AJCP / Original Article
MDM2 Copy Numbers in Well-Differentiated and Dedifferentiated Liposarcoma Characterizing Progression to High-Grade Tumors
CME/SAM
Patrick L. Ware, MD,1 Anthony N. Snow, MD,3 Maya Gvalani,2 Mark J. Pettenati, PhD,2 and Shadi A. Qasem, MD1 From the Departments of 1Pathology and 2Molecular Cytogenetics, Wake Forest School of Medicine, Winston-Salem, NC, and 3Department of Pathology, Warren Alpert Medical School of Brown University, Providence, RI. Key Words: MDM2; FISH; Well-differentiated liposarcoma; Dedifferentiated liposarcoma DOI: 10.1309/AJCPLYU89XHSNHQO
ABSTRACT Objectives: MDM2 gene amplification is associated with well-differentiated (WDL) and dedifferentiated liposarcomas (DDL). Using fluorescent in situ hybridization (FISH), we sought to characterize various patterns of MDM2 amplification among the morphologic spectrum of liposarcoma. Methods: Forty-six cases of liposarcoma in various sites were examined and included 22 WDLs, 14 DLLs, and 10 negative control subjects. Results: The MDM2 amplification ratio (MDM2/CEP12) was lower in WDL (10.2) compared with DDL (18.3) cases (P = .0000002). An amplification ratio of 16 showed optimal sensitivity (0.86) and specificity (0.96) as a cutoff point for progression to DDL. Borderline areas, defined as tumors with increased cellularity and atypia but with preserved lipomatous differentiation, showed a significantly higher MDM2 ratio (17.5; P = .0007) compared with WDL. Central (retroperitoneal and intra-abdominal) tumors also showed a significantly higher MDM2 ratio than peripheral ones (P = .02). Conclusions: Differences in MDM2 amplification profiles among liposarcomas could help further define and predict progression to high-grade neoplasia.
334 334
Am J Clin Pathol 2014;141:334-341 DOI: 10.1309/AJCPLYU89XHSNHQO
Upon completion of this activity you will be able to: • calculate the MDM2 amplification ratio and interpret the result. • describe the role of MDM2 in cell cycle regulation and progression to malignancy. • apply MDM2 amplification values to confirm or exclude malignancy. • define borderline areas and explain their suggested role in dedifferentiation. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 443. Exam is located at www.ascp.org/ajcpcme.
With the advent of cytogenetic and molecular testing, a subset of malignant lipomatous neoplasms has been characterized with amplification involving the MDM2 gene on the 12q13-15 chromosomal sequence. Both well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL) have been found to harbor this genetic alteration, distinguishing them from other benign lipomas as well as malignant lipomatous and nonlipomatous neoplasms that do not show the same amplification. Clinically, this finding has led to a better distinction between benign lipomatous neoplasms and WDLs, especially in cases with subtle cytologic atypia on routine histology. Other genes, such as CDK4, HMGA2, and SAS that are also found within this chromosomal region, have been studied and linked to WDL and DDL.1-4 Fluorescent in situ hybridization (FISH) is a practical and convenient way to detect MDM2 amplification. FISH allows © American Society for Clinical Pathology
AJCP / Original Article
the MDM2 copy number to be counted in individual cells and a ratio to be calculated in reference to a standard centromeric probe (CEP12). Other methods of testing have also been applied through immunohistochemistry, polymerase chain reaction, and comparative genomic hybridization.1,5-8 Although MDM2 amplification has become a characteristic genetic marker for these tumors, the actual process of genetic progression from WDL to DDL remains unclear. Clinically, WDL and DDL have differing treatment regimens and mortality characteristics. Even though both WDL and DDL have distinctive histologic appearances, occasional cases show overlapping morphology and lack clear distinction. Being able to accurately distinguish these two entities and better define clinical progression from one to the other could provide more accurate information from which clinical decisions are made. Several studies have commented on the trend in variation of MDM2 copy numbers between WDL and DDL2,9,10; however, none of them have focused on studying this trend or using it for diagnostic purposes. The goal of this study is to determine if the amplification of MDM2 copy numbers is higher in DDL than in WDL using FISH analysis, to determine a potential cutoff point for progression to DDL, and to observe the patterns of this amplification.
Materials and Methods Forty-six cases were chosen from our institution’s archives with original diagnoses of WDL (n = 22), DDL (n = 14), undifferentiated pleomorphic sarcoma (n = 1), myxoid liposarcomas (n = 2), pleomorphic liposarcoma (n = 1), myxoid sarcoma (n = 1), and various benign lipomatous neoplasms (n = 5). The cases were reviewed for appropriate diagnosis by a soft-tissue pathologist (S.A.Q.). Blocks were chosen and marked with tissue best representing the diagnosis. Four cases were considered to have low-grade dedifferentiation. Low-grade DDL was defined as a spindle cell neoplasm arising in conjunction with a WDL but lacking any lipomatous differentiation and showing low cellularity (approaching that of fibromatosis) with only mild atypia and low mitotic rate ❚Image 1❚.11 Four cases had an additional block chosen with morphologic features representing “borderline areas,” defined as areas within the WDL with increased cellularity and atypia in which significant lipomatous differentiation was maintained (Image 1). Overall, a total of 50 samples from the 46 cases were included in the study. FISH Procedure FISH was performed on 4-µm-thick sections of formalinfixed, paraffin-embedded tissue. Each slide was baked overnight at 56°C and then deparaffinized using Hemo-De (Scientific Safety Solvents, Keller, TX) and 100% ethanol. The © American Society for Clinical Pathology
samples underwent pretreatment using the following protocol: 0.2 N hydrochloric acid (HCL) at room temperature (RT) for 20 to 25 minutes, deionized water (H2O) at RT for 3 minutes, pretreatment solution (sodium thiocyanate) at 80°C for 30 to 35 minutes, deionized H2O at RT for 3 minutes, and standard sodium citrate (SSC) buffer at RT for 5 minutes. Protein digestion was then performed using a protease solution of 25 mg pepsin/50 mL of 0.1 N HCL at 37°C for 45 to 60 minutes with an SSC buffer rinse for 5 minutes at RT. After pretreatment, the samples were evaluated for adequate digestion and placed again in the protease solution if further digestion was required. Using 0.5 µL of MDM2 probe (Abbott Molecular, Des Plaines, IL) and 0.5 µL of CEP12 (Abbott Molecular), a probe solution was made in a base of 7 µL LSI buffer (Abbott Molecular) and 2 µL of distilled H2O. The probe was applied to the tissue slides and coverslipped. Slides were hybridized by codenaturing at 74°C for 6 minutes and then fixing for 16 hours at 37°C. Slides were then washed in SSC/0.3% NP-40 solution at 73°C for 2 minutes and air dried, and 6-diamidino-2-phenylindole was applied. Evaluation The FISH slides were evaluated by two individuals, a FISH technologist (M.G.) and a pathologist (P.L.W.), both of whom were blinded to the original diagnosis. A total of 60 cells were counted from the area of interest on each slide. The MDM2 and CEP12 signals were averaged to obtain a total score for each sample. Using the averaged totals, a ratio of MDM2 copy number to CEP12 signal and an average MDM2 copy number per cell were calculated. Calculating the ratio was important to control for chromosome 12 polysomy. An amplification ratio of 2 or more has been shown to be diagnostic of amplification and malignancy.2 The average counts from both individuals were compared and a consensus was reached when the counts were significantly different. Statistical Analysis Means of subgroups, defined by distinguishing histologic characteristics, were compared using two-tailed Student t tests assuming unequal variances. This test is robust in this setting (low sample number and nonparametric data) meaning that significant P values (
MDM2 Copy Numbers in Well-Differentiated and Dedifferentiated Liposarcoma Characterizing Progression to High-Grade Tumors
CME/SAM
Patrick L. Ware, MD,1 Anthony N. Snow, MD,3 Maya Gvalani,2 Mark J. Pettenati, PhD,2 and Shadi A. Qasem, MD1 From the Departments of 1Pathology and 2Molecular Cytogenetics, Wake Forest School of Medicine, Winston-Salem, NC, and 3Department of Pathology, Warren Alpert Medical School of Brown University, Providence, RI. Key Words: MDM2; FISH; Well-differentiated liposarcoma; Dedifferentiated liposarcoma DOI: 10.1309/AJCPLYU89XHSNHQO
ABSTRACT Objectives: MDM2 gene amplification is associated with well-differentiated (WDL) and dedifferentiated liposarcomas (DDL). Using fluorescent in situ hybridization (FISH), we sought to characterize various patterns of MDM2 amplification among the morphologic spectrum of liposarcoma. Methods: Forty-six cases of liposarcoma in various sites were examined and included 22 WDLs, 14 DLLs, and 10 negative control subjects. Results: The MDM2 amplification ratio (MDM2/CEP12) was lower in WDL (10.2) compared with DDL (18.3) cases (P = .0000002). An amplification ratio of 16 showed optimal sensitivity (0.86) and specificity (0.96) as a cutoff point for progression to DDL. Borderline areas, defined as tumors with increased cellularity and atypia but with preserved lipomatous differentiation, showed a significantly higher MDM2 ratio (17.5; P = .0007) compared with WDL. Central (retroperitoneal and intra-abdominal) tumors also showed a significantly higher MDM2 ratio than peripheral ones (P = .02). Conclusions: Differences in MDM2 amplification profiles among liposarcomas could help further define and predict progression to high-grade neoplasia.
334 334
Am J Clin Pathol 2014;141:334-341 DOI: 10.1309/AJCPLYU89XHSNHQO
Upon completion of this activity you will be able to: • calculate the MDM2 amplification ratio and interpret the result. • describe the role of MDM2 in cell cycle regulation and progression to malignancy. • apply MDM2 amplification values to confirm or exclude malignancy. • define borderline areas and explain their suggested role in dedifferentiation. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 443. Exam is located at www.ascp.org/ajcpcme.
With the advent of cytogenetic and molecular testing, a subset of malignant lipomatous neoplasms has been characterized with amplification involving the MDM2 gene on the 12q13-15 chromosomal sequence. Both well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL) have been found to harbor this genetic alteration, distinguishing them from other benign lipomas as well as malignant lipomatous and nonlipomatous neoplasms that do not show the same amplification. Clinically, this finding has led to a better distinction between benign lipomatous neoplasms and WDLs, especially in cases with subtle cytologic atypia on routine histology. Other genes, such as CDK4, HMGA2, and SAS that are also found within this chromosomal region, have been studied and linked to WDL and DDL.1-4 Fluorescent in situ hybridization (FISH) is a practical and convenient way to detect MDM2 amplification. FISH allows © American Society for Clinical Pathology
AJCP / Original Article
the MDM2 copy number to be counted in individual cells and a ratio to be calculated in reference to a standard centromeric probe (CEP12). Other methods of testing have also been applied through immunohistochemistry, polymerase chain reaction, and comparative genomic hybridization.1,5-8 Although MDM2 amplification has become a characteristic genetic marker for these tumors, the actual process of genetic progression from WDL to DDL remains unclear. Clinically, WDL and DDL have differing treatment regimens and mortality characteristics. Even though both WDL and DDL have distinctive histologic appearances, occasional cases show overlapping morphology and lack clear distinction. Being able to accurately distinguish these two entities and better define clinical progression from one to the other could provide more accurate information from which clinical decisions are made. Several studies have commented on the trend in variation of MDM2 copy numbers between WDL and DDL2,9,10; however, none of them have focused on studying this trend or using it for diagnostic purposes. The goal of this study is to determine if the amplification of MDM2 copy numbers is higher in DDL than in WDL using FISH analysis, to determine a potential cutoff point for progression to DDL, and to observe the patterns of this amplification.
Materials and Methods Forty-six cases were chosen from our institution’s archives with original diagnoses of WDL (n = 22), DDL (n = 14), undifferentiated pleomorphic sarcoma (n = 1), myxoid liposarcomas (n = 2), pleomorphic liposarcoma (n = 1), myxoid sarcoma (n = 1), and various benign lipomatous neoplasms (n = 5). The cases were reviewed for appropriate diagnosis by a soft-tissue pathologist (S.A.Q.). Blocks were chosen and marked with tissue best representing the diagnosis. Four cases were considered to have low-grade dedifferentiation. Low-grade DDL was defined as a spindle cell neoplasm arising in conjunction with a WDL but lacking any lipomatous differentiation and showing low cellularity (approaching that of fibromatosis) with only mild atypia and low mitotic rate ❚Image 1❚.11 Four cases had an additional block chosen with morphologic features representing “borderline areas,” defined as areas within the WDL with increased cellularity and atypia in which significant lipomatous differentiation was maintained (Image 1). Overall, a total of 50 samples from the 46 cases were included in the study. FISH Procedure FISH was performed on 4-µm-thick sections of formalinfixed, paraffin-embedded tissue. Each slide was baked overnight at 56°C and then deparaffinized using Hemo-De (Scientific Safety Solvents, Keller, TX) and 100% ethanol. The © American Society for Clinical Pathology
samples underwent pretreatment using the following protocol: 0.2 N hydrochloric acid (HCL) at room temperature (RT) for 20 to 25 minutes, deionized water (H2O) at RT for 3 minutes, pretreatment solution (sodium thiocyanate) at 80°C for 30 to 35 minutes, deionized H2O at RT for 3 minutes, and standard sodium citrate (SSC) buffer at RT for 5 minutes. Protein digestion was then performed using a protease solution of 25 mg pepsin/50 mL of 0.1 N HCL at 37°C for 45 to 60 minutes with an SSC buffer rinse for 5 minutes at RT. After pretreatment, the samples were evaluated for adequate digestion and placed again in the protease solution if further digestion was required. Using 0.5 µL of MDM2 probe (Abbott Molecular, Des Plaines, IL) and 0.5 µL of CEP12 (Abbott Molecular), a probe solution was made in a base of 7 µL LSI buffer (Abbott Molecular) and 2 µL of distilled H2O. The probe was applied to the tissue slides and coverslipped. Slides were hybridized by codenaturing at 74°C for 6 minutes and then fixing for 16 hours at 37°C. Slides were then washed in SSC/0.3% NP-40 solution at 73°C for 2 minutes and air dried, and 6-diamidino-2-phenylindole was applied. Evaluation The FISH slides were evaluated by two individuals, a FISH technologist (M.G.) and a pathologist (P.L.W.), both of whom were blinded to the original diagnosis. A total of 60 cells were counted from the area of interest on each slide. The MDM2 and CEP12 signals were averaged to obtain a total score for each sample. Using the averaged totals, a ratio of MDM2 copy number to CEP12 signal and an average MDM2 copy number per cell were calculated. Calculating the ratio was important to control for chromosome 12 polysomy. An amplification ratio of 2 or more has been shown to be diagnostic of amplification and malignancy.2 The average counts from both individuals were compared and a consensus was reached when the counts were significantly different. Statistical Analysis Means of subgroups, defined by distinguishing histologic characteristics, were compared using two-tailed Student t tests assuming unequal variances. This test is robust in this setting (low sample number and nonparametric data) meaning that significant P values (
