Dense Embryonal or Alveolar Rhabdomyosarcoma?
Dense Embryonal or Alveolar Rhabdomyosarcoma?
In total, 277 patients with a diagnosis of ARMS were enrolled in the D9803 study. Diagnostic pathology material archived at the Biopathology Center in Columbus, Ohio, was available for re-review from 255 (92%) patients. Of these, 213 had original central review diagnoses, whereas the remaining 42 had institutional histologic classification. In the latter cases, material was not available for central review at enrollment but was submitted at a later time. A control group of 38 randomly selected patients with an original diagnosis of ERMS was also reviewed. In total, we re-reviewed the histopathology of 293 of the 616 patients enrolled in D9803. Available material included H&E slides and select immunohistochemical stains (including myogenin for 250 of 293 cases).
Two pathologists (E.R.R. and D.M.P.) conducted the re-review. Reviewers were not in complete agreement but reached a consensus diagnosis for all cases. Cases were classified as ARMS, mixed RMS, or ERMS. Patterns of ARMS included classic and solid variants. ERMS patterns included botryoid, spindled, dense, sclerosing, epithelioid, and typical variants Appendix 1 and Image 1. A diagnosis of RMS–not otherwise specified (NOS) was made if the biopsy sample was too small, crushed, or necrotic for a definitive classification.
(Enlarge Image)
Image 1.
Rhabdomyosarcoma histologies (see Appendix 1 for a description). A, Alveolar rhabdomyosarcoma, classic pattern. B, Alveolar rhabdomyosarcoma, solid pattern. C, Embryonal rhabdomyosarcoma, typical pattern. D, Embryonal rhabdomyosarcoma, dense pattern. E, Embryonal rhabdomyosarcoma, botryoid pattern. F, Embryonal rhabdomyosarcoma, spindle cell pattern. G, Rhabdomyosarcoma, sclerosing pattern. H, Embryonal rhabdomyosarcoma, epithelioid pattern. I, Mixed rhabdomyosarcoma.
Nuclear myogenin expression was scored from 0 to 4+ based on the following percentages of tumor cells : 0 (absent expression), 1+ (<10%), 2+ (10%–50%), 3+ (>50%–90%), and 4+ (>90%). A diagnosis of ARMS (classic or solid variants) was supported by strong, diffuse (3 or 4+) myogenin expression. In the 43 cases lacking myogenin stains, a diagnosis of ARMS was based on typical histologic and cytologic features.
For 119 cases, we used a quantitative reverse transcription–polymerase chain reaction (RT-PCR) assay to assess expression of a PAX3-FOXO1 (P3F) or PAX7-FOXO1 (P7F) fusion transcript from formalin-fixed, paraffin-embedded material as described previously. Following reverse transcription from a FOXO1-specific primer, the assay incorporated consensus 5¢ PAX3/PAX7 and 3¢ FOXO1 primers and gene-specific PAX3 and PAX7 probes, thus determining both the presence and subtype of the fusion. In a second reaction, expression of the GAPDH gene was quantified to assess the quality of the RNA. A GAPDH expression level equivalent to that found in 0.5 ng of a control RMS cell line was the minimum cutoff for a satisfactory result in a sample with a negative fusion result.
For 128 cases, a fluorescence in situ hybridization (FISH) strategy was employed to detect rearrangements of FOXO1 (13q14), PAX3 (2q35), and PAX7 (1p36) loci on representative formalin-fixed, paraffin-embedded tissue sections. Studies were conducted using a FOXO1 dual-color break-apart probe (Abbott Molecular, Des Plaines, IL), per slight modification of the manufacturer's instructions, and PAX3 (2q35) and PAX7 (1p36) custom-designed break-apart probes as previously described. Hybridization signals were assessed in interphase nuclei with strong, well-delineated signals and distinct nuclear borders. A cell specimen was interpreted as abnormal if a split of flanking probe signals was detected in more than 10% of the cells evaluated (more than 2 standard deviations above the average false-positive rate). Seventy-two cases were examined by RT-PCR and FISH with concordance for all cases in which satisfactory material was available for both tests.
Event-free survival (EFS) was defined as the time from study enrollment to the first occurrence of progression, relapse after response, or death from any cause. Overall survival (OS) was defined as the time from study enrollment to death from any cause. Patients not experiencing an event were censored at their last follow-up time. Estimates of time-to-event distributions were calculated using the Kaplan-Meier method, and distributions were compared using log-rank tests.
Materials and Methods
Pathology Review
In total, 277 patients with a diagnosis of ARMS were enrolled in the D9803 study. Diagnostic pathology material archived at the Biopathology Center in Columbus, Ohio, was available for re-review from 255 (92%) patients. Of these, 213 had original central review diagnoses, whereas the remaining 42 had institutional histologic classification. In the latter cases, material was not available for central review at enrollment but was submitted at a later time. A control group of 38 randomly selected patients with an original diagnosis of ERMS was also reviewed. In total, we re-reviewed the histopathology of 293 of the 616 patients enrolled in D9803. Available material included H&E slides and select immunohistochemical stains (including myogenin for 250 of 293 cases).
Two pathologists (E.R.R. and D.M.P.) conducted the re-review. Reviewers were not in complete agreement but reached a consensus diagnosis for all cases. Cases were classified as ARMS, mixed RMS, or ERMS. Patterns of ARMS included classic and solid variants. ERMS patterns included botryoid, spindled, dense, sclerosing, epithelioid, and typical variants Appendix 1 and Image 1. A diagnosis of RMS–not otherwise specified (NOS) was made if the biopsy sample was too small, crushed, or necrotic for a definitive classification.
(Enlarge Image)
Image 1.
Rhabdomyosarcoma histologies (see Appendix 1 for a description). A, Alveolar rhabdomyosarcoma, classic pattern. B, Alveolar rhabdomyosarcoma, solid pattern. C, Embryonal rhabdomyosarcoma, typical pattern. D, Embryonal rhabdomyosarcoma, dense pattern. E, Embryonal rhabdomyosarcoma, botryoid pattern. F, Embryonal rhabdomyosarcoma, spindle cell pattern. G, Rhabdomyosarcoma, sclerosing pattern. H, Embryonal rhabdomyosarcoma, epithelioid pattern. I, Mixed rhabdomyosarcoma.
Nuclear myogenin expression was scored from 0 to 4+ based on the following percentages of tumor cells : 0 (absent expression), 1+ (<10%), 2+ (10%–50%), 3+ (>50%–90%), and 4+ (>90%). A diagnosis of ARMS (classic or solid variants) was supported by strong, diffuse (3 or 4+) myogenin expression. In the 43 cases lacking myogenin stains, a diagnosis of ARMS was based on typical histologic and cytologic features.
Determining Fusion Status
For 119 cases, we used a quantitative reverse transcription–polymerase chain reaction (RT-PCR) assay to assess expression of a PAX3-FOXO1 (P3F) or PAX7-FOXO1 (P7F) fusion transcript from formalin-fixed, paraffin-embedded material as described previously. Following reverse transcription from a FOXO1-specific primer, the assay incorporated consensus 5¢ PAX3/PAX7 and 3¢ FOXO1 primers and gene-specific PAX3 and PAX7 probes, thus determining both the presence and subtype of the fusion. In a second reaction, expression of the GAPDH gene was quantified to assess the quality of the RNA. A GAPDH expression level equivalent to that found in 0.5 ng of a control RMS cell line was the minimum cutoff for a satisfactory result in a sample with a negative fusion result.
For 128 cases, a fluorescence in situ hybridization (FISH) strategy was employed to detect rearrangements of FOXO1 (13q14), PAX3 (2q35), and PAX7 (1p36) loci on representative formalin-fixed, paraffin-embedded tissue sections. Studies were conducted using a FOXO1 dual-color break-apart probe (Abbott Molecular, Des Plaines, IL), per slight modification of the manufacturer's instructions, and PAX3 (2q35) and PAX7 (1p36) custom-designed break-apart probes as previously described. Hybridization signals were assessed in interphase nuclei with strong, well-delineated signals and distinct nuclear borders. A cell specimen was interpreted as abnormal if a split of flanking probe signals was detected in more than 10% of the cells evaluated (more than 2 standard deviations above the average false-positive rate). Seventy-two cases were examined by RT-PCR and FISH with concordance for all cases in which satisfactory material was available for both tests.
Statistics
Event-free survival (EFS) was defined as the time from study enrollment to the first occurrence of progression, relapse after response, or death from any cause. Overall survival (OS) was defined as the time from study enrollment to death from any cause. Patients not experiencing an event were censored at their last follow-up time. Estimates of time-to-event distributions were calculated using the Kaplan-Meier method, and distributions were compared using log-rank tests.
