Conventional chondrosarcoma with focal clear cell change:a clinicopathological and molecular analysis

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

Clinical, Histologic, and Molecular Characteristics of Anaplastic Lymphoma Kinase-positive Primary Cutaneous Anaplastic Large Cell Lymphoma

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

No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone

Background: Chondrosarcoma is a malignant cartilage forming bone tumour for which no effective systemic treatment is available. Previous studies illustrate the need for a better understanding of the role of the IGF pathway in chondrosarcoma to determine if it can be a target for therapy, which was therefore explored in this study. Methods: Expression of mediators of IGF1R signalling and phosphorylation status of IRS1 was determined in chondrosarcoma cell lines by qRT-PCR and western blot. The effect of activation and inhibition of IGF1R signalling on downstream targets was assessed by western blot. Ten chondrosarcoma cell lines were treated with OSI-906 (IGF1R and IR dual inhibitor) after which cell proliferation and migration were determined by a viability assay and the xCELLigence system, respectively. In addition, four chondrosarcoma cell lines were treated with a combination of doxorubicin and OSI-906. By immunohistochemistry, IGF1R expression levels were determined in tissue microarrays of 187 cartilage tumours and ten paraffin embedded cell lines. Results: Mediators of IGF1R signalling are heterogeneously expressed and phosphorylated IRS1 was detected in 67 % of the tested chondrosarcoma cell lines, suggesting that IGF1R signalling is active in a subset of chondrosarcoma cell lines. In the cell lines with phosphorylated IRS1, inhibition of IGF1R signalling decreased phosphorylated Akt levels and increased IGF1R expression, but it did not influence MAPK or S6 activity. In line with these findings, treatment with IGF1R/IR inhibitors did not impact proliferation or migration in any of the chondrosarcoma cell lines, even upon stimulation with IGF1. Although synergistic effects of IGF1R/IR inhibition with doxorubicin are described for other cancers, our results demonstrate that this was not the case for chondrosarcoma. In addition, we found minimal IGF1R expression in primary tumours in contrast to the high expression detected in chondrosarcoma cell lines, even if both were derived from the same tumour, suggesting that in vitro culturing upregulates IGF1R expression. Conclusions: The results from this study indicate that the IGF pathway is not essential for chondrosarcoma growth, migration or chemoresistance. Furthermore, IGF1R is only minimally expressed in chondrosarcoma primary tumours. Therefore, the IGF pathway is not expected to be an effective therapeutic target for chondrosarcoma of bone. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2522-8) contains supplementary material, which is available to authorized users

MYD88 mutations identify a molecular subgroup of diffuse large B-cell lymphoma with an unfavorable prognosis

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

Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from a

Frequent mutated B2M, EZH2, IRF8, and TNFRSF14 in primary bone diffuse large B-cell lymphoma reflect a GCB phenotype

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 DLBCL with 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)-NanoString/Lymph2Cx analysis. Mutational profiles were identified with 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) compared with NO-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)

Benign and low-grade cartilaginous tumors: an update on differential diagnosis

Cartilaginous neoplasms hold considerable share in the routine diagnostics of bone tumor pathology. Benign cartilage forming tumors, that are in general more frequent than their malignant counterparts, may often pose diagnostic challenges. Distinction of a benign cartilaginous lesion from a low-grade malignant tumor can render difficulties especially in a small biopsy material. Considering the divergent therapeutic and prognostic implications, a careful assessment of histologic, radiologic and clinical findings/features is very important, and an experienced multidisciplinary team is indispensable to achieve an accurate diagnosis. This mini review focuses on the histomorphological findings of benign, locally aggressive and low-grade cartilaginous tumors referring to the latest edition of WHO Classification of Tumors of Soft Tissue and Bone Tumors (fifth edition, 2020). The focus of this article is on the differential diagnosis of benign cartilaginous lesions and their distinction from other cartilaginous lesions, especially their malignant counterparts. Histomorphology, in correlation with imaging findings, serve as the cornerstone in the diagnosis of cartilaginous tumors. Based on overlapping morphological and radiological features between the tumor types, including benign and malignant ones, their diagnosis can be challenging. Emerging molecular findings play a notable role in understanding the underlying mechanisms involved in their development, though their routine use in molecular diagnostics is limited to few indications. Likewise, the role of immunohistochemistry remains limited in the differential diagnosis of the cartilaginous tumors, emphasizing the need for joined histological and radiological evaluation

Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas

Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1) in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis

An experimental guideline for the analysis of histologically heterogeneous tumors by MALDI-TOF mass spectrometry imaging

MTG4Molecular tumour pathology - and tumour genetic