Diagnosis and management of tropomyosin receptor kinase (TRK) fusion sarcomas: expert recommendations from the World Sarcoma Network.

Sarcomas are a heterogeneous group of malignancies with mesenchymal lineage differentiation. The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as tissue-agnostic oncogenic drivers has led to new personalized therapies for a subset of patients with sarcoma in the form of tropomyosin receptor kinase (TRK) inhibitors. NTRK gene rearrangements and fusion transcripts can be detected with different molecular pathology techniques, while TRK protein expression can be demonstrated with immunohistochemistry. The rarity and diagnostic complexity of NTRK gene fusions raise a number of questions and challenges for clinicians. To address these challenges, the World Sarcoma Network convened two meetings of expert adult oncologists and pathologists and subsequently developed this article to provide practical guidance on the management of patients with sarcoma harboring NTRK gene fusions. We propose a diagnostic strategy that considers disease stage and histologic and molecular subtypes to facilitate routine testing for TRK expression and subsequent testing for NTRK gene fusions.

Is Peritoneal Tumor Penetration of Prognostic Importance in Gastrointestinal Stromal Tumors?

BACKGROUND: Peritoneal tumor penetration (PP) strongly affects prognosis in gastrointestinal carcinomas. In gastrointestinal stromal tumor (GIST), its significance in the absence of tumor rupture has not been subjected to detailed analysis. METHODS: Patients undergoing complete resection for non-metastatic GIST from 2000 to 2017 were identified in the regional sarcoma database at Oslo University Hospital. Patients with extraperitoneal tumors (esophagus, rectum) or ruptured tumors were excluded from the study. Rupture was defined according to the Oslo criteria, and PP was assessed via routine histopathologic examination by sarcoma pathologists. RESULTS: The study enrolled 341 patients. The median follow-up period was 51 months (range 0-175) months. In 82 (24%) of the 341 patients, PP was recorded. There were 32 recurrences, 9 in patients with PP and 23 in patients without PP. Despite statistically significant associations between PP and established risk factors (size, mitotic index, non-gastric location), the 5-year recurrence-free survival rate did not differ between the patients with PP (86%) and those without PP (90%) (hazard ratio 1.25; 95% confidence interval 0.58-2.70; P = 0.577). Adjuvant imatinib was administered to 53 of 97 patients in the high-risk category. The recurrence rates did not differ between the PP-positive and PP-negative patients in either group. CONCLUSIONS: In GIST, PP without tumor rupture appears not to influence prognosis. This lack of prognostic significance may reflect unexplored differences between epithelial and mesenchymal malignancies.

Relationship between R1 resection, tumour rupture and recurrence in resected gastrointestinal stromal tumour.

BACKGROUND: According to guidelines, adjuvant treatment or re-excision should be considered after R1 resection of gastrointestinal stromal tumours (GISTs). However, the prognostic significance of R1 resection is uncertain and tumour rupture confounds its assessment. Here, the impact of positive margins was examined and related to rupture in a population-based cohort. METHODS: Patients undergoing surgery for non-metastatic GIST since 2000 were identified in the sarcoma database of Oslo University Hospital. Margins were coded according to the residual tumour (R) classification and tumour rupture defined according to the Oslo criteria. RESULTS: Among 410 patients, there were 47 who underwent R1 resection and 52 had tumour rupture. The relative risk of R1 resection with rupture was 3·55 (95 per cent c.i. 2·09 to 6·03; P < 0·001). In patients without rupture, there was no difference in estimated 5-year recurrence-free survival after R0 versus R1 resection (87·6 versus 93 per cent; hazard ratio (HR) 0·71, 95 per cent c.i. 0·17 to 2·98; P = 0·638); nor was there any difference among patients with rupture (37 versus 31 per cent; HR 1·31, 0·68 to 2·54; P = 0·420). In multivariable analysis, tumour rupture but not R1 resection was independently associated with recurrence. Twenty-four patients at very low, low or intermediate risk did not receive adjuvant imatinib after R1 resection and remained recurrence-free. CONCLUSION: Positive resection margins are strongly associated with tumour rupture. R1 resection does not independently influence prognosis. Adjuvant imatinib may not be justified after R1 resection in the absence of tumour rupture or other high-risk features.

Genotype and risk of tumour rupture in gastrointestinal stromal tumour.

BACKGROUND: Tumour rupture is a strong predictor of poor outcome in gastrointestinal stromal tumours (GISTs) of the stomach and small intestine. The objective was to determine whether tumour genotype was associated with risk of rupture. METHODS: Rupture was classified according to the definition proposed by the Oslo Sarcoma Group. Since January 2000, data were registered retrospectively for all patients at Oslo University Hospital undergoing surgery for localized GIST of the stomach or small intestine. Tumour genotype was analysed by Sanger sequencing. RESULTS: Two hundred and nine patients with mutation data available were identified. Tumour rupture occurred in 37 patients. Among the 155 patients with KIT exon 11 mutations, an increased risk of rupture was observed with a deletion or insertion-deletion (25 of 86, 29 per cent) compared with substitutions (5 of 50, 10 per cent) or duplications/insertions (2 of 19, 11 per cent) (P = 0·014). Notably, rupture occurred in 17 of 46 tumours (37 per cent) with deletions involving codons 557 and 558 (del557/558) versus 15 of 109 (13·8 per cent) with other exon 11 mutations (P = 0·002). This association was confined to gastric tumours: 12 of 34 (35 per cent) with del557/558 ruptured versus six of 77 (8 per cent) with other exon 11 mutations (P = 0·001). In multivariable logistic regression analysis, del557/558 and tumour size were associated with an increased likelihood of tumour rupture, but mitotic count was not. CONCLUSION: Gastric GISTs with KIT exon 11 deletions involving codons 557 and 558 are at increased risk of tumour rupture. This high-risk feature can be identified in the diagnostic evaluation and should be included in the assessment when neoadjuvant imatinib treatment is considered.

Definition and clinical significance of tumour rupture in gastrointestinal stromal tumours of the small intestine.

BACKGROUND: Tumour rupture is a risk factor for recurrence of gastrointestinal stromal tumour (GIST). In this study, patterns of recurrence after potential tumour seeding were investigated, and a new definition of tumour rupture, based on major and minor defects of tumour integrity, is proposed. METHODS: Patients undergoing surgery for non-metastatic small intestinal GIST from 2000 to 2012 were included in the study. Tumour spillage, tumour fracture or piecemeal resection, bowel perforation at the tumour site, blood-tinged ascites, microscopic tumour infiltration into an adjacent organ, and surgical biopsy were defined as major defects of tumour integrity. Peritoneal tumour penetration, iatrogenic peritoneal laceration and microscopically involved margins were defined as minor defects. RESULTS: Seventy-two patients were identified. Median follow-up was 58 (range 7-122) months. Radical surgery was performed in 71 patients. A major defect was recorded in 20 patients, and a minor defect in 21. The 5-year recurrence rate was 64, 29 and 31 per cent in patients with major, minor and no defect respectively (P = 0·001). The hazard ratio (HR) for major defect versus no defect was 3·55 (95 per cent c.i. 1·51 to 8·35). Peritoneal recurrence rates for major, minor and no defect were 52, 25 and 19 per cent respectively (P = 0·002), and the HR for major defect versus no defect was 4·98 (1·69 to 14·68). On multivariable analysis, mitotic index, major defect of tumour integrity, tumour size and age were independently associated with risk of recurrence. CONCLUSION: Recurrence rates were increased after major, but not minor tumour ruptures.

Why do patients with radiation-induced sarcomas have a poor sarcoma-related survival?

BACKGROUND: This study aims to provide reasons for the poor sarcoma-related survival in patients with radiation-induced sarcoma (RIS). METHODS: We performed a case-control study comparing sarcoma-related survival of 98 patients with RIS to that of 239 sporadic high-grade malignant sarcomas. RESULTS: The cumulative sarcoma-related 5-year survival was 32% (95% confidence interval (CI): 22-42) for patients with RIS vs 51% (95% CI: 44-58) for controls (P<0.001). Female gender, central tumour site and incomplete surgical remission were significantly more frequent among RIS patients than in controls. In multivariate analysis incomplete surgical remission (hazard ratio (HR) 4.48, 95% CI: 3.08-6.52), metastases at presentation (HR 2.93, 95% CI: 1.95-4.41), microscopic tumour necrosis (HR 1.88, 95% CI: 1.27-2.78) and central tumour site (HR 1.71, 95% CI: 1.18-2.47) remained significant adverse prognostic factors, but not sarcoma category (RIS vs sporadic). CONCLUSION: The poor prognosis of RIS patients are not due to the previous radiotherapy per se, but related to the unfavourable factors - central tumour site, incomplete surgical remission, microscopic tumour necrosis and the presence of metastases, the two former factors overrepresented in RIS.

Identification of low-risk tumours in histological high-grade soft tissue sarcomas.

In more than one-third of patients with a histological high-grade malignant soft tissue sarcoma metastasis develops despite local control of the primary tumour. Hence, adjuvant chemotherapy is increasingly used for these relatively chemoresistant tumours which requires improved prognostication to exclude low-risk patients from overtreatment. We assessed the value of stepwise prognostication in a series of 434 histological high-grade STS of the extremity and trunk wall. Vascular invasion was used as the first discriminator whereafter the risk factors tumour necrosis, size (>8cm) and infiltrating growth pattern were used to discriminate high- and low-risk tumours. We identified a high-risk group with a cumulative incidence of metastasis >0.4 at 5 years, and a low-risk group, comprising half of the tumours, with a cumulative incidence of metastasis <0.15. The model was validated in an independent material of 175 patients. This model improved prognostication in STS and is of value for identifying patients who probably should not receive adjuvant chemotherapy.

Fusion of the RBP56 and CHN genes in extraskeletal myxoid chondrosarcomas with translocation t(9;17)(q22;q11).

Although most extraskeletal myxoid chondrosarcomas (EMC) are cytogenetically characterized by the translocation t(9;22)(q22;q12), another subset has recently been identified carrying a t(9;17)(q22;q11). Whereas the t(9;22) is known to result in fusion of the CHN (TEC) gene from 9q22 with the EWS gene from 22q12, creating a chimeric EWS/CHN, the genes involved in the t(9;17) of EMC are unknown. We examined two EMC with t(9;17)(q22;q11) and found that the CHN gene was recombined with the RBP56 gene from 17q11 to generate a chimeric RBP56/CHN. RBP56 has not previously been shown to be involved in tumorigenesis but it encodes a putative RNA-binding protein similar to the EWS and FUS (TLS) proteins known to play a pathogenetic role in several sarcomas. The presence of the RBP56/CHN chimeric gene in EMC with t(9;17)(q22;q11) shows that the N-terminal parts of EWS and RBP56 have similar oncogenic potential making them pathogenetically equivalent in oncoproteins arising from fusions with certain transcription factors.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Extraskeletal myxoid chondrosarcoma: multimodal diagnosis and identification of a new cytogenetic subgroup characterized by t(9;17)(q22;q11).

Extraskeletal myxoid chondrosarcoma is a rare malignant soft tissue tumour that can be difficult to diagnose correctly, especially preoperatively. We describe four cases of extraskeletal myxoid chondrosarcoma of the extremities diagnosed by a multimodal approach. The cytological examination of fine-needle aspirates showed small and round, mildly pleomorphic cells lying in sheets and cords, but also dispersed within a myxoid and metachromatic intercellular substance. Histological, electron microscopic and immunocytochemical examination also yielded findings compatible with the diagnosis of extraskeletal myxoid chondrosarcoma. Cytogenetic analysis demonstrated a t(9;22)(q22;q12) in two tumours and a t(9;17)(q22;q11) in the third and fourth. The translocation t(9;22)(q22;q12) has been described repeatedly in extraskeletal myxoid chondrosarcoma but never in other tumours; hence, the detection of this pathognomonic chromosome abnormality in short-term cultured cells from fine-needle aspirates verified the diagnosis in two of the cases. The t(9;17)(q22;q11) found in the last two cases probably represents a new cytogenetic subgroup of extraskeletal myxoid chondrosarcoma as it, too, is unknown in other contexts. The multimodal approach taken in these four cases enabled a definite diagnosis of a rare malignant tumour whose cytological and histological features alone are usually not sufficiently distinct to rule out other differential diagnostic possibilities.

A well-differentiated liposarcoma with a new type of chromosome 12-derived markers.

Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14 approximately 15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14 approximately 15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.

Dedifferentiation of a well-differentiated liposarcoma to a highly malignant metastatic osteosarcoma: amplification of 12q14 at all stages and gain of 1q22-q24 associated with metastases.

Well-differentiated liposarcomas (WDLPS), especially those located in the retroperitoneum, may occasionally undergo dedifferentiation. Although this process is associated with a more aggressive clinical course, dedifferentiated liposarcomas rarely produces metastases. The case reported here is rather uncommon: A retroperitoneal WDLPS gave lung metastases that were diagnosed as highly malignant osteosarcomas. We used comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and Southern blot analyses to characterize the copy number changes and genetic aberrations occurring at different stages of the disease. In the primary tumor, the only detectable aberration was amplification of 12q13-q14, which was present only in a fraction of the cells and revealed by FISH analysis. High-level amplification of 12q13-q14, involving CDK4, MDM2, and HMGIC, was seen both in the relapse and the metastases. The second most common change, gain or high-level amplification of 1q22-q24, was detectable by CGH only in the osteogenic metastases, as was loss of the distal 2q. FISH analyses revealed considerable heterogeneity in the samples, and the percentage of cells showing aberrations was significantly higher in the metastatic samples. In particular, increased copy numbers of 789f2, a marker for 1q21 amplification in sarcomas, was observed in more than 65% of the cells in the metastatic samples, but in less than 10% of the cells from the recurrent samples. These observations could indicate that 1q amplification, in particular, may be indicative of a more malignant phenotype and ability of metastasis in WDLPS, as has also been suggested by others.

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma.

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.

Immunohistochemical profile of synovial sarcoma with emphasis on the epithelial-type differentiation. A study of 49 primary tumours, recurrences and metastases.

The relationship between biphasic (BSS) and monophasic (MSS) subtypes of synovial sarcoma (SS) as well as the relationship between cells of solid/glandular areas and the spindle cells of BSS remain controversial. In order to further evaluate the immunohistochemical phenotype of SS we studied 34 primary tumours (15 BSS; 19 MSS), 7 recurrences (4 from primary BSS; 3 from primary MSS) and 8 metastases (7 BSS; one MSS), using several antibodies (EMA, CEA, keratins 1, 4, 5/6, 7, 8, 13, 18, 19, 20, vimentin, collagen IV and laminin) that work in paraffin-embedded material. Spindle cells outside solid/glandular areas of BSS and in MSS showed immunoreactivity for keratins 5/6, 7, 8, 18 and 19. The transition of solid/glandular areas to surrounding spindle cells also showed keratin staining and failed to show a distinct separation regarding the immunoreactivity for laminin and collagen IV. Peripheral cells of solid/glandular areas were immunoreactive for vimentin. No major differences were observed between immunophenotypical cell profiles of BSS and MSS, apart from the exclusive immunostaining of solid/glandular areas of BSS for keratin 13 and CEA. Downgrading of keratin and extracellular matrix antigens immunoreactivity was observed when primary tumours were compared to recurrent and/or metastatic tumours of both subtypes (MSS and BSS). We conclude that SS should be regarded as carcinomas of soft tissues with an immunohistochemical phenotype depending on the degree of epithelial differentiation: spindle cells (MSS and BSS) predominantly expressing simple keratins, and poorly differentiated (solid/glandular) as well as well-differentiated (glandular) areas (BSS) expressing, in addition, complex epithelial-type keratins.

Synovial sarcoma: immunohistochemical expression of P-glycoprotein and glutathione S transferase-pi and clinical drug resistance.

Our purpose was to study the role of the expression of P-glycoprotein (Pgp) and glutathione S transferase-pi (GST-pi) in predicting the response to chemotherapy, relapse-free interval, and survival of patients with synovial sarcoma (SS). Thirty-seven cases of primary SS, without regional lymph node or distant metastases, were studied. There were 17 females and 20 males, ranging in age from 7 to 81 years (median, 31 years) with tumors located in the lower extremity (n = 24) upper extremity (n = 5) and trunchus (n = 8). The cases were retrospectively studied without knowledge of clinical course to compare the immunohistochemical expression of Pgp and GST-pi, flow cytometry parameters (ploidy and % of cells in S+G2 phases), and PCNA and Ki-67 labeling of primary tumors before any therapy, with that observed in local recurrences and metastases after chemotherapy. The relationship of the aforementioned parameters with clinicopathological features (gender, age, and histo-blood group of the patients, size, location, histological subtype. TNM stage, and clinical response to chemotherapy of the tumors) was also evaluated. Results revealed that Pgp and GST-pi were expressed in 29.7% and 40.5% of the cases, respectively. In 48.6% of the tumors there was expression of a least one of the drug resistance markers. The markers were coexpressed in 25.0% of the tumors. The prevalence of Pgp expression was lower, but not significantly, in stage I-II (17.6%) than in stage III (40.0%) tumors, and also in cases without clinical progression (16.7%), than in cases with (36.0%). No such differences were observed for GST-pi expression. Pgp and GST-pi expressions were significantly associated with biphasic SS and were particularly noticeable in solid/glandular areas of biphasic SS. The expression of the drug resistance markers was not significantly associated with gender, age, and histo-blood group of the patients, dimension, location, and proliferative activity of the tumors; it was also not significantly related to relapse-free interval and survival of the patients. The expression of Pgp and GST-pi was not significantly associated either to response to chemotherapy or influenced by chemotherapy. We conclude that Pgp and GST-pi expressions are not good predictors response to of the chemotherapy in patients with localized SS. Other drug resistance mechanisms may be active in SS.

Primary spinal chondrosarcoma: radiologic findings with pathologic correlation.

PURPOSE: To describe the radiologic appearance of the four types of primary spinal chondrosarcoma (CHS) (conventional intramedullary, juxtacortical, clear cell, and mesenchymal) and to correlate with histopathologic findings. MATERIAL AND METHODS: A retrospective review was carried out of 5 patients with histopathologically confirmed primary spinal CHS; 3 F and 2 M ranging in age between 27 and 66 years (mean 40.2; median 39). Charts, conventional radiographs, computed tomography scans, and magnetic resonance images were reviewed. All the patients underwent surgical excision, followed by postoperative chemotherapy (1 patient) and radiotherapy (3 patients). Follow-up was available for all patients but one. The mean follow-up was 42 months (14-120 months). Histopathological specimens for all patients were available for review. RESULTS: Vertebral column distribution was 3 thoracic (60%), 1 cervical (20%), and 1 lumbar (20%). Neurological deficits were present in 3 (60%) cases. The radiological appearance of the four types of primary spinal CHS varies with specific lesion type. Imaging findings suggest diagnosis of the conventional intramedullary and juxtacortical types. While the clear cell and mesenchymal types show some distinctive features, these do not allow confident radiologic diagnosis. CONCLUSION: The radiologist must be aware of imaging features of these tumors in order to improve diagnostic accuracy, treatment planning, and prognosis.

[Puncture cytology]. / Punksjonscytologi.

Limited public medical resources make it important to consider any diagnostic test which is inexpensive and reliable and without major side effects. The presented data are based on retrieval from the medical databases Medline and Cancerline, as well as the authors' clinical experience. The role of fine needle aspiration cytology for the diagnosis of solid and non-solid tumours is well established in the literature. The technique is simple and cost-effective and without major side effects. The sensitivity and specificity concurs that of histology in different tumours and may reduce the need for histopathology. In this review we discuss the combined use of fine needle aspiration cytology and modern immunocytochemistry and molecular techniques which may enhance diagnostic precision. We present the role of ancillary techniques which may prevent unnecessary extensive examinations and influence the therapeutic management of the patient.

Carcinoma of the renal pelvis. Experience of 80 cases.

The clinical course and association with other urothelial carcinomas was studied in 80 patients with carcinoma of the renal pelvis. At the time of diagnosis macroscopic haematuria was the most common symptom. Urography and retrograde pyelography gave the most accurate diagnoses. Sixty-eight patients (85%) had transitional cell carcinomas, 39 had a history of another urothelial carcinoma either before or after the diagnosis of the renal tumour, usually in the urinary bladder. A previous urothelial carcinoma together with a small renal tumour were predictive of the subsequent development of another urothelial tumour. Regular cystoscopy is recommended during follow-up because of the short observed interval between the development of carcinoma of the renal pelvis and a subsequent urothelial malignancy. The overall five-year survival was 20%. Survival did not depend on the operation, but was significantly related to the extent of locoregional or distant metastases at the time of diagnosis. Patients with such renal tumours that were preceded by or associated with other urothelial carcinomas had a more favourable median survival (42 months) than patients whose renal tumour was their first urothelial carcinoma (19 months).

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH.

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.