Search

Primer sequences and PCR conditions.

Univariate HRs of 24 single genes overall and for the stage 2, MSS, BRAF-wild type subgroup and stage 3, MSS, BRAF-wild type subgroup within the study population.

Purpose: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations.Experimental Methods: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations.Results: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P = P < 0.01). Subgroup analysis for tumor stage, MSI, and BRAF status showed no statistically significant differences in survival between CL1, CL2, and CL3 nor between CIMP and non-CIMP CRCs. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, microsatellite stability (MSS), BRAF wild-type (WT) CRCs: multivariate Cox proportional HR = 3.89 [95% confidence interval (CI), 1.58–9.60, P < 0.01; n = 66] and HR = 2.11 (95% CI, 0.95–4.69, P = 0.068, n = 136) in a second independent population-based study.Conclusions:CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs. Clin Cancer Res; 20(12); 3261–71. ©2014 AACR.

CIMP and CL1, CL2 and CL3 in relation to clinicopathological characteristics of the study population.

Epi)genetic alterations in relation to clinicopathological characteristics of the study population.

Psammomatoid ossifying fibroma (PsOF), also known as juvenile PsOF, is a benign fibro-osseous neoplasm predominantly affecting the extragnathic bones, particularly the frontal and ethmoid bones, with a preference for adolescents and young adults. The clinical and morphologic features of PsOF may overlap with those of other fibro-osseous lesions, and additional molecular markers would help increase diagnostic accuracy. Because identical chromosomal breakpoints at bands Xq26 and 2q33 have been described in 3 cases of PsOF located in the orbita, we aimed to identify the exact genes involved in these chromosomal breakpoints and determine their frequency in PsOF using transcriptome sequencing and fluorescence in situ hybridization (FISH). We performed whole RNA transcriptome sequencing on frozen tissue in 2 PsOF index cases and identified a fusion transcript involving SATB2, located on chromosome 2q33.1, and AL513487.1, located on chromosome Xq26, in one of the cases. The fusion was validated using reverse transcription (RT)-PCR and SATB2 FISH. The fusion lead to a truncated protein product losing most of the functional domains. Subsequently, we analyzed an additional 24 juvenile PsOFs, 8 juvenile trabecular ossifying fibromas (JTOFs), and 11 cemento-ossifying fibromas (COFs) for SATB2 using FISH and found evidence of SATB2 gene rearrangements in 58% (7 of 12) of the evaluable PsOF cases but not in any of the evaluable JTOF (n = 7) and COF (n = 7) cases. A combination of SATB2 immunofluorescence and a 2-color SATB2 FISH in our index case revealed that most tumor cells harboring the rearrangement lacked SATB2 expression. Using immunohistochemistry, 65% of PsOF, 100% of JTOF, and 100% of COF cases showed moderate or strong staining for SATB2. In these cases, we observed a mosaic pattern of expression with >25% of the spindle cells in between the bone matrix, with osteoblasts and osteocytes being positive for SATB2. Interestingly, 35% (8 of 23) of PsOFs, in contrast to JTOFs and COFs, showed SATB2 expression in

Background. With soft-tissue sarcoma of the extremity (ESTS) representing a heterogenous group of tumors, management decisions are often made in multidisciplinary team (MDT) meetings. To optimize outcome, nomograms are more commonly used to guide individualized treatment decision making. Purpose. To evaluate the influence of Personalised Sarcoma Care (PERSARC) on treatment decisions for patients with high-grade ESTS and the ability of the MDT to accurately predict overall survival (OS) and local recurrence (LR) rates. Methods. Two consecutive meetings were organised. During the first meeting, 36 cases were presented to the MDT. OS and LR rates without the use of PERSARC were estimated by consensus and preferred treatment was recorded for each case. During the second meeting, OS/LR rates calculated with PERSARC were presented to the MDT. Differences between estimated OS/LR rates and PERSARC OS/LR rates were calculated. Variations in preferred treatment protocols were noted. Results. The MDT underestimated OS when compared to PERSARC in 48.4% of cases. LR rates were overestimated in 41.9% of cases. With the use of PERSARC, the proposed treatment changed for 24 cases. Conclusion. PERSARC aids the MDT to optimize individualized predicted OS and LR rates, hereby guiding patient-centered care and shared decision making.

Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma subtype. This retrospective study elucidates the currently unknown genetic background of a large clinically well-annotated cohort of DLBCLwith osseous localizations (O-DLBCL), including PB-DLBCL. A total of 103 patients with O-DLBCL were included and compared with 63 (extra)nodal non-osseous (NO)-DLBCLs with germinal center B-cell phenotype (NO-DLBCL-GCB). Cell-of-origin was determined by immunohistochemistry and gene-expression profiling (GEP) using (extended)-Nano-String/Lymph2Cx analysis. Mutational profileswere identifiedwith targeted next-generation deep sequencing, including 52 B-cell lymphoma-relevant genes. O-DLBCLs, including 34 PB-DLBCLs, were predominantly classified as GCB phenotype based on immunohistochemistry (74%) and NanoString analysis (88%). Unsupervised hierarchical clustering of an extended-NanoString/Lymph2Cx revealed significantly different GEP clusters for PB-DLBCL as opposed to NO-DLBCL-GCB (P < .001). Expression levels of 23 genes of 2 different targeted GEP panels indicated a centrocyte-like phenotype for PB-DLBCL, whereas NO-DLBCL-GCB exhibited a centroblast-like constitution. PB-DLBCL had significantly more frequent mutations in four GCB-associated genes (ie, B2M, EZH2, IRF8, TNFRSF14) comparedwithNO-DLBCL-GCB (P = .031, P = .010, P = .047, and P = .003, respectively). PB-DLBCL, with its corresponding specific mutational profile, was significantly associated with a superior survival compared with equivalent Ann Arbor limited-stage I/II NO-DLBCL-GCB (P = .016). This study is the first to show that PB-DLBCL is characterized by a GCB phenotype, with a centrocyte-like GEP pattern and a GCB-associated mutational profile (both involved in immune surveillance) and a favorable prognosis. These novel biology-associated features provide evidence that PB-DLBCL represents a distinct extranodal DLBCL entity, and its specific mutational landscape offers potential for targeted therapies (eg, EZH2 inhibitors).

Cementoblastomas are rare odontogenic tumors developing in close proximity to the roots of teeth. Due to their striking morphologic resemblance to osteoblastomas of the peripheral skeleton, we set out to determine whether cementoblastomas harbor the same FOS rearrangements with overexpression of c-FOS as has recently been described for osteoblastomas. In total, 16 cementoblastomas were analyzed for FOS expression by immunohistochemistry and for FOS rearrangements by fluorescence in situ hybridization (FISH). We observed strong and diffuse staining of c-FOS in 71% of cementoblastomas and identified a FOS rearrangement in all cases (n=3) applicable for FISH. In the remaining cases, FISH failed due to decalcification. Cementoblastomas harbor similar FOS rearrangements and show overexpression of c-FOS like osteoblastomas, suggesting that both entities might represent parts of the spectrum of the same disease.

Chondrogenic or cartilaginous tumours represent the most common bone tumours and can be diagnostically challenging especially on scarce biopsy material. As morphologically they may overlap, correlation between radiological and pathological findings and discussion in a multidisciplinary tumour board is mandatory for a final diagnosis. The aim of this review is to discuss clinical and pathological features that are important in the differential diagnosis of benign (chondromesenchymal hamartoma of chest wall, osteochondroma, enchondroma, periosteal chondroma) and intermediate (synovial chondromatosis, central or secondary peripheral atypical cartilaginous tumour) or malignant cartilaginous tumours (central or secondary peripheral chondrosarcoma grades 1–3, periosteal chondrosarcoma, dedifferentiated chondrosarcoma, clear cell chondrosarcoma and mesenchymal chondrosarcoma). The 5th edition WHO classification proposed a new concept for cartilage tumours in which the term atypical cartilaginous tumour is used for low grade tumours located in the long and short tubular bones of the appendicular skeleton, as they behave as locally aggressive lesions and therefore should not be considered full blown malignant or treated aggressively. Histologically similar tumours in the axial skeleton (including pelvis, scapula and skull base) are now diagnosed as chondrosarcoma grade 1. Immunohistochemistry is of limited value in the distinction between the different cartilaginous tumours. Molecular diagnostics can be helpful when used on small biopsies to distinguish mesenchymal chondrosarcoma (with HEY1-NCOA2 fusion) from other undifferentiated round cell sarcomas, or dedifferentiated chondrosarcoma (often with IDH mutation) from other high grade sarcomas.

Contains fulltext : 288341.pdf (Publisher’s version ) (Open Access) BACKGROUND: In all giant-cell-rich lesions (GCRL) occurring in bone, a common underlying excessive RANKL expression is held responsible for the osteolytic activity. Apart from giant cell tumour of bone (GCTB), systematic outcome analysis of RANKL inhibition in other GCRL is unavailable. The aim of this study is to assess the efficacy and safety of a 1-year denosumab protocol in giant cell lesions of the jaw (GCLJ). METHODS: A retrospective cohort study was conducted compromising patients treated with a 1-year protocol of monthly subcutaneously administered 120 mg denosumab. Objective tumour response based on histology and imaging was used to calculate objective tumour response rate, progression-free survival (PFS) and time to progression. Type, severity and frequency of adverse events were recorded in a standardised way to assess safety. RESULTS: Twenty patients, predominantly female (90%), were included. Fifty-five per cent of lesions were located in the mandible; most classified as aggressive lesions (90%). Thirty-five per cent (7/20) of cases were either recurrent after prior treatment or progressive, while on other drug treatment. Objective tumour response rate was 100% after 12 months of treatment. Median PFS was 50.4 months (95% CI 38.0-62.8) with a cumulative PFS rate of 22.6% (95% CI 1.8-43.4) at 5 years follow-up. Median time to progression was 38.4 months (95% CI 26.0-50.8). Treatment was well tolerated, and none of the patients had to interrupt therapy for toxicity. CONCLUSION: High-dose denosumab is effective and safe in achieving a complete response in GCLJ within 12 months. The high long-term relapse rate after treatment cessation is the main obstacle for denosumab to become standard treatment for GCLJ.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens., Preservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of translocations in preserved samples.

A simple bone cyst (SBC) is a cystic bone lesion predominantly affecting young males. The cyst is lined by a fibrous membrane and filled with serosanguinous fluid. EWSR1/FUS-NFATC2 rearrangements were recently identified in SBC. We here report exactly the same rearrangement in 3 lesions diagnosed as vascular malformations of 2 elderly patients. In total, through Archer FusionPlex, fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction the EWSR1-NFATC2 rearrangement was identified in 6 of 9 SBC, 3 of 12 benign vascular tumors, and none of 5 aneurysmal bone cyst lacking USP6 fusion. Using fluorescence in situ hybridization, it was apparent that amplification of the fusion, as seen in EWSR1-NFATC2 round cell sarcomas, was absent, and that in the vascular tumors the fusion was present both in the lining cells as well as in the surrounding spindle cells. Of note, not all of the spaces in the vascular malformations were lined by endothelial cells. Aggrecan was positive in all cases but was not specific. NKX2-2 and NKX3-1 staining were negative in all cases. Thus, even though the overlap between the 2 entities is limited to the presence of few thick-walled cysts lacking endothelial lining in the benign vascular malformations, the spectrum of benign tumors containing NFATC2 fusions should be expanded and contains not only SBC in the young, but also vascular malformation/hemangioma in elderly patients.

According to the WHO, mesenchymal tumours of the maxillofacial bones are subdivided in benign and malignant maxillofacial bone and cartilage tumours, fibro-osseous and osteochondromatous lesions as well as giant cell lesions and bone cysts. The histology always needs to be evaluated considering also the clinical and radiological context which remains an important cornerstone in the classification of these lesions. Nevertheless, the diagnosis of maxillofacial bone tumours is often challenging for radiologists as well as pathologists, while an accurate diagnosis is essential for adequate clinical decision-making. The integration of new molecular markers in a multidisciplinary diagnostic approach may not only increase the diagnostic accuracy but potentially also identify new druggable targets for precision medicine. The current review provides an overview of the clinicopathological and molecular findings in maxillofacial bone tumours and discusses the diagnostic value of these genetic aberrations.

Aims Clear cell chondrosarcomas are known to occasionally contain areas of low-grade conventional chondrosarcoma; however, the opposite phenomenon has not yet been described. We identified five cases of conventional chondrosarcoma alongside clear cell chondrosarcoma. Here, we report on their clinicopathological and molecular characteristics, and investigate whether these hybrid lesions should be considered to be a collision tumour, conventional chondrosarcoma with clear cell change, or clear cell chondrosarcoma with extensive areas of conventional chondrosarcoma, as this has clinical implications. Methods and results Clinicohistopathological features were characterised, immunohistochemistry was performed for H3 histone family member 3B (H3F3B), histone H3 trimethylated on lysine 27 (H3K27me3), and p53, and genetic alterations of IDH1 (encoding isocitrate dehydrogenase 1), IDH2 (encoding isocitrate dehydrogenase 2), TP53 and H3F3B were evaluated. All five chondrosarcomas consisted predominantly of areas with conventional chondrosarcoma. Different grades were found [grade I (n = 1), grade II (n = 2), and grade III (n = 2)]. Up to 20% of the tumour consisted of classic features of clear cell chondrosarcoma. Gradual merging between both components was observed. Molecular analysis of conventional chondrosarcoma components revealed an IDH1 c.395G>T, p.(Arg132Leu) mutation in two cases, and an IDH1 c.394C>T, p.(Arg132Cys) mutation in one case, with identical IDH mutations in the clear cell chondrosarcoma counterpart (100%). Two cases were IDH wild-type. In all cases, none of the components harboured H3F3B mutations. High-grade tumours had an aggressive course, as three patients died of the disease. Conclusion On the basis of clinicopathological characterisation and genetic alterations, it is suggested that these lesions should be considered to be conventional chondrosarcoma, with clear cell change. Pathologists should be aware of their existence to avoid confusion with clear cell chondrosarcoma, dedifferentiated chondrosarcoma, or chondroblastic osteosarcoma.

Editor's Note.-RadioGraphics continues to publish radiologic-pathologic case material selected from the "best case" presentations of the American Institute for Radiologic Pathology (AIRP), a program of the American College of Radiology. The AIRP conducts a 4-week Radiologic Pathology Correlation Course, which is offered five times per year. On the penultimate day of the course, the best case presentation is held at the American Film Institute Silver Theater and Cultural Center in Silver Spring, Md. The AIRP faculty identifies the best cases, from each organ system, brought by the resident attendees. One or more of the best cases from each of the five courses are then solicited for publication in RadioGraphics. These cases emphasize the importance of radiologic-pathologic correlation in the imaging evaluation and diagnosis of diseases encountered at the institute and its predecessor, the Armed Forces Institute of Pathology (AFIP).

Aims Because of the efficacy of tropomyosin receptor kinase (Trk) inhibitor therapy in tumours with rearrangements of the neurotrophic tyrosine kinase receptor genes (NRTK genes), there has been a surge in demand for NTRK fusion screening. To date, most studies involving mesenchymal tumours have focused on soft tissue tumours, and data on bone tumours are sparse. Hence, we aimed to explore the frequency of NTRK fusions in a large series of primary bone tumours. Methods and results Immunohistochemical expression of pan-Trk was successfully assessed in 354 primary bone tumours by the use of tissue microarrays. In a selection of positive cases, additional molecular analysis for NTRK fusions was performed with anchored multiplex polymerase chain reaction-based targeted next-generation sequencing. Positivity was found in 19 cases (5%), which comprised Ewing sarcoma (n = 6, 33%), osteosarcoma (n = 11, 13%), and giant-cell tumour of bone (n = 2, 3%). In all except one case, cytoplasmic staining was observed. Weak staining was most often observed (n = 13), although five cases showed moderate staining and one case showed focal strong staining. Molecular analysis was successful in six cases, all of which were negative for NTRK fusions. Conclusion The likelihood of finding an NTRK fusion in bone tumours in clinical practice is extremely low. This may imply that, if more comprehensive large-scale molecular studies confirm this, routine predictive NTRK testing in bone tumour patients with advanced disease may be reconsidered.

In the cranio-facial skeleton, a heterogeneous group of well characterized fibro-osseous lesions can be distinguished. Whereas fibrous dysplasia can affect any skeletal bone, ossifying fibroma and cemento-osseous dysplasia exclusively develop in the cranio-facial region, with most subtypes restricted to the tooth bearing areas of the jaws. Herein we present a series of 20 fibro-osseous lesions that developed mostly in the frontal bone and in the mandible, presenting as expansile intramedullary tumors with a unique histologic appearance and an indolent clinical course. We provide evidence that these tumors are distinct from the categories included in the WHO classification and are therefore currently unclassifiable. The definition of cemento-ossifying fibroma as an odontogenic neoplasm developing only in close proximity to teeth should be re-considered and incorporate also extragnathic lesions as shown here.

Malignant transformation of fibrous dysplasia lesions has been reported in patients with fibrous dysplasia/McCune-Albright syndrome (FD/MAS). Recently, we have observed an increased risk for breast cancer. In this study, the prevalence of skeletal and extraskeletal malignancies in patients with FD/MAS in the Netherlands was assessed by analyzing data from our cohort of FD/MAS patients, the Dutch Pathology Registry (PALGA), and the Netherlands Cancer Registry (NCR). We extracted data on sex, age at diagnosis of FD/MAS, type of FD/MAS, type of malignancy, and age at diagnosis of malignancy and histology of bone and malignant tissue when available, including GNAS-mutation analysis from patients’ medical records. Standardized Morbidity Ratios (SMRs) with 95% confidence intervals were calculated. Twelve malignancies were identified in the LUMC FD/MAS cohort and 100 in the PALGA cohort. In this cohort, SMR was increased for osteosarcoma (19.7, 95% CI 3.5–48.9), cervical cancer (4.93, 95%CI 1.7–8.2), thyroid cancer (3.71, 95% CI 1.1–7.8), prostate cancer (3.08, 95% CI 1.8–4.6), and melanoma (2.01, 95%CI 1.2–3.1). SMRs for pancreatic cancer or hepatocellular carcinoma could not be calculated due to low numbers. The small number of malignancies identified in our FD/MAS cohort precluded the calculation of SMRs for our cohort specifically. Our findings show that patients with FD/MAS appear to have an increased risk for osteosarcoma, cervical, thyroid, and prostate cancer and melanoma. However, these data should be interpreted with caution, as true incidence rates of the identified malignancies may be influenced by the inclusion of only patients with histologically confirmed FD/MAS. The etiology of this increased risk for malignancies still needs to be elucidated.

Unlike systemic anaplastic large cell lymphoma, the vast majority of primary cutaneous anaplastic large cell lymphomas (C-ALCL) do not carry translocations involving the ALK gene and do not express ALK. Expression of ALK protein therefore strongly suggests secondary cutaneous involvement of a systemic anaplastic large cell lymphoma. Recent studies described a small subgroup of ALK-positive C-ALCL, but information on frequency, prognosis, and translocation partners is virtually lacking. A total of 6/309 (2%) C-ALCL patients included in the Dutch registry for cutaneous lymphomas between 1993 and 2019 showed immunohistochemical ALK expression. Clinical and histopathologic characteristics, immunophenotype and disease course were evaluated. Underlying ALK translocations were analyzed with anchored multiplex polymerase chain reaction-based targeted next-generation sequencing. Median age at diagnosis was 39 years (range: 16 to 53 y). All patients presented with a solitary lesion. Treatment with radiotherapy (n=5) or anthracycline-based chemotherapy (n=1) resulted in complete responses in all 6 patients. Three patients developed a relapse, of whom 2 extracutaneous. After a median follow-up of 41 months, 5 patients were alive without disease and 1 patient died of lymphoma. Immunohistochemically, 3 cases (50%) showed combined nuclear and cytoplasmic ALK expression with underlying NPM1-ALK fusions, while 3 cases (50%) showed solely cytoplasmic ALK expression with variant ALK fusion partners (TRAF1, ATIC, TPM3). ALK-positive C-ALCL is extremely uncommon, has a comparable favorable prognosis to ALK-negative C-ALCL, and should be treated in the same way with radiotherapy as first-line treatment.

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) is an indolent cutaneous CD30-positive T-cell lymphoma with no signs of extracutaneous localizations at the time of diagnosis. C-ALCL is clinically characterized by single or localized, sometimes ulcerating tumors and histopathologically by diffuse sheets of large anaplastic cells.1 Spitzoid neoplasms are melanocytic tumors usually occurring in young individuals. They are characterized clinically by pigmented or spherical reddish lesions and histopathologically by spindle-shaped and epithelioid melanocytes.2 In a small proportion of patients, both diseases can be driven by or harbor rearrangements involving the anaplastic lymphoma kinase (ALK) gene.2,3

TO THE EDITOR: Intravascular large B-cell lymphoma (IVLBCL) is a rare variant of extranodal diffuse large B-cell lymphoma (DLBCL). It is characterized by proliferation of blastic, neoplastic B cells within the lumina of small- or intermediate-sized blood vessels and capillaries.[1][1] IVLBCL may

Hematologic neoplasms that primarily present in bone are rare; this article describes the most common examples of hematologic tumors primarily presenting in bone, including plasma cell myeloma, solitary plasmacytoma of bone, primary non-Hodgkin lymphoma of bone, acute lymphoblastic leukemia/lymphoma, and Langerhans cell histiocytosis. The macroscopic and microscopic features, differential diagnosis, diagnostic workup, and prognosis of all these different entities are discussed, with special emphasis on common differential diagnosis.

Osteoid osteoma and osteoblastoma are bone-forming tumors shown to harbor FOS (87%) and FOSB (3%) rearrangements. The aim was to evaluate the immunohistochemical expression of FOS and FOSB in these tumors in comparison to other bone tumors, to evaluate the influence of decalcification, and to correlate immunohistochemical findings with the underlying genetic alteration using fluorescence in situ hybridization (FISH). Immunohistochemistry using whole sections was performed on osteoid osteoma (n=23), osteoblastoma (n=22), osteoblastoma-like osteosarcoma (n=3), reactive (n=3), and proliferative (n=11) bone lesions. Immunoreactivity in giant cell tumor of bone (n=74), aneurysmal bone cyst (n=6), chondromyxoid fibroma (n=20), osteosarcoma (n=85), chondroblastoma (n=17), and clear cell chondrosarcoma (n=20) was assessed using tissue micro arrays. Strong nuclear expression of FOS in > 50% of the tumor cells was observed in all osteoid osteomas (22/22), in 57% of osteoblastomas (12/21) and in 3/197 control cases. FOS immunoreactivity disappeared after > 3 days decalcification. FOS rearrangements were present in 94% of osteoid osteomas and osteoblastomas, with a concordance of 86% between FISH and immunohistochemistry. Two osteoblastomas (5%) were positive for FOSB, as opposed to 8/177 control cases. Additional FISH revealed no FOSB rearrangements in these cases. To conclude, in short decalcified biopsies, FOS immunohistochemistry can be used to diagnose osteoid osteoma and osteoblastoma, as overexpression is seen in the majority, being rare in their mimics. FOS immunohistochemistry should not be used after long decalcification. Moreover, low level of focal expression found in other lesions and tissues might cause diagnostic problems, in which case FISH could be employed.

International audience; Rhabdomyosarcomas with TFCP2 fusions represent an emerging subtype of tumors, initially discovered by RNA-sequencing. We report herein the clinicopathological, transcriptional, and genomic features of a series of 14 cases. Cases were retrospectively and prospectively recruited and studied by immunohistochemistry (MYF4, MYOD1, S100, AE1/E3, ALK), fluorescence in situ hybridization with TFCP2 break-apart probe (n = 10/14), array-comparative genomic hybridization (Agilent), whole RNA-sequencing (Truseq Exome, Illumina), or anchored multiplex PCR-based targeted next-generation sequencing (Archer® FusionPlex® Sarcoma kit). Patient's age ranged between 11 and 86 years, including 5 pediatric cases. Tumors were located in the bone (n = 12/14) and soft tissue (n = 2/14). Most bone tumors invaded surrounding soft tissue. Craniofacial bones were over-represented (n = 8/12). Median survival was 8 months and five patients are currently alive with a median follow-up of 20 months. Most tumors displayed a mixed spindle cell and epithelioid pattern with frequent vesicular nuclei. All tumors expressed keratins and showed a rhabdomyogenic phenotype (defined as expression of MYF4 and/or MYOD1). ALK was overexpressed in all but three cases without underlying ALK fusion on break-apart FISH (n = 5) nor next-generation sequencing (n = 14). ALK upregulation was frequently associated with an internal deletion at genomic level. TFCP2 was fused in 5' either to EWSR1 (n = 6) or FUS (n = 8). EWSR1 was involved in both soft tissue cases. FISH with TFCP2 break-apart probe was positive in all tested cases (n = 8), including one case with unbalanced signal. On array-CGH, all tested tumors displayed complex genetic profiles with genomic indexes ranging from 13 to 107.55 and recurrent CDKN2A deletions. FET-TFCP2 rhabdomyosarcomas clustered together and distinctly from other rhabdomyosarcomas subgroups. Altogether, our data confirm and expand the spectrum of the new family of FET-TFCP2 rhabdomyosarcomas, which are associated with a predilection for the craniofacial bones, an aggressive course, and recurrent pathological features. Their association with ALK overexpression might represent a therapeutic vulnerability.

The 2016 World Health Organization classification defines diffuse large B-cell lymphoma (DLBCL) subtypes based on Epstein-Barr virus (EBV) infection and oncogenic rearrangements of MYC/BCL2/BCL6 as drivers of lymphomagenesis. A subset of DLBCL, however, is characterized by activating mutations in MYD88/CD79B. We investigated whether MYD88/CD79B mutations could improve the classification and prognostication of DLBCL. In 250 primary DLBCL, MYD88/CD79B mutations were identified by allele-specific polymerase chain reaction or next-generationsequencing, MYC/BCL2/BCL6 rearrangements were analyzed by fluorescence in situ hybridization, and EBV was studied by EBV-encoded RNA in situ hybridization. Associations of molecular features with clinicopathologic characteristics, outcome, and prognosis according to the International Prognostic Index (IPI) were investigated. MYD88 and CD79B mutations were identified in 29.6% and 12.3%, MYC, BCL2, and BCL6 rearrangements in 10.6%, 13.6%, and 20.3%, and EBV in 11.7% of DLBCL, respectively. Prominent mutual exclusivity between EBV positivity, rearrangements, and MYD88/CD79B mutations established the value of molecular markers for the recognition of biologically distinct DLBCL subtypes. MYD88-mutated DLBCL had a significantly inferior 5-year overall survival than wild-type MYD88 DLBCL (log-rank; P=0.019). DLBCL without any of the studied aberrations had superior overall survival compared to cases carrying .1 aberrancy (log-rank; P=0.010). MYD88 mutations retained their adverse prognostic impact upon adjustment for other genetic and clinical variables by multivariable analysis and improved the prognostic performance of the IPI. This study demonstrates the clinical utility of defining MYD88-mutated DLBCL as a distinct molecular subtype with adverse prognosis. Our data call for sequence analysis of MYD88 in routine diagnostics of DLBCL to optimize classification and prognostication, and to guide the development of improved treatment strategies.

BACKGROUND Anatomic and surgical complexity make pelvic and sacral bone sarcoma resections challenging. Positive surgical margins are more likely to occur in patients with pelvic and sacral bone sarcomas than in those with extremity sarcomas and are associated with an increased likelihood of local recurrence. Intraoperative navigation techniques have been proposed to improve surgical accuracy in achieving negative margins, but available evidence is limited to experimental (laboratory) studies and small patient series. Only one small historically controlled study is available. Because we have experience with both approaches, we wanted to assess whether navigation improves our ability to achieve negative resection margins. QUESTIONS/PURPOSES Are navigated resections for pelvic and sacral primary bone sarcomas better able to achieve adequate surgical margins than nonnavigated resections? METHODS Thirty-six patients with pelvic or sacral sarcomas treated with intraoperative navigation were retrospectively compared with 34 patients undergoing resections without navigation. All patients underwent resections between 2000 and 2017 with the intention to achieve a wide margin. Patients in the navigation group underwent surgery between 2008 and 2017; during this period, all resections of pelvic and sacral primary bone sarcomas with the intention to achieve a wide margin were navigation-assisted by either CT fluoroscopy or intraoperative CT. Patients in the control group underwent surgery before 2008 (when navigation was unavailable at our institution), to avoid selection bias. We did not attempt to match patients to controls. Nonnavigated resections were performed by two senior orthopaedic surgeons (with 10 years and > 25 years of experience). Navigated resections were performed by a senior orthopaedic surgeon with much experience in surgical navigation. The primary outcome was the bone and soft-tissue surgical margin achieved, classified by a modified Enneking system. Wide margins (≥ 2 mm) and wide-contaminated margins, in which the tumor or its pseudocapsule was exposed intraoperatively but further tissue was removed to achieve wide margins, were considered adequate; marginal (0-2 mm) and intralesional margins were considered inadequate. RESULTS Adequate bone margins were achieved in more patients in the navigated group than in the nonnavigation group (29 of 36 patients [81%] versus 17 of 34 [50%]; odds ratio, 4.14 [95% CI, 1.43-12.01]; p = 0.007). With the numbers available, we found no difference in our ability to achieve adequate soft-tissue margins between the navigation and nonnavigation group (18 of 36 patients [50%] versus 18 of 34 [54%]; odds ratio, 0.89 [95% CI, 0.35-2.27]; p = 0.995). CONCLUSIONS Intraoperative guidance techniques improved our ability to achieve negative bony margins when performing surgical resections in patients with pelvic and sacral primary bone sarcomas. Achieving adequate soft-tissue margins remains a challenge, and these margins do not appear to be influenced by navigation. Larger studies are needed to confirm our results, and longer followup of these patients is needed to determine if the use of navigation will improve survival or the risk of local recurrence. LEVEL OF EVIDENCE Level III, therapeutic study.

Tumor-induced osteomalacia is a rare acquired metabolic bone disorder characterized by isolated renal phosphate wasting due to abnormal tumor production of fibroblast growth factor 23. We report the case of a 59 year old woman referred to our department with a long history of progressive diffuse muscle weakness and pain, generalized bone pains and multiple insufficiency fractures of heels, ankles and hips due to a hypophosphatemic osteomalacia. A fibroblast growth factor 23-producing phosphaturic mesenchymal tumor localized in the left quadriceps femoris muscle was identified 7 years after onset of symptoms. Excision of the tumor resulted in normalization of serum phosphate and fibroblast growth factor 23 levels and in complete resolution of the clinical picture with disappearance of all musculoskeletal symptoms. This case illustrates the diagnostic difficulties in establishing a diagnosis tumor-induced osteomalacia and in identifying the responsible tumor. Our case underscores the clinical need to investigate all patients with persistent musculoskeletal symptoms for hypophosphatemia. A systematic approach is of pivotal importance because early recognition and treatment of the metabolic abnormality can prevent deleterious effects of osteomalacia on the skeleton.

The combination of high heterogeneity, both intratumoral and intertumoral, with their rarity has made diagnosis, prognosis of high-grade sarcomas difficult. There is an urgent need for more objective molecular biomarkers, to differentiate between the many different subtypes, and to also provide new treatment targets. Mass spectrometry imaging (MSI) has amply demonstrated its ability to identify potential new markers for patient diagnosis, survival, metastasis and response to therapy in cancer research. In this study, we investigated the ability of MALDI-MSI of proteins to distinguish between high-grade osteosarcoma (OS), leiomyosarcoma (LMS), myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) (Ntotal = 53). We also investigated if there are individual proteins or protein signatures that are statistically associated with patient survival. Twenty diagnostic protein signals were found characteristic for specific tumors (p ≤ 0.05), amongst them acyl-CoA-binding protein (m/z 11 162), macrophage migration inhibitory factor (m/z 12 350), thioredoxin (m/z 11 608) and galectin-1 (m/z 14 633) were assigned. Another nine protein signals were found to be associated with overall survival (p ≤ 0.05), including proteasome activator complex subunit 1 (m/z 9753), indicative for non-OS patients with poor survival; and two histone H4 variants (m/z 11 314 and 11 355), indicative of poor survival for LMS patients.

Chromosomal translocations with immunoglobin (IG) loci are the classic drivers in a large subset of B-cell lymphomas. Detection of these translocations is important for confirmation of diagnosis and for prognosis and therapy decisions. Currently, molecular diagnosis of translocations in lymphomas is not addressed well by next-generation sequencing (NGS). The standard method for detection of translocations is fluorescence in situ hybridization (FISH), which is labor intensive and can be difficult to interpret. There is a need for a robust technology that can be standardized. Targeted Locus Capture (TLC) selectively enriches and sequences entire genes based on the crosslinking of physically proximal sequences, and thereby enables complete sequencing of genes of interest, including detection of large structural variants. Because the technology is based on the crosslinking and fragmenting of DNA, it has particular advantages in the analysis of formalin-fixed, paraffin-embedded (FFPE) samples in which DNA is inherently crosslinked and fragmented. In order to validate the FFPE-TLC technology as a novel approach for translocation detection in lymphoma samples, we have developed a panel assay containing genes with frequent translocations (MYC, BCL2, BCL6, IG loci). With this assay we have analyzed >140 lymphoma and control FFPE samples of variable input amounts and qualities that had previously been analyzed with FISH, and a subset also with standard targeted NGS. Good concordance with FISH results was observed for both translocation-positive and -negative samples. In 10 cases for which FFPE-TLC analysis resulted in a different finding than FISH, discordance could be explained by higher sensitivity of FFPE-TLC or by inconclusive FISH results. In a specific case, FFPE-TLC detected a small-distance rearrangement on chromosome 3 that caused a BCL6 fusion but led to insufficient and therefore undetectable break-apart with FISH. Secondly, the FFPE-TLC approach was tested on a set of 19 B-cell lymphoma FFPE samples that had previously been analyzed using standard targeted NGS and FISH and was enriched for discordant results between these methods. FFPE-TLC-based NGS enables more robust translocation calling as the detection relies on broad sequencing coverage across the translocation partner rather than on breakpoint sequences only. In 3 cases, FFPE-TLC could proof false negative calls in standard targeted NGS due to breakpoints located in regions difficult to capture or to sequence. In 1 case, standard targeted NGS had made a false positive call on a breakpoint sequence that was shown to be caused by a small insertion rather than a genuine translocation. This study shows that FFPE-TLC promises to be a robust alternative for FISH analysis and standard targeted NGS procedures in lymphoma diagnostics and in other cancers with frequent structural variants. The FFPE-TLC approach enables a single, DNA-based NGS test detecting both small mutations and translocations. Citation Format: Amin Allahyar, Mark Pieterse, Joost Swennenhuis, Tjitske Los-de Vries, Mehmet Yilmaz, Roos Leguit, Ruud Meijers, Nathalie Hijmering, Daphne de Jong, Bauke Ylstra, Robert van der Geize, Joost Vermaat, Arjen Cleven, Tom van Wezel, Arjan Diepstra, Leon van Kempen, Karima Hajo, Harma Feitsma, Marieke Simonis, Max van Min, Erik Splinter, Wouter de Laat. Robust detection of translocations in lymphoma FFPE samples using Targeted Locus Capture-based sequencing [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-45.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

Background: Immunotherapy may be a rational strategy in leiomyosarcoma (LMS), a tumor known for its genomic complexity. As a prerequisite for therapeutic applications, we characterized the immune microenvironment in LMS, as well as its prognostic value. Methods: CD163+ macrophages, CD3+ T-cells, PD-L1/PD-L2 and HLA class I expression (HCA2, HC10 and β2m) were evaluated using immunohistochemistry in primary tumors (n = 75), local relapses (n = 6) and metastases (n = 19) of 87 LMS patients, as well as in benign leiomyomas (n = 7). Correlation with clinicopathological parameters and survival analyses were assessed. Effect of LMS cells on macrophage differentiation was investigated using coculture of CD14+ monocytes with LMS cell lines or their conditioned media (CM). Results: 58% and 52% of the tumors were highly infiltrated with CD163+ macrophages and T-cells, respectively, with HLA class I expression observed in almost all tumors and PD-L1 expression in 30%. PD-L2 expression was also detected in some PD-L1+ tumors. All these immune markers correlated with high tumor grade but only CD163 associated with overall survival (p = 0.003) and disease-specific survival (p = 0.041). In vitro, CD163 was upregulated in the presence of LMS cells producing M-CSF, suggesting that this tumor drives macrophages towards the M2 phenotype. Conclusion: The clinical significance of M2 macrophages, possibly induced by LMS cell-secreted factors, suggests that 2/3 of high-grade LMS patients might benefit from macrophage-targeting agents. Furthermore, PD-L1 expression together with high T-cell infiltrate and HLA class I expression in around 30% of high grade LMS reflects an active immune microenvironment potentially responsive to immune checkpoint inhibitors.

Synovial sarcoma expresses multiple cancer testis antigens that could potentially be targeted by T-cell receptor (TCR) gene therapy. In this study we investigated whether PRAME-TCR-gene therapy could be an effective treatment for synovial sarcoma by investigating the potential of PRAME-specific T-cells to recognize sarcoma cells and by evaluating the expression patterns of PRAME and HLA class I (HLA-I) in synovial sarcoma tumor samples. All PRAME expressing sarcoma cell lines, including 2 primary synovial sarcoma cell cultures (passage

Molecular assays for translocation detection in bone and soft tissue tumors have gradually been incorporated into routine diagnostics. However, conventional methods such as fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR come with several drawbacks. In this study, the applicability of a novel technique termed anchored multiplex PCR (AMP) for next-generation sequencing (NGS), using the Archer FusionPlex Sarcoma kit, aimed at 26 genes, was evaluated and compared with FISH and reverse transcriptase-PCR. In case of discrepant results, further analysis occurred with a third independent technique. Eighty-one samples were subjected to AMP-based targeted NGS, and 86% (n = 70) were successfully conducted and were either fusion positive (n = 48) or fusion negative, but met all criteria for good quality (n = 22). A concordance of 90% was found between NGS and conventional techniques. AMP-based targeted NGS showed superior results, as in four cases reverse transcriptase-PCR and FISH were false negative. Moreover, because the assay targets one partner of a gene fusion, novel or rare fusion partners can be identified. Indeed, it revealed COL1A1 and SEC31A as novel fusion partners for USP6 in nodular fasciitis. Despite the fact that fusions involving genes outside the selectively captured region cannot be detected and false-negative results due to poor quality samples can be encountered, this method has demonstrated excellent diagnostic utility for translocation detection in sarcomas.

Introduction: Chondrosarcoma is a malignant cartilage-forming bone tumour in which mutations in IDH1 and IDH2 frequently occur. Previous studies suggest an increased dependency on glutaminolysis in IDH1/2 mutant cells, which resulted in clinical trials with the drugs CB-839, metformin and chloroquine. In this study, the preclinical rationale for using these drugs as a treatment for chondrosarcoma was evaluated. Methods: Expression of glutaminase was determined in 120 cartilage tumours by immunohistochemistry. Ten chondrosarcoma cell lines were treated with the metabolic compounds CB-849, metformin, phenformin (lipophilic analogue of metformin) and chloroquine. Results: A difference in glutaminase expression levels between the different tumour grades (p = 0.001, one-way ANOVA) was identified, with the highest expression observed in high-grade chondrosarcomas. Treatment with CB-839, metformin, phenformin or chloroquine revealed that chondrosarcoma cell lines are sensitive to glutaminolysis inhibition. Metformin and phenformin decreased mTOR activity in chondrosarcoma cells, and metformin decreased LC3B-II levels, which is counteracted by chloroquine. Conclusion: Targeting glutaminolysis with CB-839, metformin, phenformin or chloroquine is a potential therapeutic strategy for a subset of high-grade chondrosarcomas, irrespective of the presence or absence of an IDH1/2 mutation.

Identification of p53-positive cells by immunohistochemistry in bone marrow from primary myelodysplastic syndrome patients correlates with the presence of TP53 mutations and poor prognosis. Mutations in the tumor suppressor gene TP53 are more frequent in therapy-related acute myeloid leukemia and myelodysplastic syndrome than in de novo disease, but the role of p53 immunohistochemistry in the therapy-related setting has not been specifically investigated. We studied p53 protein immunoreactivity in bone marrow biopsies of therapy-related myeloid neoplasms and correlated protein expression with TP53 mutation status, clinicopathologic features and outcome. We first studied 32 patients with therapy-related acute myeloid leukemia and 63 patients with therapy-related myelodysplastic syndrome/chronic myelomonocytic leukemia from one institution and then validated our results in a separate group of 32 patients with therapy-related acute myeloid leukemia and 56 patients with therapy-related myelodysplastic syndrome from a different institution. Strong p53 immunostaining in ≥1% of bone marrow cells was highly predictive of a TP53 gene mutation (P

The aseptic lymphocyte vasculitis-associated lesion (ALVAL) score and the modified Oxford ALVAL score are frequently used scoring methods to evaluate the morphologic features of periprosthetic tissues around metal-on-metal (MoM) hip implants. Except for the initial studies of these two morphology scoring methods, to our knowledge, no other studies have reported on intraclass correlation coefficient (ICC) values for interobserver reliability of these scoring methods. Are the ALVAL and Oxford ALVAL scores reproducible? The periprosthetic tissue of 37 revisions of 36 patients with failed MoM THAs were independently scored by three experienced pathologists using ALVAL and Oxford ALVAL scoring methods. All patients were included who underwent revision surgery in our hospital until January 2013, with a large-head MoM prosthesis and also met the criteria: blood serum cobalt levels, available MRI scan, and intraarticular cobalt levels. The population included 26 patients with pseudotumors diagnosed by two radiologists using the method described by Matthies et al. The ALVAL describes morphologic features of the synovial lining, tissue organization, and inflammatory cell infiltrate in periprosthetic tissues. The Oxford-ALVAL score uses a semiquantitative measure of the immune response which should be easier to score. The ALVAL score showed an ICC of 0.38 (95% CI, 0.18–0.58) (fair) for the sum score and this improved up to 0.50 (95% CI, 0.31–0.68) (moderate) using the modified Oxford ALVAL score. The individual parameters of the ALVAL score showed an ICC for the scoring of inflammatory infiltrate of 0.37 (95% CI, 0.17–0.57), an ICC of 0.32 (95% CI, 0.12–0.53) for the scoring of tissue organization, and an ICC of 0.14 (95% CI, −0.04 to 0.34) for synovial lining. Scoring morphologic features of MoM tissue is not reproducible using the ALVAL score or the Oxford ALVAL score. This may reflect heterogeneous morphologic features in tumor tissue and between different tumor tissue samples that cannot be reliably quantified by pathologists using the parameters of these two scoring methods. An alternative, simplified scoring system should be developed to improve the interrater agreement. Level III, diagnostic study.

Indeterminate cell histiocytosis is a rare variant of histiocytosis. The diagnosis is currently based on presence and absence of immunohistochemical markers. The current case described an indeterminate cell histiocytosis with ETV3-NCOA2 translocation.

Mass spectrometry imaging (MSI) has been widely used for the direct molecular assessment of tissue samples and has demonstrated great potential to complement current histopathological methods in cancer research. It is now well established that tissue preparation is key to a successful MSI experiment; for histologically heterogeneous tumor tissues, other parts of the workflow are equally important to the experiment's success. To demonstrate these facets here we describe a matrix-assisted laser desorption/ionization MSI biomarker discovery investigation of high-grade, complex karyotype sarcomas, which often have histological overlap and moderate response to chemo-/radio-therapy. Multiple aspects of the workflow had to be optimized, ranging from the tissue preparation and data acquisition protocols, to the post-MSI histological staining method, data quality control, histology-defined data selection, data processing and statistical analysis. Only as a result of developing every step of the biomarker discovery workflow was it possible to identify a panel of protein signatures that could distinguish between different subtypes of sarcomas or could predict patient survival outcome. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. (C) 2016 Elsevier B.V. All rights reserved.

Background Mutations in isocitrate dehydrogenase (IDH)1 or -2 are found in ~50% of conventional central chondrosarcomas and in up to 87% of their assumed benign precursors enchondromas. The mutant enzyme acquires the activity to convert α-ketoglutarate into the oncometabolite d-2-hydroxyglutarate (d-2-HG), which competitively inhibits α-ketoglutarate dependent enzymes such as histone- and DNA demethylases. Methods We therefore evaluated the effect of IDH1 or -2 mutations on histone modifications (H3K4me3, H3K9me3 and H3K27me3), chromatin remodeler ATRX expression, DNA modifications (5-hmC and 5-mC), and TET1 subcellular localization in a genotyped cohort (IDH, succinate dehydrogenase (SDH) and fumarate hydratase (FH)) of enchondromas and central chondrosarcomas (n = 101) using immunohistochemistry. Results IDH1 or -2 mutations were found in 60.8% of the central cartilaginous tumours, while mutations in FH and SDH were absent. The mutation status did not correlate with outcome. Chondrosarcomas are strongly positive for the histone modifications H3K4me3, H3K9me3 and H3K27me3, which was independent of the IDH1 or -2 mutation status. Two out of 36 chondrosarcomas (5.6%) show complete loss of ATRX. Levels of 5-hmC and 5-mC are highly variable in central cartilaginous tumours and are not associated with mutation status. In tumours with loss of 5-hmC, expression of TET1 was more prominent in the cytoplasm than the nucleus (p = 0.0001). Conclusions In summary, in central chondrosarcoma IDH1 or -2 mutations do not affect immunohistochemical levels of 5-hmC, 5mC, trimethylation of H3K4, -K9 and K27 and outcome, as compared to wildtype. Electronic supplementary material The online version of this article (doi:10.1186/s13569-017-0074-6) contains supplementary material, which is available to authorized users.

Introduction Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma comprising 1-2% of all malignant lymphomas. This study aims to elucidate the genetic background of a homogeneous cohort of PB-DLBCL. Methods This retrospective study consists of primary DLBCL-patients with bone localization(s) of which pretreatment fresh frozen or formalin-fixed paraffin-embedded bone tissue samples were available. Patients were diagnosed (2003-2019) at Leiden University Medical Center (LUMC), a center of expertise for bone tumors, Amsterdam University Medical Center (AUMC), Erasmus MC and affiliated Dutch hospitals. Based on strict definitions regarding radiological assessment of anatomical disease localizations at diagnosis three subgroups were categorized: solely osseous involvement (single or multiple bone lesions; PB-DLBCL), osseous involvement and locoregional lymphadenopathy (locoregional disease), and osseous and (multiple) extra-osseous localizations (disseminated disease). Cell-of-origin (COO) was determined by immunohistochemistry (BCL6, CD10, and MUM1) and classified according to the Hans' algorithm. Additionally, COO was confirmed with NanoString and the Lymph2Cx assay (Scott et al., Blood 2014), in a subset of patients. With similar procedures (Vermaat et al., Haematologica 2019), molecular profiles were determined with an in-house developed and validated targeted next-generation sequencing (tNGS) panel, comprising 52 DLBCL-specific genes, for sequencing with the Ion S5TM System. Obtained results were compared to sequencing data of (1) an independent 'in-house' cohort of 23 primary GCB (Germinal Center B-Cell)-DLBCL patients without bone localization ('non-osseous') and (2) pooled data of 651 GCB-DLBCL patients from literature (Chapuy et al., Nature Medicine 2018, Karube et al., Leukemia 2018, Reddy et al., Cell 2017, Schmitz et al., NEJM 2018). Results Our cohort contained 56 patients (males, N=33, (59%)) with a median age at diagnosis of 62 years (range 13-92). Twenty-four patients had PB-DLBCL (45%), 8 had locoregional disease (14%), and 23 had disseminated disease (41%). In general, immunohistochemistry and Lymph2Cx identified a GCB subtype for the majority of all DLBCL with bone localizations (Figure-1A) and these results for the hitherto unperformed cases will follow shortly. tNGS identified 48 genes with 'pathogenic' mutations, with on average four mutated genes per patients (range 0-10; Figure-1A). Overall, high mutation frequencies were observed in TNFRSF14 (33%), KMT2D (27%), EZH2 (25%), CREBBP (22%), B2M (22%), and TP53 (20%) in DLBCL with bone localizations and mainly genes involved in epigenetic machinery. In PB-DLBCLs, high frequency of mutated EZH2 (38%) and IRF8 (25%) were identified. Both are epigenetic genes that regulates tumor suppression and type I interferon, respectively. In four PB-DLBCLs EZH2 and IRF8 were concomitantly mutated. Locoregional disease showed a similar molecular profile as PB-DLBCL. Association with clinical characteristics will be performed shortly. Compared to our cohort of non-osseous GCB-DLBCL (Figure-1B) and pooled data of GCB-DLBCL in large sequencing studies (Figure-1C), EZH2 (Chi-square; P=0.046 and P=0.005, respectively) was significantly more frequently mutated in PB-DLBCL, though IRF8 did not attain this significance (Chi-square; P=0.121 and P=0.111, respectively; Figure-1D). Conclusion This study is the first that provides integrative analyses of immunohistochemistry, Lymph2Cx, and tNGS of a homogeneous cohort of PB-DLBCL, demonstrating the importance of epigenetic genes in lymphomagenesis. In contrast to (non-osseous) GCB-DLBCLs, the molecular profile of PB-DLBCL is characterized by significantly frequent mutations in EZH2 and frequent mutations in IRF8 and other epigenetic genes, which is indicative for a GCB phenotype (Scherer F. et al., Sci Transl Med 2016) and supported by immunohistochemistry and Lymph2Cx data. These results suggest that PB-DLBCL is a specific DLBCL-entity with a unique molecular profile and provide a rationale for exploration of novel targeted treatment with EZH2 (and IRF8) inhibitors for PB-DLBCL patients. Disclosures Lugtenburg: Genmab: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; Celgene: Consultancy, Honoraria; Janssen Cilag: Honoraria; Takeda: Consultancy, Honoraria, Research Funding. Kersten:Gilead: Honoraria; Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda Oncology: Research Funding; Miltenyi: Honoraria; Kite Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Metabolites can be an important read-out of disease. The identification and validation of biomarkers in the cancer metabolome that can stratify high-risk patients is one of the main current research aspects. Mass spectrometry has become the technique of choice for metabolomics studies, and mass spectrometry imaging (MSI) enables their visualization in patient tissues. In this study, we used MSI to identify prognostic metabolite biomarkers in high grade sarcomas; 33 high grade sarcoma patients, comprising osteosarcoma, leiomyosarcoma, myxofibrosarcoma, and undifferentiated pleomorphic sarcoma were analyzed. Metabolite MSI data were obtained from sections of fresh frozen tissue specimens with matrix-assisted laser/desorption ionization (MALDI) MSI in negative polarity using 9-aminoarcridine as matrix. Subsequent annotation of tumor regions by expert pathologists resulted in tumor-specific metabolite signatures, which were then tested for association with patient survival. Metabolite signals with significant clinical value were further validated and identified by high mass resolution Fourier transform ion cyclotron resonance (FTICR) MSI. Three metabolite signals were found to correlate with overall survival (m/z 180.9436 and 241.0118) and metastasis-free survival (m/z 160.8417). FTICR-MSI identified m/z 241.0118 as inositol cyclic phosphate and m/z 160.8417 as carnitine. Graphical Abstractᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1544-4) contains supplementary material, which is available to authorized users.

Introduction Immunotherapy may be an excellent choice for treating osteosarcoma given its exceptionally high genomic instability, potentially generating neoantigens. In this study, we aim to investigate the HLA class I expression, PD-L1 and tumour-infiltrating lymphocytes in primary osteosarcomas and relapses/metastases, as well as their changes during disease progression. Materials and methods Tumour samples from multiple stages of the disease (pretreatment biopsies, surgical resections of primary osteosarcomas, relapses and metastases) were collected and stained for HLA-A (HCA2), HLA-B/C (HC10), β2-microglobulin and PD-L1 using immunohistochemistry on whole sections. Density and type of T-cell infiltrate were characterised by a triple immunofluorescent staining CD3-CD8-FOXP3. Results Overall, 85 formalin-fixed, paraffin-embedded blocks from 25 osteosarcoma patients were included. HLA class I expression was detected in 94% of osteosarcomas (strongly positive in 56%, heterogeneous in 38%) and negative or weakly positive in 6%, without differences between the stages of the disease. HLA-A expression was more frequently negative than HLA-B/C. Tumour-infiltrating lymphocytes were highly heterogeneous and mainly observed in tumour areas with expression of HLA class I. Density of T cells was significantly higher in metastases than in primary tumours and local relapses (p = 0.0003). Positive PD-L1 expression was found in 13% of primary tumours, 25% of relapses and 48% of metastases and correlated with a high T-cell infiltrate (p = 0.002). Conclusion An increased number of tumour-infiltrating T cells and PD-L1 expression in metastases compared with primary tumours, suggesting accessibility for T cells, could imply that osteosarcoma patients with metastatic disease may benefit from T-cell-based immunotherapy. Electronic supplementary material The online version of this article (doi:10.1007/s00262-016-1925-3) contains supplementary material, which is available to authorized users.