Multifocal myxoid liposarcoma, metastasis or second primary tumor Multifocal myxoid liposarcoma, metastasis or second primary tumor? A molecular biological analysis. Ronald de Vreeze, research physician Daphne de Jong Petra Nederlof Henrique Ruijter Rick Haas Frits van Coevorden

Introduction Introduction Multifocality 1% soft tissue sarcomas Liposarcoma 20% of all malignancies in adults The multifocal (lipo)sarcoma = Myxoid round cell liposarcoma 1% of all sarcomas is assumed to be multifocal. There are 50 histologic subtypes, liposarcoma has the highest incidence 50% of liposarcomas are myxoid liposarcomas The liposarcoma which is known for its multifocality is myxoid liposarcoma Introduction

Definition Introduction Multifocal (myxoid lipo)sarcoma: The presence of sarcoma at two separate sites, before the manifestation of disease in sites where sarcomas most commonly metastasizes, in particular the lungs Multifocal myxoid liposarcoma is defined as the presence of sarcoma at two separate sites, before the manifestation of disease in sites where sarcomas most commonly metastasizes, such as the lungs Introduction

Purpose Molecular biological differentiation between metastasis and 2nd primary tumors In case of 2nd primary: Optimal local treatment: Radiotherapy Surgery In case of metastasis: Systemic treatment : Chemotherapy Marginal resection Differentiation between second primary and monoclonal MRLS has major clinical consequences. Treatment options for patients with second primary MRLS are different from those for metastasized MRLS. A resectable second primary could imply optimal surgical approach combined with radiotherapy, whereas metastatic disease in general justifies palliative systemic therapy or limited surgical treatment. Introduction

Methods Methods N=15 Clinical data afterwards inconclusive Histopatologic classification not distinctive Translocation analysis type translocation: difference or similarity in subsequent lesion can give information: clonal or 2nd primary Loss of heterozygosity analysis difference in pattern can give information: clonal or 2nd primary From a series of 331 liposarcoma patients who were treated in the Netherlands Cancer Institue, the fifteen multifocal patients were retrieved Histologic classifications were reviewed Translocation analysis and LOH analysis was performed from representative sections of the formalin fixed paraffin embedded specimens To distuinguish between second primaty and metastasis, Clinical data were informative but not conclusive. Histologic classification was was used for inclusion but could not discriminate between 2nd primary and metastasis. Translocation and LOH analysis however can. I will expain this issue in the next slides. Methods

Translocation frequency 7-2 (type I) 20% 8-2 (type III) 10% Other types 10% There are several known fusion transcripts correlating to multiple different breakpoints, of which I have shown the FUS-CHOP fusion transcripts. These fusion transcripts have very different incidences in MRLS. Therefore, this detailed molecular analysis of breakpoints can be used as an additional marker in selected cases. If a similar rare fusion transcript is found in subsequent lesions, the patient was considered clonally related. Due to the incidence of the 5-2 fusion transcript. Repeated occurence of this fusion transcripts was not considered informative. Methods

Translocation frequency 7-2 (type I) 20% 5-2 (type II) 60% 8-2 (type III) 10% Other types 10% There are several known fusion transcripts correlating to multiple different breakpoints, of which I have shown the FUS-CHOP fusion transcripts. These fusion transcripts have very different incidences in MRLS. Therefore, this detailed molecular analysis of breakpoints can be used as an additional marker in selected cases. If a similar rare fusion transcript is found in subsequent lesions, the patient was considered clonally related. Due to the incidence of the 5-2 fusion transcript. Repeated occurence of this fusion transcripts was not considered informative. Methods

Loss of heterozygosity Clinics already introduced at Head / neck & lung tumors Multifocal breast cancer Furthermore LOH is a technique which is used in our clinic in head and neck tumors and pulmonary metastasis, and is used in multi focal breast cancer. Methods

Loss of heterozygosity (LOH) 12 markers spread over 11 chromosomes Patterns of loss of heterozygosity LOH ratio < 0,5 It is an easy to perform, analysis on paraffin embedded tissue and, thus, potentially very useful in everyday clinical practice, as well as for the analysis of retrospective series. For MRLS we analysed 12 markers distributed over 11 chromosome arms. The peak intensity of the PCR products was measured and used to calculate the intensity ratio between the two allele peaks. Ratios of more than 0.50 were scored as LOH Methods

Strategy of analysis of clonality 1)Translocation 2)Loss of heterozygosity For the strategy of analysis of clonality translocation was considered before LOH analysis. Methods

Strategy of analysis of clonality Translocation Concordant rare fusion transcript (i.e.7-2 8-2, 5-3) Different fusion transcripts at different sites Fusion transcript 5-2 LOH analysis Concordant LOH/AI (LOHx – LOHx) Discordant LOH/AI (LOHx – LOHy) Discordant LOH As can be seen in the flowchart: The presence of concordant fusion products with the exception of the most frequent 5-2 fusion transcript was considered as proof of clonality. In case of a 5-2 fusion transcript loss of heterozygosity was used as clonal marker. Concordant losses (losses of similar alleles) were scored. Discordant losses were scored. If there was a covincing amount of concordant losses, patients were consdered clonal Discordant losses were similarly scored. And if there was a convincing amount of these, tumor were considered 2nd primary. And as can be seen in the top right, different fusion products at different sites in a single patient was considered as a strong argument for absence of clonal relation ≥ 2 LOH or ≥ 3 AI ≥ 1 LOH + ≥ 1 AI ≤ 1 LOH or ≤ 2 AI ≤1 LOH or ≤2 AI ≥1 LOH + ≥ 1 AI ≥2 LOH or ≥ 3 AI Second primary Metastasis Metastasis? Second Primary? Metastasis Second primary Methods

Results Results N=15 Primary localization Localization 2nd lesion Lower extremity 14 Upper extremity 1 Localization 2nd lesion Extremity 4 Abdominal wall 4 Retroperitoneal 3 Head/Neck 1 Other 3 Median time to 2nd lesion 25 (0-82) 15 patients were analysed All patients had a primary localisation in the extremities And median time to second lesion was 25 months Results

Patients Results Time (mnts) Translocation Time (mnts) Translocation 1a  Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 Here we can see all 15 patients Results

1 15 Patients Results Time (mnts) Translocation Time (mnts) 1a  Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 1 Here we can see all 15 patients 15 Results

Patients Results Time (mnts) Translocation Time (mnts) Translocation 1a  Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 The time interval, Results

Patients Results Time (mnts) Translocation Time (mnts) Translocation 1a  Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 And the type of translocations, which are all similar in subsequent lesions per patient Results

Translocation Results Time (mnts) Translocation 1a Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 The first 6 patients in red all had subsequent rare fusion transcripts, defining for clonality Results

Loss of Heterozygosity Time (mnts) Translocation 1a  Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 The green patients had a convincing amount of concordant LOH patterns, also defining for clonality. Which confirmed 2 patients that were alraedy considered clonal due to translocation analysis Results

Loss of Heterozygosity Time (mnts) Translocation 1a  Thigh 1b Neck 1c Chest wall 38 44 5-3 2a  Thigh 2b Th Spine 2c Lung 2d Abd wall 21 30 7-2 3a  Upper arm 3b Back 3c Forearm 20 64 8-2 4a  Thigh 4b Chest wall 4c Retroperitoneum 18 5a  Thigh 5b Retroperitoneal 5c Neck 5d Buttock 5e Hip 14 19 32 6a  Calf 6b Subclavia 6c Axilla 6d Lung 2  7a  Thigh 7b Retroperitoneal 7c Lung 7d Th Spine 2 5-2 Time (mnts) Translocation 8a  Thigh 8b Upper arm R 8c Shoulder L 8d Buttock 74 75 82 5-2 9a  Thigh 9b Abd. wall 9c Omentum 9d Omentum 9e Hip 12 22 36 10a  Thigh 10b Retroperitoneal 28 11a  Calf 11b Hip 12a  Thigh 12b Buttock 14 13a  Thigh 13b Upper arm 13c Retroperitoneal     65 71 14a  Ankle 14b Chest wall 58 15a  Thigh 15b Chest wall 15c Back 15d Retroperitoneal 20 27 43 The green patients had a convincing amount of concordant LOH patterns, also defining for clonality. Which confirmed 2 patients that were alraedy considered clonal due to translocation analysis Results

Suggestive for clonal relation Results Patient Translocation LOH Decision 1 Clonal Unconvincing 2 3 4 Suggestive for clonal relation 5 6 7 8 9 10 11 12 13 14 15 So, although a high suspicion of clonal relation was found in all patients, a definitive conclusion considering translocation alone, was established in six patients and 4 patients were considered clonal by means of LOH. Results

Results Results Clonal 8/15 Suggestive for clonal relation 7/15 Not in a single case development of independent second primary tumors was found In total there were 8 patients considered clonal. And 7 were suggestive for clonal relation. In no single case development of independent second primary tumors was found Results

Conclusions Conclusions It is highly suggestive that: multifocal myxoid/ round cell liposarcoma are metastasized tumors Primary curative surgical approach is probably not rational Treatment strategy designed for metastasized disease is more appropriate Therfore It is highly suggestive that: multifocal myxoid/ round cell liposarcoma are metastasized tumors Basis for primary curative surgical approach is probably not rational Treatment strategy designed for metastasized disease is more appropriate Thank you for your attention Conclusions