair V, et al. Cancer in the families of children with soft tissue sarcoma. Cancer. 1990;66:2239-2248.

11. Hartley AL, Birch JM, Blair V, et al. Patterns of cancer in the families of children with soft tissue sarcoma. Cancer. 1993;72:923-930.

12. Ruymann FB, Maddux HR, Ragab A, et al. Congenital anomalies associated with rhabdomyosarcoma. Med Pediatr Oncol. 1988;16:33-39.

13. Coffin CM. The new International Rhabdomyosarcoma Classification, its progenitors, and consideration beyond morphology. Adv Anal Pathol. 1997;4:1-16.

14. Dishop MK, Anderson JR, Weiner E, Crier H, Crist W, Qualman SJ. Inflammatory infiltrate in paratesticular rhabdomyosarcoma: a comparison of histologic subtypes and correlation with survival. Mod Pathol. 2001;14:1P.

15. Kodet R, Newton WA Jr, Hamoudi A, Asmar L, Jacobs DL, Maurer H. Childhood rhabdomyosarcoma with anaplastic (pleomorphic) features: a report of the lntergroup Rhabdomyosarcoma Study. Am J Surg Pathol. 1993;17:433-453.

16. Wexler LH, Garvin JH Jr. Pediatric nonrhabdomyosarcoma soft tissue sarcomas: progress on clinical and biologic fronts. J Pediatr. 1997;131:508-509.

17. Parham DM, Webber BL, Jenkins JJ, Cantor AB, Maurer HM. Nonrhabdomyosarcomatous soft tissue sarcomas of childhood: formulation of a simplified system for grading. Mod Pathol. 1995;8:705-710.

18. Shives TC. Biopsy of soft-tissue tumors. Clin Orthop. 1993;289:32-35.

19. Conrad EU, Bradford L, Chonsky HA. Pediatric soft tissue sarcomas. Orthop Clin North Am. 1996;27:655-664.

20. Marcus KC, Crier HE, Shamberger RC, et al. Childhood soft tissue sarcoma: a 20-year experience. J Pediatr. 1997;131:603-607.

21. Fletcher C, Kempson RL, Weiss S. Recommendations for reporting soft tissue sarcomas. Am J Clin Pathol. 1999;111:594-598.

Bibliography

Coffin CM, Dehner LP, O'Shea PA, eds. Pediatric Soft Tissue Tumors. Philadelphia, Pa: Williams & Wilkins; 1997.

Stephen J. Qualman, MD; Jay Bowen, MS; David M. Parham, MD; Philip A. Branton, MD; William H. Meyer, MD; for the Members of the Cancer Committee, College of American Pathologists

Accepted for publication May 13, 2003.

From the Department of Laboratory Medicine, Children's Hospital, Columbus, Ohio (Drs Qualman and Bowen); Department of Pediatric Pathology, Arkansas Children's Hospital, Little Rock (Dr Parham); Department of Pathology, Inova Fairfax Hospital, Falls Church, Va (Dr Branton); and Section of Pediatric Hematology/Oncology, University of Oklahoma Health Sciences Center, Oklahoma City (Dr Meyer).

Reprints: Stephen J. Qualman, MD, Department of Laboratory Medicine, Children's Hospital, 700 Children's Dr, 313 B Ross Hall, Columbus, OH 43205 (e-mail: qualmans@pediatrics.ohio-state.edu).

Copyright College of American Pathologists Oct 2003
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