| Myofibroblasts, which share morphologic and functional features with fibroblast and smooth-muscle cell, especially in relation to matrix production and contractility, were initially described in granulation tissue in 1971 by Gabbiani. Myofibroblasts bearing ultrastructural features of both fibroblast and smooth-muscle cell are present in various reactive conditions including reparative granulation, granulomas and inflammatory pseudotumor. It is evident that various benign and malignant soft tissue tumors may contain myofibroblastic cells in varying degrees. It was used to be a controversial issue that if the myofibroblastic tumors exist.However, with the development of recognition about myofibroblast, the myofibroblastic tumor was gradually accepted. Inflammatory myofibroblastic tumor (IMT), a well-known true myofibroblastic neoplasm, was reported in the literature recently. Myofibroblastoma and myofibromatosis were also documented in the literature. Malignant mesenchymal neoplasm, composed of a cell population with myofibroblastic characteristics, was uncommon. Low-grade myofibroblastic sarcoma was classified as a distinct entity in the newly-published World Health Organization (WHO) classification of soft tissue tumors.With increasing case reports, it has come to be clear that myofibroblastic sarcoma (MS), as a distinct entity in soft tissue sarcomas, is different from fibrosarcoma and leiomyosarcoma, as well as other kinds of spindle cell lesions.MS was used to be a controversial neoplasm, which was suspected in defining a distinct entity. MS was easy to be misdiagnosed as reactive lesions owing to myofibrolasts' common existence in the reactive granuloma; and was easy to be misinterpreted as other spindle cell sarcomas due to its morphologic similality with the fibroblasts and smooth muscle cells and overlap immunophynotype. Even though MS was sparsely reported in the international literature, more systemic and detailed research on the disease is still needed to be carried out, in order to elucidate the clinicopathologic features of MS thoroughly.Despite MS was classified as an entity, the molecular cytogenetic data about the tumor is presently limited.Generally speaking, nearly all human malignant tumors are characterized by genomic changes.Classical cytogenetic analysis using chromosome banding technique over decades, was the "gold standard" for detection of genomic changes of solid tumor. However, as a well-known fact, only a limited portion of solid tumors examined by classical cytogenetic technique yielded a sufficient number of evaluable mitoses for establishing the karyotype. The main reasons were low mitotic yield, low number of evaluable metaphases, low quality of chromosome banding, and sometimes very complex karyotypes not allowing definite analysis. All collections of classical tumor cytogenetic data, therefore, had to be based on a selected fraction of evaluable tumors and can not carry out on the archival material as retrospective research.The advent of comparative genomic hybridization (CGH), which was established in 1992, had opened a reliable way for the detection of all genomic imbalances (copy number alterations of DNA) in each tumor (including archival material) without selection.Different patterns of chromosomal aberrations, including gains and losses of tumor DNA sequences, were detected in various human malignant neoplasms by CGH,which not only provided a method to detecte aberration genes, but also found some gene aberrations correlated with the clinicopathologic parameters, and was used as tumorigenesis, differential diagnosis, tumor development, tumor prognosis and treatment research. CGH is an idea method to study a new tumor, whose cytogenetic aberration is unknown and needed to be screened, in order to finally set up the target gene for research. With the CGH, we can detect gene aberrations (gains or losses), then define the onconge and the tumor suppressor gene, finally revealing the tumorigenesis of the tumor. On the other hand,we can find the DNA aberrations that correlated with the clinicopathologic parameters during the development of the tumor. We can also decide if different tumors belong to the same entity by comparing their gene aberration profile.To date,few gene imbalance study of MS have ever been documented in the literature.Karyotype analysis of 3 cases of MS was documented in the literature in 1999 by Fletcher,and recently, one case report of intermediate myofibroblastic sarcoma ocurring in the lung,which was detected by CGH, was reported by Morawietz in 2005.In this study, We studied 29 cases of MS using light microscopy, immunohistochemistry and electron microscopy with review of the literature, in order to elucidate the clinicopathologic features of MS and discuss the differential diagnosis from other spindle cell sarcomas showing fibroblastic and smooth muscle cell differentiation. All cases of MS included in the study were demonstrated to exhibit the ultrastructural features of moyfibroblast. Considering the fact that MS, especially the low grade MS, is easy to be morphologically confused with the fibromatosis, nodular fasciitis and leiomyosarcoma, which is the difficulty in surgical pathology diagnosis, we included a cohort of the three lesions into the research as comparative study with the aim of investigating the differential diagnosis between MS and the three lesions.After that, we detected 29 cases of MS, 5 cases of fibromatosis, nodular fasciitis and leiomyosarcoma respectively by CGH,in order to investigate the DNA copy number changes that may harbor the potential oncogenes and tumor suppressor genes involving in tumorigenesis of MS.By camparing the DNA imbalance profile of MS with that of fibromatosis, nodular fasciitis and leiomyosarcoma respectively, we investigated the differential diagnosis on the level of molecular cytogenetics between MS and the three lesions. The study was composed of two parts, which included the clinicopathologic and the molecular cytogenetic study of MS.Part 1: The clinicopathologic study of MSThe paraffin-embedded blocks of 29 cases of MS were studied using light microscopy, immunohistochemistry and electron microscopy, in order to elucidate the clinicopathologic characteristics of MS. At the same time, 7 cases of nodular fasciitis, 8 cases of leiomyosarcoma and 10 cases of fibromatosis were studied morphologically and immunohistochemically in order to investigate the differential diagnosis between MS and the three lesions.The results were as followings: MS can occur in any range of age, and locates preferentially in the head and neck region, as well as in the bone. The painless enlarging mass is the most common clinical symptoms. All cases were single nodular except one case showing multicentric lesions in the bone. Clinical follow-up information of 28 patients revealed that: 8 cases (28.6%) died of tumors; 7 cases (25.0%) recurred locally and developed distant metastasis after initial operation; 10 cases (35.7%) recurred locally after initial excision, and 9 cases (32.1%) are alive with no evidence of disease.Grossly, 23 out of 29 cases of MS (79.3%) showed diffusely infiltrative growth pattern. Microscopically,the neoplastic myofibroblasts were spindled to stellate with enlarged round to oblong nuclei, variable nucleoli and abundant eosinophilic to basophilic appearing fibrillar cytoplasm with indistinct cell boundary. Some tumor cells may exhibit a more epithelioid or histiocytoid appearance, that is round to oval nuclei, prominent nucleoli and rich cytoplasm. The other tumor cells may appear as slender axonal cells with elongated nuclei, inappearent nucleoli and long cytoplasmic extensions creating a tadpole-like appearance. The degree of cellularity and pleomorphism of tumor cells was variable from areas to areas in the same tumor or in different cases of tumor. In short, hypercellular areas and hypocellular areas, both of which distributed irregularly and transitioned to each other, can be identified in the same tumor. In the hypercellular areas, the spindle or plump oval tumor cells with abundant pale or eosinophilic cytoplasm and ovoid vesicular nuclei were arranged in an intersecting fascicle or vaguely storiform pattern,exhibiting moderate nuclear atypia.In other hypercellular areas, the tumor cells were slender spindle-shaped with eosinophilic ill-defined cytoplasm, and tapering and wavy nucleis with finely distributed chromatin and a small inconspicuous nucleoli, showing mild nuclear atypia. In hypocellular areas, the spindle, or feathery tumor cells with indistinct cell boundary arranged in fascicle or intersecting fascicle pattern, were scattered haphazardly in abundant myxoid matrix with infiltration of lymphocytes, plasma cells and eosinophils, bearing some resemblance to the tissue culture-like growth pattern. In other hypocellular areas, the spindle tumor cells with tapering nuclei were arranged in fascicles pattern and deposited in a hyalinized collagenous matrix. Low grade MS showed no nuclear atypia or mild nuclear atypia with rare mitotic figures.Intermediate grade MS showed moderate nuclear atypia with mean mitotic rate of 6-10/10HPF.High grade MS was pleomormophic tumor.The current study demonstrated that myxoid stroma, the infiltration of a large number of lymphocytes and plasma cells and collagenous stroma were present in most of cases.Immunohistochemically, SMA staining in MS was accentuated beneath the cell membrane in a linear pattern, as compared with diffuse staining in the cytoplasm of leiomyosarcoma.The fibronectin showed cell membrane and extracellular matrix positive staining. The tumor cells were extensively positive for calponin (86%),SMA (96%), fibronectin (100%) and MSA (55%). A small number of cases were positive for CD68 (27%). Desmin (6.8%), type IV collagen (17%) and laminin (24%) were rarely positive staining;while h-caldesmon and ALK1 were consistently negative staining.The result demonstrated that MS was mainly express SMA, calponin and fibronectin; MSA was not so sensitive than SMA in MS. In contrast, the leiomyosarcoma showed diffusely strong positive staining for desmin, MSA, h-caldesmon and laminin. Nodular fasciitis was negative staining for calponin, while positive for laminin. Fibromatosis was positive for laminin,occationally focally positive for SMA, but negative for calponin. The average Ki-67 labeling index in indermediate grade MS was significantly higher than that in low grade MS,P=0.000.The average Ki-67 labeling index in low grade MS was also significantly lower than that in low grade leiomyosarcoma, P=0.000.The average Ki-67 labeling index in fibromatosis was significantly lower than that in low grade MS,P=0.000. The average Ki-67 index in low grade MS was lower than that in nodular fasciitis,P=0.001.The findings demonstrated that ki-67 index was helpful in grading of MS,as well as in differential diagnosis of MS from the three lesions.Ultrastructurally, spindle-shaped tumor cells and abundant intercellular collagen were seen under low-power magnification. Under high-power magnification, abundant rough endoplasmic reticulum (rER) and longitudinally arranging myofilaments with focal densities which distributed primarily at the peripheral cytoplasm were present in all cases examined. Fibronexus junctions, which were roughly parallel with extracellular fibrils, and diverged into the outside collagenous matrix, were present at the cell surface of 17 out of 29 cases of MS (58.6%), and not identified in other 12 cases. Nuclear indentation was present in some tumor cells.MS showing diverse and bland histologic appearance was easy to be misinterpreted as benign lesions. In contrast to MS, leiomyosarcomas exhibited mainly a well delineated pushing margin and generally lacked a diffusely infiltrative growth pattern, and showed more eosinophilic and longitudinally fibrillary cytoplasm and cigar-shaped vesicular nuclei with paranuclear vacuolation. Immunohistochemically, leiomyosarcoma was extensively positive for h-caldesmon, desmin and laminin,showing higher Ki-67 labeling index. Ultrastructurally, leiomyosarcomas showed poor rER in the cytoplsam,with the myofilememts distributing throughout the cytoplasm and exhibiting no fibronexus junction. In contrast to MS,fibromatosis contained a large proportion of fibroblasts and small proportion of focal myofibroblasts. Additionally, fibromatosis differentiated from MS in that the cells in fibromatosis did not touch one another, but are frequently separated by collagen owing to its richer hyalinization collagen. Furthermore, fibromatosis exhibits lower cellularity with absence of nuclear atypia, atypical mitotic figure and necrosis. Immunohistochemically, fibromatosis showing very low Ki-67 labeling index as compared with MS (P=0.000),was diffuse positive for laminin, and occationally focal positive for SMA, but negative for calponin. In contrast to nodular fasciitis, MS is more cellular, fascicle, and infiltrates into the muscle than nodular fasciitis, which exhibits no nuclear hyperchromatin, pleomorphism and atypical mitoses. Additionally, nodular fasciitis is usually characterized by short duration and smaller size (smaller than 5cm), and by locating in some specific regions such as the subcutaneous of forearm. Immunohistochemically, nodular fasciitis was positive for laminin, but negative for calponin (P=0.000).Additionally,MS should be distinguished from other myofibroblastic tumors such as IMT,myofibroblastoma and myofibromatosis.MS was demonstrated to have a relatively indolent course with frequent recurrence and rare metastasis. Low grade MS was local aggressive lesion with frequent recurrence; while intermediate grade MS exhibited highrecurrence rate and frequent metastasis.Part 2 The molecular cytogenetic study of MSDNA copy number changes were analyzed by CGH in 29 cases of MS and 5 cases of nodular fasciitis, fibromatosis and leiomyosarcoma respectively with purpose to investigate the DNA copy number imbalance in MS, and to correlate the frequency of genomic alterations with histopathologic parameters. At the same time,we compared the DNA imbalance profile in MS with that in the three lesions in order to find the differential evidence in term of molecular cytogenetics. Tumor genomic DNA was isolated from paraffin-embedded blocks of tumors by a standard phenol-chloroform extraction procedure after proteinase K digestion. Reference DNA was obtained from blood samples of healthy male and female donors. We labeled 1μg of each tumor and reference DNA by nick translation using a kit according to the manufacture's instructions (Vysis, Germany). Tumor DNA was labeled with spectrum Green-conjugated deoxyuridine triphosphate and reference DNA with spectrum Red-Conjugated deoxyuridine triphosphate (Vysis).Then hybridization, image acquisition and analysis were performed. Gains or losses were considered recurrent if they were detected in two or more cases.The DNA copy number changes in 29 cases of MS detected by CGH were as followings. Out of 29 samples studied by CGH, 23 cases (79.3%) showed DNA sequence copy number changes, the mean number of aberrations was 6.4 per sample. Gains were more frequent than losses(gains:losses=3:2).High-level amplication was not detected. The minimal common regions for the most frequent gains were:1p11→p36.3 (65.5%),12p12.2→p13.2(44.8%),5p13.2→p15.3(31%),22(27.5%),2p11.2→p 25.1 (20.6%),2q34→q37.3 (17%),3q24.3→p25 (17%) and 5q31.3→35.3 (17%); the minimal overlap regions were: 1p35→36.3 ( 3/29 ) ,1p32.3→34.3(3/29),1p21 ( 3/29 ) ,1p13.3 ( 3/29 ) ,1p13.2 ( 4/29 ) ,1p11→13.1 ( 3/29 ) ,1p31.1→36.3 (2/29) ,5p15.2(3/29),5p15.3(2/29),5p14(2/29),5p13.2→13.3(3/29),12p13.1( 4/29),12p13.2(3/29),12p12.3 ( 3/29 ) ,12p12.2(3/29),22p11.2(3/29) and 22q13.1(3/29).The common regions for the most frequent losses were: 15q25-26.2 (24%) ,3p14.3→25 (13.8%) ,6q27 (13.8%) ,17pl3 (13.8%) and 18p11.3 (13.8%) ;the minimal overlap regions were: 15q26.2(5/29) and 15q25(2/29). CGH analysis in MS showed multiple and complex DNA copy number changes, which included a number of characteristic chromosomal imbalances such as gains at 1p (65.5%),12p(44.8%),5p(31.0%),and 22(27.6%);loss at 15q(24.1%).The recurrent genetic changes in MS affecting chromosomes sites that may harbour genes participating in tumorigenesis of MS provided evidence for the localization of potential oncogenes and tumor suppressor genes active in MS genomes.Correlating the total number of aberrations and the most frequent aberrations with clinicopathological parameters, we found that larger number of aberrations was associated with higher tumor grade,P=0.000.The gain of 1p was associated with higher tumor grade,P=0.025,as well as with larger tumor size,P=0.027.The gain of 12p was associated with higher tumor grade,P=0.000. DNA gain at 1p may be important for tumor development,as it was seen preferentially in larger tumors; whereas, gains in 1p and 12p appeared to be associated with tumor progression,as they were seen preferentially in higher grade tumor.The chromosome aberrations of gains at 10p14→p15, 10q26.3 and 20q12→q13.3 were detected in one out of five cases of nodular fasciitis examined by CGH. But no loss was detected. The average number of aberrations in nodular fasciitis (0.6) was significant lower than that in MS (6.4), P=0.000. The recurrent gain at 1q32.2→41(2/5), the recurrent losses at 20p11.2(2/5) and 21p12(2/5) were found in 5 cases of leiomyosarcoma. For the fibromatosis, recurrent gain was: 16p13.1(2/5); and the recurrent losses were 17p13(3/5), 18p11.3(3/5), 2q37.3 (2/5) ,5q35.3 (2/5) ,6q27 (2/5),14q32.1→32.3( 2/5) and 15q26.1→26.3q( 2/5). The above-mentioned data showed that there was no overlap recurrent gains or losses between MS and nodular fasciitis, fibromatosis and leiomyosarcoma respectively, which demonstrated that MS showed different cytogenetic aberrations as compared with the three lesions, and was cytogenetically different from the nodular fasciitis, fibromatosis and leiomyosarcoma by sharing no range of common chromosome abnormalities, indicating that despite the morphologic and phenotypic similarity, they should be regarded as different disease entity.The study investigated systematically the clinicopathologic and molecular cytogenetic characteristics of MS in a large series,which will be helpful for the pathologists to recognize the clinicopathologic features of MS.The present study demonstrated that MS showed characteristic cytogenetic aberrations profile, and should be regarded as a desease entity.The findings in the current study provided some useful evidence,on the base of which, by more exact molecular cytogenetic methods such as FISH,gene chip and array-CGH, oncogenes and tumor suppressor genes,as well as other genetic aberrations including mutation and translocation, are expected to be uncovered,and finally elucidate the tumorigenesis of MS. |
PDF Full Text Request