Evidence for alternative lengthening of telomeres in liposarcomas in the absence of ALT-associated PML bodies.

Immortalized and cancer cells maintain their telomeres by activation of a telomere maintenance mechanism (TMM). In approximately 85% of cancers telomerase is activated (TA) but in some tumours, in particular sarcomas, an alternative lengthening of telomeres (ALT) pathway is used. Liposarcomas are the most common soft-tissue sarcoma in adults and they activate ALT or telomerase with equal frequency, however no TMM has been identified in approximately 50% of liposarcomas. In our study, we have shown that instability at the minisatellite MS32, usually associated with ALT activation, aids the identification of liposarcomas that have recombination-like activity at telomeres in absence of ALT associated PML-bodies (APBs). Furthermore, using single molecule telomere analysis, we have detected complex telomere mutations directly in ALT positive liposarcomas and interestingly in some liposarcomas with an unknown TMM but high MS32 instability. We have shown by sequence analysis that some of these complex telomere mutations must arise by an inter-molecular recombination-like process rather than by deletion caused by t-loop excision or by unequal telomere-sister-chromatid-exchange (T-SCE), which is known to be elevated in ALT cell lines. Preliminary evidence also suggests that inter-molecular recombination events may be processed differently in liposarcomas with APBs compared to those without. In conclusion, we have shown for the first time, that some telomerase negative liposarcomas without APBs have other features associated with ALT, indicating that the incidence of ALT in these tumours has previously been under-estimated. This has major implications for the use of cancer treatments targeted at TMMs.

ALT-associated promyelocytic leukaemia body (APB) detection as a reproducible tool to assess alternative lengthening of telomere stability in liposarcomas.

Most cancers maintain telomeres by activating telomerase, but a significant minority, mainly of mesenchymal origin, utilize an alternative lengthening of telomeres (ALT) mechanism. We previously showed the presence of ALT, as detected by ALT-associated promyelocytic leukaemia bodies (APBs) by combined promyelocytic leukaemia immunofluorescence and telomere fluorescence-in situ hybridization, in approximately 25% of frozen specimens obtained from adult patient liposarcomas and proved that ALT negatively affects patient prognosis. In the present study, we assessed the reproducibility of APB detection on frozen versus formalin-fixed, paraffin-embedded specimens from the same liposarcoma specimens and investigated the eventual stability of ALT in 103 different lesions from 40 adult patients followed during their disease. Irrespective of liposarcoma subtype, we (1) confirmed the presence of ALT in 21.4% of tumours; (2) demonstrated the reliability of ALT-associated promyelocytic leukaemia body detection in formalin-fixed, paraffin-embedded sections (with qualitative concordance between matched frozen and formalin-fixed, paraffin-embedded samples in 29/30 specimens, and high quantitative agreement, as indicated by a Spearman correlation coefficient of 0.85); and (3) suggested the stability of ALT status during disease evolution, since the ALT mechanism was never acquired in the 29 patients with initially ALT-negative lesions and lost over time in only two of 11 patients with initially ALT-positive liposarcomas. In conclusion, these results confirm the possibility of investigating the ALT mechanism in archival specimens to obtain biologically relevant information on liposarcoma progression, even when the primary lesion is not available.

Multiple mechanisms of telomere maintenance exist in liposarcomas.

PURPOSE: Telomeres are specialized nucleoprotein complexes that protect and confer stability upon chromosome ends. Loss of telomere function as a consequence of proliferation-associated sequence attrition results in genome instability, which may facilitate carcinogenesis by generating growth-promoting mutations. However, unlimited cellular proliferation requires the maintenance of telomeric DNA; thus, the majority of tumor cells maintain their telomeres either through the activity of telomerase or via a mechanism known as alternative lengthening of telomeres (ALT). Recent data suggest that constitutive telomere maintenance may not be required in all tumor types. Here we assess the role and requirement of telomere maintenance in liposarcoma. EXPERIMENTAL DESIGN: Tumor samples were analyzed with respect to telomerase activity, telomere length, and the presence of ALT-specific subcellular structures, ALT-associated promyelocytic leukemia nuclear bodies. This multi-assay assessment improved the accuracy of categorization. RESULTS: Our data reveal a significant incidence (24%) of ALT-positive liposarcomas, whereas telomerase is used at a similar frequency (27%). A large number of tumors (49%) do not show characteristics of telomerase or ALT. In addition, telomere length was always shorter in recurrent disease, regardless of the telomere maintenance mechanism. CONCLUSIONS: These results suggest that approximately one half of liposarcomas either employ a novel constitutively active telomere maintenance mechanism or lack such a mechanism. Analysis of recurrent tumors suggests that liposarcomas can develop despite limiting or undetectable activity of a constitutively active telomere maintenance mechanism.

Primitive round cell liposarcoma of the omentum: diagnostic value of ultrastructural study.

A 25-year-old woman presented with abdominal distension first observed 1 month earlier. She had a rapidly growing omental tumor that was eventually diagnosed as round cell liposarcoma by ultrastructural examination. This case illustrates the importance of ultrastructural study and the limitations of immunohistochemistry in the diagnosis of such tumors, particularly when they grow in unusual locations.

Epithelioid variant of pleomorphic liposarcoma: a comparative immunohistochemical and ultrastructural analysis of six cases with emphasis on overlapping features with epithelial malignancies.

Pleomorphic liposarcoma (PL) is the least common subtype of liposarcoma, displaying a lipoblastic, malignant fibrous histiocytoma (MFH)-like and, less frequently, an epithelioid growth pattern. The epithelioid morphology in PL is still underrecognized and may closely simulate other epithelial neoplasms, mainly adrenal cortical carcinoma (ACC). No electron microscopic (EM) studies of the epithelioid variant of PL have been previously described, nor have there been studies of its immunoreactivity with A103 or alpha-inhibin. The purpose of this study is to analyze the histological, immunohistochemical, and EM features of epithelioid PL in an attempt to better explore the distinction from their epithelial mimickers, such as ACC. A panel of 5 antibodies was studied, including A103, alpha-inhibin, smooth muscle actin (SMA), AE1/AE3, and Cam 5.2. Out of 22 cases of PLs, 6 cases characterized by the presence of both epithelioid phenotype and pleomorphic lipoblasts were identified from the EM archives. There were 4 females and 2 males, with a mean age of 58 (range, 39-78). Two lesions arose in the thigh and 1 each in the abdominal wall, chest wall, anterior mediastinum, and retroperitoneum, with tumor size ranging from 7 to 17 cm (mean, 13 cm). Histologically, 2 PLs were pure epithelioid, whereas the other 4 had a mixed epithelioid and MFH-like appearance. Immunohistochemically, A103 (4/6), SMA (4/6), and AE1/AE3 (1/6) revealed a various degree of positive reactions. No immunolabeling for alpha-inhibin or Cam5.2 was detected in any case. By EM, the epithelioid areas revealed round or polyhedral cells with lipid droplets of various sizes and number, intimately apposed cell surfaces, occasional junction-like structures (4/6), and micropinocytotic vesicles (4/6). Interestingly, the ribosome-lamellar complexes, once thought to be characteristic of hairy cell leukemia but rarely seen in solid tumors, were noted in one pure epithelioid PL. When compared to the MFH-like area, rough endoplasmic reticula (RER) were less well developed, but mitochondria were more prominent in the epithelioid components. Neither mitochondria with tubulovesicular cristae nor prominent smooth endoplasmic reticula indicative of ACC were seen. Well-formed external lamina was not present. Other features to support a higher level of epithelial differentiation, such as lumen formation, microvilli, and tonofilaments, were not found. In conclusion, focal A103 reactivity in epithelioid undifferentiated tumors should be interpreted with caution before rendering the diagnosis of a primary or metastatic ACC, especially when examining biopsy specimens. The possibility of an epithelioid variant of PL must be excluded; alpha-inhibin can serve as a useful adjunct in this regard. In addition to variable intracytoplasmic fat droplets, the distinctive ultrastructural features of epithelioid variant of PL include numerous mitochondria, pinocytotic vesicles, junction-like structures, and, rarely, ribosome-lamellar complex. Despite some overlapping features, electron microscopy remains a useful tool to distinguish between epithelioid PL and ACC.

Ultrastructure of oral sarcoma.

Sarcoma of the oral region is extremely rare and ultrastructural studies of the tumor are limited in number. We collected oral sarcomas, such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, alveolar soft-part sarcoma, solitary plasmacytoma, and osteosarcoma, and performed ultrastructural studies of these tumors. The value of these studies for an understanding of the biological behavior of the tumors was then investigated. In these studies, electron microscopic examinations of oral sarcoma were of assistance in our attempt to establish correct diagnosis and histogenesis. Data from the studies of oral sarcoma by light microscopy, electron microscopy, and immunohistochemistry should be accumulated.

Morphologic-cytogenetic analysis of dedifferentiated liposarcomas with an extensive misleading leiomyosarcomatous component.

This report describes two cases of recurrent retroperitoneal dedifferentiated liposarcoma characterized by an extensive leiomyomatous component that prevented the correct diagnosis before the last recurrence. Strong immunoreactivity with smooth muscle and desmin antibodies and ultrastructural features consistent with leiomyosarcoma were observed in the spindle-cell and/or myxoid-like components in all four recurrences in case 1, and in the spindle-cell component of the primary tumor and the first recurrence in case 2. In case 1, the correct diagnosis was suggested by the cytogenetic evidence of ring markers, a hallmark of well-differentiated/dedifferentiated liposarcoma. In case 2, tumor type was yielded mainly by the morphology of the second recurrence, which consisted entirely of a well-differentiated liposarcoma, a sclerosing inflammatory variant, as confirmed by the karyotype. Reevaluation of the first two surgical specimens of each case revealed small areas consistent with well-differentiated liposarcoma that had been previously overlooked. Despite the smooth-muscle antigen profile, both cases retained an mdm2+/p53+/cdk4+ immunophenotype consistent with the genotype.

Primary liposarcoma of the stomach: a case report and a review of the literature.

Primary liposarcoma of the stomach is rare and only seven cases have been described in the English literature. Here we report the eighth case, which occurred in a 68-year-old woman who presented with repeated tarry stools and hematemesis. Endoscopic examination revealed a large ulcerated submucosal mass at the gastric angle. The patient was treated by total gastrectomy. On microscopic examination, the tumor showed the features of a well differentiated sclerosing liposarcoma. Immunohistochemically, many spindle to stellate tumor cells were diffusely positive for vimentin and CD34. Positivity for S-100 protein was found in the adipocytic component, including lipoblasts, in addition to some spindle-shaped tumor cells. On ultrastructural examination, the spindle to stellate cells had features characteristic of fibroblasts. No recurrence or metastasis was seen during 13 months. Liposarcoma of the stomach has to be considered in the differential diagnosis with other submucosal lesions, such as gastric lipoma and gastrointestinal stromal tumor.

The ultrastructure of liposarcomas with attention to "dedifferentiation".

Liposarcomas are among the most common soft tissue sarcomas. It is recognized that dedifferentiation can occur within a well-differentiated liposarcoma, but there is limited information concerning the ultrastructure of the dedifferentiated cells. A series of 8 cases has been studied by light and electron microscopy and compared with well-differentiated, myxoid, and pleomorphic liposarcomas. No definite evidence of lipoblastic differentiation could be found in the dedifferentiated cases. The tumor cells resembled atypical cells in the well-differentiated liposarcomas, supporting the close relationship between these two types of tumors. However, since no conclusive line of differentiation could be found in the dedifferentiated cases, this study supports the contention that these neoplasms are undifferentiated counterparts of well-differentiated liposarcomas.

Progressive approach to the cytologic diagnosis of retroperitoneal spindle cell tumors.

OBJECTIVE: To assess the utility of fine needle aspiration biopsy (FNAB) in retroperitoneal spindle cell tumors with difficult tumor typing. STUDY DESIGN: Thirty-six cases of spindle cell tumors of the retroperitoneum were studied. Cytological diagnoses were set progressively: first exclusively by morphologic criteria on routinely stained slides, and then with the aid of immunocytochemistry (ICC), electron microscopy (EM) and clinical data. RESULTS: The morphologic diagnosis of benignity or malignancy was first made in double blind fashion by two researchers; it permitted an exact diagnosis in 31 cases (86%) by one examiner and 27 cases (75%) by the second. Using a progressive approach, benignity or malignancy was definitively determined in 35 cases (97%). As far as cytologic tumor typing is concerned, first it proved to be possible in 27 cases (75%) and then in 30 cases (83%) with the progressive approach. CONCLUSION: FNAB proved to be a useful tool in the diagnosis of retroperitoneal spindle cell tumors. A progressive approach to cytopathologic diagnosis, correlating morphology with ICC and EM results and matching them with clinical data, permitted a better differential diagnosis between benign and malignant spindle cell tumors and increased the possibility of correct tumor typing.

Mesenteric pleomorphic liposarcoma in an adolescent.

We report a case of pleomorphic liposarcoma arising in the root of the mesentery of an adolescent girl. Pleomorphic liposarcoma is an extremely rare tumor in the pediatric age group, and few well-documented cases are found in the literature. To the best of our knowledge, none have been described in the abdomen. The histologic and ultra-structural features of this tumor are described, and the literature concerning pediatric pleomorphic liposarcoma is reviewed.

A well-differentiated giant liposarcoma originating from the buccal fat pad.

Although liposarcoma is one of the most common soft-tissue sarcomas, facial localization is extremely rare. The buccal fat pad is an important anatomic structure located in the face that recently gained interest as a result of increasing research on facial anatomy. In this paper, we report a case of giant liposarcoma originating from the buccal fat pad. The precise localization of the tumor was determined preoperatively with computed tomography examination. The liposarcoma that invaded the body and the extensions of the buccal fat pad was resected completely. The pathological examination revealed a sclerosing, well-differentiated liposarcoma, which is known to be very rare in the head and neck region. Chemotherapy and radiotherapy were not necessary because of the favourable histological type of the tumor and the advanced age and poor general condition of the patient. Local recurrence and distant metastasis were not observed during the 1-year follow-up.

Image cytometry analysis of Feulgen-stained nuclei in 72 lipomatous lesions including atypical lipomas and well-differentiated liposarcomas.

Well-differentiated lipomatous tumors constitute a histopathologic category whose nomenclature has been controversial, particularly with respect to the distinction between atypical lipomas of the extremities and well-differentiated liposarcomas of the retroperitoneum. To determine whether there were differences in image analytic parameters between these neoplasms, 72 lesions including 21 typical lipomas, 7 atypical lipomas, 16 retroperitoneal and 5 nonretroperitoneal well-differentiated, 9 dedifferentiated, and 14 pleomorphic liposarcomas were submitted to the computer-assisted microscopic analysis of Feulgen-stained nuclei. This methodology enabled four groups of variables to be calculated. These included: (1) quantitative chromatin pattern description (14 variables); (2) the measurement of proliferative activity (1 variable); (3) nuclear DNA content (DNA ploidy level, 5 variables); and (4) the measurement of cell density and topographical cell nuclei organization (2 variables). The results strongly suggest that atypical lipomas, whether superficial or deep, and well-differentiated liposarcomas, whether retroperitoneal or not, belong to the same category in terms of the variables analyzed.

Study on the nucleolar organiser regions in soft tissue liposarcoma.

The authors studied 24 cases of liposarcoma of soft tissue included in two categories: myxoid liposarcoma and pleomorphic liposarcoma. The 24 tumors were stained by Ploton's method for revealing the nucleolar organiser regions (AgNORs). AgNORs were counted in the nuclei of 100 cells/case. AgNORs in the cells of some cases showed hyperchromasia and an increased size. The results suggest a role of AgNORs as a possible prognostic discriminator of different histological types.

[Ultrastructural analysis of liposarcoma].

9 cases of liposarcoma, including 5 myxoid type, 3 pleomorphic type and 1 well differentiated type, were studied with light, electron microscopy and immunohistochemistry. Comparative observations revealed similarities between liposarcoma cells and cells of developing fat tissue. Liposarcoma cells resemble primary mesenchymal cells, fibroblasts, early, midstage and late lipoblasts or mature lipocytes. But certain differences also exist: First, atypia in liposarcoma cells, such as the appearance of mono- or multinuclei giant lipoblasts in some cases. Second, certain types of liposarcoma are predominated by lipoblasts of a specific stage. Under electron microscope, transitional morphology from both primary mesenchymal cell and fibroblast to lipoblast can be observed, which is an indication that lipoblasts may originate from these two types of cells. Differential diagnosis by electron microscopy is also discussed.

Sarcomas with combined features of liposarcoma and leiomyosarcoma. Study of two cases of an unusual soft-tissue tumor showing dual lineage differentiation.

Two cases are described of a soft-tissue sarcoma characterized histologically by the intimate admixture of areas displaying the features of liposarcoma and leiomyosarcoma. Both cases occurred in men, 70 and 77 years of age. The lesions were located in the left scrotum and abdominal cavity, respectively. Histologically, the lipomatous component in both cases consisted predominantly of well-differentiated liposarcoma with myxoid areas. The smooth-muscle component was characterized by intersecting fascicles of spindle cells displaying nuclear atypicality and scattered mitotic figures; the spindle cells in these areas were strongly immunoreactive with actin and desmin antibodies. The above tumors must be distinguished from angiomyolipoma, spindle-cell lipoma, myolipoma of soft tissue, and more importantly, from "dedifferentiated" liposarcoma. The development of dual lineage differentiation within adipose tissue tumors as exemplified by these two cases may be more prevalent than has been generally recognized, and may require the application of immunohistochemical markers for specific identification of the spindle-cell component.

Cytogenetic and fluorescence in situ hybridization investigation of ring chromosomes characterizing a specific pathologic subgroup of adipose tissue tumors.

Cytogenetic analysis of 184 adipose tissue tumors, 175 lipomas, and nine liposarcomas (LPS) showed the presence of a ring chromosome and/or a long marker chromosome in 10 cases with common histologic features such as atypical stromal cells with or without lipoblasts. In five of the cases, this appeared to be the sole cytogenetic abnormality. Fluorescence in situ hybridization (FISH) analysis with a microclone library specific for chromosome region 12q13-q15 showed extensive staining of the ring and long marker chromosomes, indicating that genetic sequences of this particular region of chromosome 12 are present in these marker chromosomes, most likely in an amplified form.

Chromosome abnormalities in liposarcomas.

We performed a cytogenetic study of short-term cultures from fresh surgical specimens obtained from four patients with liposarcoma. Myxoid liposarcomas (cases 1-3) were associated with a specific translocation between chromosomes 12 and 16. Trisomy 8, a nonrandom secondary aberration in myxoid liposarcoma, was observed in the third case as the only additional change. Round cell liposarcoma (case 4) showed complex chromosomal aberrations affecting chromosomes 1, 2, 5, 6, 7, 13, 14, 17, 19, and 22. Neither band 12q13 nor 16p11 was visibly rearranged. Three subgroups of liposarcomas are proposed. The first group is characterized by t(12;16)(q13;p11), the second group by ring chromosomes, telomeric associations, and giant markers, and the last by complex numerical and structural aberrations.