Immunohistochemical analysis of PD-L1, CD4, CD8, CD20, and PU.1 expression in a dedifferentiated chondrosarcoma: the clinical and prognostic value

<div><p>Dedifferentiated chondrosarcoma (DDCS) is a rare high-grade chondrosarcoma characterized by a well-differentiated chondrosarcoma (WDCS) component that abruptly transitions to a high-grade, noncartilaginous sarcomatous component. To date, the molecular pathogenesis of DDCS and its distinction from conventional chondrosarcoma remain poorly understood. By targeted sequencing, we examined the mutational and copy-number profiles of 18 DDCS, including macrodissected WDCS components, compared with 55 clinically sequenced conventional chondrosarcomas. In conjunction with publicly available external data, we analyzed the methylation and expression profiles of 34 DDCS and 94 conventional chondrosarcomas. Isocitrate dehydrogenase 1/isocitrate dehydrogenase 2 (<i>IDH1/IDH2)</i> mutations were present in 36% conventional chondrosarcomas and 71% DDCS. Compared with conventional chondrosarcomas, DDCS had higher frequencies of <i>TP53</i> and <i>TERT</i> promoter mutations and <i>CDKN2A/B</i> copy-number losses. Paired analysis of macrodissected WDCS and the high-grade components revealed <i>TERT</i> promoter mutations as early events. Despite phenotypic similarities, the percentage of genome with copy-number alterations in DDCS was significantly lower than that in other high-grade sarcomas. Differential methylation analysis revealed reduction of <i>IDH1/IDH2</i>-associated global hypermethylation characteristically seen in conventional chondrosarcoma and a distinct methylation profile in DDCS. The WDCS and high-grade components in DDCS showed similar methylation profiles. These CpG sites were associated with upregulated expression of genes involved in G<sub>2</sub>–M checkpoints and E2F targets. Genomic profiling revealed enrichment of <i>TP53</i>, <i>TERT</i> promoter, and <i>CDKN2A/B</i> alterations in DDCS. Integrated methylation and gene expression analysis revealed distinct <i>IDH1/IDH2</i>-associated methylation and transcriptional profiles as early events in DDCS, which may underlie the pathogenesis of dedifferentiation in chondrosarcomas.</p>Significance:<p>DDCS is a rare, high-grade chondrosarcoma with a dismal prognosis. About 50%–80% of DDCS harbor IDH1/IDH2 mutations. We uncover a significant alteration of IDH-associated methylation profile in DDCS, which we propose is key to the progression to dedifferentiation. In this context, the potential effect of the use of IDH inhibitors is unclear but important to address, as clinical trials of selective IDH1 inhibitors showed worse outcome in DDCS.</p></div>

Dedifferentiated chondrosarcoma (DDCS) is a rare high-grade chondrosarcoma characterized by a well-differentiated chondrosarcoma (WDCS) component that abruptly transitions to a high-grade, noncartilaginous sarcomatous component. To date, the molecular pathogenesis of DDCS and its distinction from conventional chondrosarcoma remain poorly understood. By targeted sequencing, we examined the mutational and copy-number profiles of 18 DDCS, including macrodissected WDCS components, compared with 55 clinically sequenced conventional chondrosarcomas. In conjunction with publicly available external data, we analyzed the methylation and expression profiles of 34 DDCS and 94 conventional chondrosarcomas. Isocitrate dehydrogenase 1/isocitrate dehydrogenase 2 (IDH1/IDH2) mutations were present in 36% conventional chondrosarcomas and 71% DDCS. Compared with conventional chondrosarcomas, DDCS had higher frequencies of TP53 and TERT promoter mutations and CDKN2A/B copy-number losses. Paired analysis of macrodissected WDCS and the high-grade components revealed TERT promoter mutations as early events. Despite phenotypic similarities, the percentage of genome with copy-number alterations in DDCS was significantly lower than that in other high-grade sarcomas. Differential methylation analysis revealed reduction of IDH1/IDH2-associated global hypermethylation characteristically seen in conventional chondrosarcoma and a distinct methylation profile in DDCS. The WDCS and high-grade components in DDCS showed similar methylation profiles. These CpG sites were associated with upregulated expression of genes involved in G2–M checkpoints and E2F targets. Genomic profiling revealed enrichment of TP53, TERT promoter, and CDKN2A/B alterations in DDCS. Integrated methylation and gene expression analysis revealed distinct IDH1/IDH2-associated methylation and transcriptional profiles as early events in DDCS, which may underlie the pathogenesis of dedifferentiation in chondrosarcomas.Significance:DDCS is a rare, high-grade chondrosarcoma with a dismal prognosis. About 50%–80% of DDCS harbor IDH1/IDH2 mutations. We uncover a significant alteration of IDH-associated methylation profile in DDCS, which we propose is key to the progression to dedifferentiation. In this context, the potential effect of the use of IDH inhibitors is unclear but important to address, as clinical trials of selective IDH1 inhibitors showed worse outcome in DDCS.

The immune system plays an important role in tumor development and treatment. In this study, we aimed to determine the relationships among the expressions of PD-L1, CD3, CD8, MMR proteins, clinicopathological features, and prognosis of CRC. Immunohistochemistry was used to determine the expression of PD-L1, CD3, and CD8 in 771 patients with CRC. The expression of PD-L1 in TC was related to the right colon, adenocarcinoma, and dMMR, and in IC, it was related to younger CRC patients and the TNM stage. The expression of CD3 and CD8 in tumor-infiltrating lymphocytes was related to lymph node metastasis and the TNM stage. The expression of PD-L1 in TC and IC was correlated with the infiltration of CD3+ and CD8+ lymphocytes. Univariate survival analysis showed that the expression of PD-L1 in TC, IC, and dMMR was related to a better prognosis. Multivariate survival analysis showed that age, TNM stage, and dMMR were independent prognostic factors for CRC. The OS of the chemotherapy was significantly higher than that of the non-chemotherapy in III-IV TNM stage patients; CRC patients with positive PD-L1 expression in TC or IC and dMMR did not benefit from chemotherapy. PD-L1 expression in TC and IC was closely related to the density of CD3 and CD8 infiltration in tumor-infiltrating lymphocytes. The expression of CD3 and CD8 in tumor-infiltrating lymphocytes and the expression of PD-L1 in IC were linked to the TNM stage of CRC patients. PD-L1 expression in TC and IC and MMR status may act as an important biomarker for guiding the postoperative treatment of III-IV TNM stage CRC patients.

Dedifferentiated chondrosarcoma (DDCS) comprises approximately 10% of all chondrosarcomas and has the worst outcome with a 5-year survival of 10%.The preferred Iocalizations are the femur,humerus and pelvis.DDCS represents a special form of chondrosarcoma characterized by the presence of well-differentiated cartilaginous component in juxtaposition with malignant mesenchymal tumor of high-malignancy grade.The diagnosis of DDCS is highly complicated,requiring detailed radiological and histopathological evaluation as well as precise bioptic technique.The dedifferentiated component is typically a high-grade sarcoma (usually grade 3 or 4),which can be either an osteosarcoma,a malignant fibrous histiocytoma or an anaplastic spindle cell sarcoma.In approximately one-third of the radiographs,one-third of the MR images, and one-half of the CT scans,the tumors demonstrates bimorphic features.Recently,array-based comparative genomic hybridization (array-CGH) studies have been performed on frozen chondrosarcoma (including DDCS) specimens.There is a statistically significant association between high-grade tumor (grade Ⅱ and dedifferentiated) and the recurrent genetic deletions at 5q14.2～q21.3,6q16～q25.3,9p24.2～q12,and 9p21.3. One of the most commonly deleted regions of DDCS involved chromosome 9.Earlier investigations of DDCS showed p53 mutation and p53-LOH in the anaplastic component.It is also accompanied by Rb-LOH. P161NK4 and E-cadherin promotor methylation were observed in the low grade chondroid compartment of DDCS.While p161NK4,FHIT,and E-cadherin were methylated in highly malignant osteosarcomatous compartment of the tumor.Surgical resection of the tumor within wide or radical margins is the most important treatment.The value of neoadjuvant or adjuvant therapy remain uncertain.Several new drug targets have been identified and phase Ⅱ studies are currently ongoing.Current phase Ⅱ trials open for DDCS patients used the following medicine:apomab (proapoptotic selective agonist of Apo2L/TRAIL death receptor),perifosine (serine/threonine kinase Akt inhibitor),dasatinib (multitargeted small-molecule tyrosine kinase inhibitor), and the combination of gemcitabine and docetaxel.More recently,several phase Ⅰ studies have reported incidental responses of DDCS to newer targeted agents,such as histone deacetylase and vascular endothelial growth factor antisense oligodeoxynucleotide.The prognosis for patients with DDCS remains poor.The poor prognosis of the DDCS is determined by nonchondroid high grade component caused by invasive growth and formation of metastases.Therefore,early diagnosis and prompt surgical treatment may improve the outcome.

Dedifferentiated chondrosarcomas are primary bone tumors characterized by the presence of both low-grade cartilaginous and high-grade sarcomatous components. The high-grade component usually shows histologic features of either malignant fibrous histiocytoma or fibrosarcoma. We are aware of only 10 published cases in which the high-grade component showed rhabdomyosarcomatous differentiation. To further clarify the clinical, radiographic, and pathologic features of this unusual variant, we report three additional cases of dedifferentiated chondrosarcoma with rhabdomyosarcomatous differentiation. The patients included two men and one woman; their mean age was 63 years. Tumors originated in the pelvis (ilium), scapula, and tibia. Two patients presented with radiographic findings typical of dedifferentiated chondrosarcoma, including a geographic, lytic lesion with areas of mineralization suggestive of cartilage in close association with a permeative component. The third patient presented with a primarily lytic, destructive lesion of the right iliac wing. Histologically, the tumors contained lobules of well-differentiated chondrosarcoma associated with a high-grade sarcoma with prominent rhabdomyoblasts. Immunohistochemical stains for actin and desmin were positive in all three tumors, and electron microscopy revealed evidence of skeletal muscle differentiation. All three patients died with metastatic disease, 1, 6, and 12 months postoperatively. This histologic variant of dedifferentiated chondrosarcoma is rare, but it shows radiographic and clinical features similar to "conventional" dedifferentiated chondrosarcoma, including a very poor prognosis.

Background: Immune checkpoint inhibitors such as PD-L1 are promising therapy targets in triple negative breast cancer (TNBC). In the present study, we examined the clinical relevance of PD-L1 and CD8 expression in TNBC patients treated with adjuvant chemotherapy. Methods: FFPE tumor material from 118 patients with early TNBC treated between 1999 and 2012 was included in our analysis. Immunohistochemical (IHC) expression of PD-L1 was determined by immune score using the diagnostic antibody SP142 (Ventana). CD8 IHC was performed using the SP57 antibody (Ventana). Follow-up data were available for all 118 patients with a median follow up time of 19.4 years. Biomarkers were examined as continuous or categorical variable (predefined cutoffs). Invasive disease-free survival (IDFS) and overall survival (OS) were analyzed using Cox regression models. Results: The median PD-L1 immune score was 1% and PD-L1 expression was classified as positive in 79/116 (68.1%, cutoff ≥1%) cases. Median CD8 expression was 10% and was scored as positive in 51/115 (44.3%, cutoff >10%) samples. Significant associations were observed between PD-L1 expression and tumor grade, Ki67, and CD8 expression. PD-L1-positive tumors were more frequently in the G3 group (p=0.006) and had higher Ki67 (p< 0.001) and higher CD8 expression (p< 0.001). PD-L1 but not CD8 expression was associated with IDFS or OS. Univariate Cox regression analyses showed that PD-L1-negative patients had a shorter IDFS (HR 0.52, 95%CI 0.30-0.89, p=0.02) and showed a trend towards shorter OS (HR 0.56, 95%CI 0.30-1.03, p=0.06). CD8 expression was neither associated with IDFS (HR 0.74, 95%CI 0.43-1.27, p=0.27) nor with OS (HR 0.87, 95%CI 0.48-1.60, p=0.66). In multivariate analyses, PD-L1 expression was an independent prognostic factor for IDFS (HR 0.51, 95%CI 0.29-0.88, p=0.016) but not for OS (HR 0.53, 95%CI 0.28-1.01, p=0.052). Conclusions: Our results suggest that PD-L1 but not CD8 expression assessed by IHC predicted outcome in TNBC. Further analysis of larger, suitable patient cohorts is warranted to further assess the prognostic and predictive value of PD-L1 expression. Citation Format: Zsuzsanna Bago-Horvath, Maximilian Marhold, Ulrike Heber, Rupert Bartsch, Florian Fitzal, Christian F. Singer, Martin Filipits. Clinical Relevance of PD-L1 and CD8 Expression in Triple Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-04.

Ten cases of dedifferentiated chondrosarcoma (DCS) were immunohistochemically and histochemically compared with 12 de novo malignant fibrous histiocytomas, 10 osteoblastic osteosarcomas, 9 conventional chondrosarcomas, and 4 fibrosarcomas (all of bone or soft tissues), in order to discern similarities and differences in the immunophenotypes of these neoplasms. All cases of DCS and malignant fibrous histiocytoma were reactive for alpha-1-antichymotrypsin, and several examples of both tumor types bound peanut agglutinin, and expressed positivity for alpha-1-antitrypsin and lysozyme. None of these four cellular markers was observed in de novo osteosarcoma and fibrosarcoma; in addition, conventional chondrosarcoma lacked all of them except for peanut agglutinin receptors. S100 protein reactivity and binding of wheat germ agglutinin were detectable in conventional chondrosarcomas and in rare cells of the anaplastic components of primary DCS, but not in malignant fibrous histiocytoma arising ab initio and the other sarcomas. These results suggest the evolution of a second neoplastic cellular clone in DCS, with primitive morphological and phenotypic characteristics.

Diagnostic utility of IDH1/2 mutations to distinguish dedifferentiated chondrosarcoma from undifferentiated pleomorphic sarcoma of bone

Aberrant PD-L1 Expression in CLL As a Result of Adaptive Immune Resistance Mediated By Tumor-Secreted Circulating miRNA Binding to Toll-like Receptor 7

Dedifferentiated chondrosarcoma of bone with prominent rhabdoid component.

Undifferentiated pleomorphic sarcoma (UPS) is a sarcoma with a poor prognosis. A clinical trial, SARC028, revealed that treatment with anti-PD-1 drugs was effective against UPS. Studies have reported that UPS expresses PD-L1, sometimes strongly (≥ 50%). However, the mechanism of PD-L1 expression in UPS has remained unclear. CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) was identified as a novel regulator of PD-L1 expression. The positive relationship between PD-L1 and CMTM6 has been reported in several studies. The aim of this study was thus to examine CMTM6 expression in UPS and evaluate the relationship between PD-L1 and CMTM6 in this disease. Fifty-one primary UPS samples were subjected to CMTM6 and PD-L1 immunostaining. CMTM6 expression was assessed using proportion and intensity scores. CMTM6 gene copy number was also evaluated using a real-time PCR-based copy number assay. We also analyzed the mRNA expression and copy number variation of PD-L1 and CMTM6 in The Cancer Genome Atlas (TCGA) data. TCGA data indicated that the mRNAs encoded by genes located around 3p22 were coexpressed with CMTM6 mRNA in UPS. Both proportion and intensity scores of CMTM6 positively correlated with strong PD-L1 expression (≥ 50%) (both p = 0.023). CMTM6 copy number gain increased CMTM6 expression. Patients with UPS with a high CMTM6 intensity score had a worse prognosis for overall survival. UPS showed variation in CMTM6 copy number and CMTM6 expression. CMTM6 expression was significantly correlated with PD-L1 expression, especially with strong PD-L1 expression.

Background: Undifferentiated pleomorphic sarcoma (UPS) are genetically complex sarcomas often showing a relatively higher immune infiltration among sarcomas, and a demonstrated benefit for immunotherapy in a subset of patients. This study characterizes the immune microenvironment of UPS including tumor infiltrating lymphocytes (TILs), as well as immune checkpoint (IC) and related biomarkers. Methods: We used FFPE surgical resected UPS from 90 patients (43 primary, 28 recurrence and 19 metastasis) placed in a tissue microarray and stained with immunohistochemistry for the following biomarkers: IC (stimulatory: OX40 & ICOS; inhibitory: PD-L1, LAG3, IDO1, & PD1), TILs (CD3 & CD8), monocytes-macrophages (CD163), Adenosine Pathway (CD73 & CD39), and pSTAT3. We scored TIL and IC biomarkers as positive cell densities, and PD-L1, pSTAT3, CD73 and CD39 as percentage of expression in malignant cells. Clinicopathological data and clinical outcome were available for all patients. Spearman correlation was used to assess the correlation between two biomarkers. Cox proportional hazard regression models were used to identify the prognostic biomarkers for overall survival (OS), recurrence-free survival (RFS), and metastasis-free survival (MFS). Results: In our cohort, PD-L1, pSTAT3, CD73 and CD39 positive expression (cut-off ≥1%) was 19, 73 78 and 68% respectively. These biomarkers were positively correlated with immune infiltrates: CD3 (p=0.013, 0.009, 0.009, <.001), CD8 (p=0.004, 0.016, <.001, <.001) and ICOS (p=0.005, 0.222, 0.007, 0.022). CD73 and CD39 were also positively correlated with both CD163 (p=0.001, <.001) and PD-1 (p=0.012, <.001). In recurrent tumors, smaller tumors had higher CD3, CD8, ICOS and PD-1 (p=0.011, 0.045, 0.013, 0.045). In metastatic tumor, CD163 and OX40 were positively correlated with age (p=0.045, 0.019). OX40 was inversely correlated with tumor size (p=0.04). ICOS positively correlated with PD-1, PD-L1 and CD39 (p<.001, 0.002, 0.003) in primary tumors; with IDO1 and PD1 (p=0.012, <.001) in recurrent tumors; and with PD-L1 expression (p=0.031) in metastatic tumors. Low CD3, CD8, CD163 densities (cut-off median) were associated with inferior OS (HR: 3.19 [1.37, 7.42]; p=0.007), RFS (HR: 2.79 [1.21, 6.42], p=0.016), and MFS (HR: 2.42 [1.05, 5.56]; p=0.038) after adjusting for size, respectively. Low CD73 and PD-L1 (cut-off median) were associated with inferior OS (HR 3.03 [HR: 1.21, 7.61], p=0.018), RFS (HR: 2.59 [1.06, 6.36]; p=0.037), and MFS (HR: 2.43 [1.00, 5.89], p=0.049), respectively. Conclusions: In this study, we characterized the immune cell infiltrates and the expression of PD-L1, pSTAT3, CD73 and CD39 in UPS. These biomarkers differentially correlated with clinicopathological characteristics in primary, recurrent and metastatic tumors. Lower TILs and low CD73/PD-L1 expression were associated with inferior survival outcomes. Citation Format: Carmelia M. Barreto, Luisa M. Solis-Soto, Ruth Salazar, Swati Gite, Edwin R. Parra, Barbara Mino, Davis Ingram, Khalida M. Wani, Cheuk H. Leung, Heather Lin, Ignacio I. Wistuba, Alexander Lazar, Wei-Lien Wang. Characterization of immune cell biomarkers in undifferentiated pleomorphic sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3857.

Conjunctival melanoma (CM) iss a rare and aggressive tumour that is increasing in frequency. The prognostic value of PD-L1 expression, alone or in combination with CD8 and PD-1 expression and the BRAF and NRAS status, has not been determined in CM to date. We evaluated the expression of PD-L1, CD8, PD-1 in CM and investigated whether there was an association between the expression of these markers and the BRAF and NRAS molecular profile as well as some clinico-pathological criteria. A total of sixty-five CM were assessed for PD-L1, PD-1, and CD8 expression by immunohistochemistry (IHC) and for BRAF and NRAS genomic alterations using molecular biology techniques and anti-BRAF and anti-NRAS antibodies. PD-L1 expression in tumour cells (TC) was very low or absent but detected in tumour-infiltrating immune cells (IC). A correlation was observed between the expression of PD-L1, CD8, and PD-1 in IC. No correlation between PD-L1 expression (in tumour and/or immune cells) and BRAF or NRAS mutations was observed. PD-L1 expression in IC correlated with a higher pTNM stage and PD-L1 expression in TC with worse disease-specific survival. PD-L1 expression is a potential prognostic biomarker that correlates with poor prognosis in CM patients.

Dedifferentiated chondrosarcoma (DCS) is a biphasic tumor, comprising a low-grade chondrosarcoma juxtaposed to an anaplastic sarcoma with a high degree of malignancy. DCS of the spine arising from osteochondromas is extremely rare. We report a very unusual case of a 28-year-old female with DCS originating from a cervical spine osteochondroma. At the age of 12 the patient was diagnosed as having multiple osteochondromas. At the age of 28, she consulted our clinic complaining of rapid increase of the mass and dull pain. Radiological examination revealed a 12 cm × 8 cm sized mass with irregular calcification and cauliflower-like surface, arising from the second to the sixth cervical spines. The tumor was removed with the suspicion of secondary chondrosarcoma arising from the predisposing osteochondroma of the spine. Histological examination revealed the tumor was composed of a biphasic pattern, one with well differentiated chondrosarcoma and the other with high grade spindle cell sarcoma (malignant fibrous histiocytoma). The tumor was diagnosed as DCS in the spine. The patient died of pulmonary metastasis at 6 years after surgery.

Neoplastic PD-L1 (nPD-L1, clone SP142) expression remains unclear in cutaneous T-cell lymphoma (CTCL), although it is well-documented in classic Hodgkin lymphoma (CHL). Here, we report two cases of primary cutaneous large T-cell lymphoma (PCLTCL) with CD30 expression that developed secondary nodal lesions morphologically mimicking CHL, and describe their PD-L1 expression. Our two cases (52- and 60-year-old males) had long-standing clinical courses of CTCL. Their PCLTCL with CD30 expression developed nodal lesions, having a nodular growth pattern containing scattered CD30+ Hodgkin and Reed-Sternberg-like and/or lacunar cells that expressed CD15 but did not harbor Epstein-Barr virus. Their differential diagnosis from CHL was challenging. A diagnosis of PCLTCL with secondary nodal involvement featuring CHL mimicry was based on comparison of the primary and secondary lesions. In one case, shared expression of the same T-cell antigen was revealed by immunohistochemistry, and in the other, identical clonal TCR rearrangement was demonstrated by polymerase chain reaction (PCR). Interestingly, nPD-L1 was expressed on more than 50% of the tumor cells in the secondary nodal lesions, but on very few in the primary cutaneous lesions, in both cases. This is the first report of nPD-L1 expression greatly increasing with PCLTCL tumor progression to nodal involvement.