VISTA in Soft Tissue Sarcomas: A Perspective for Immunotherapy?

(1) Background: V domain immunoglobulin suppressor of T cell activation (VISTA) plays a critical role in antitumor immunity and may be a valuable target in cancer immunotherapy. To date, it has never been studied in a large and well-characterised cohort of soft tissue sarcomas (STS). (2) Methods: Using immunohistochemistry, we examined VISTA expression in tumour tissues of 213 high-risk STS. We then analysed whether VISTA was associated with other clinicopathological parameters, including tumour-infiltrating lymphocyte (TIL) counts, programmed death receptor-1 (PD1), programmed death ligand-1 (PDL1), CD3, grading, and long-term survival. (3) Results: We observed VISTA expression in 96 (45%) of 213 specimens with distinct patterns ranging from 26 to 63% for histological subtypes. VISTA was associated with higher grade (G3 vs. G2, p = 0.019), higher TIL counts (p = 0.033), expression of PD1 (p = 0.046), PDL1 (p = 0.031), and CD3+ (p = 0.023). In patients without CD3+ TILs, 10-year survival was higher when VISTA was expressed compared to when there was no VISTA expression (p = 0.013). In a multivariate analysis, VISTA expression was independently associated with prolonged survival (p = 0.043). (4) Conclusions: VISTA is expressed in different STS subtypes and is associated with increased TILs, PD-1, PD-L1, and CD3 expression. Patients with VISTA+ tumours show improved survival. These results may help define future immunotherapeutic approaches in STS.

Cancer Testis Antigens and Immunotherapy: Expression of PRAME Is Associated with Prognosis in Soft Tissue Sarcoma.

(1) Background: PRAME, NY-ESO-1, and SSX2 are cancer testis antigens (CTAs), which are expressed in testicular germ cells with re-expression in numerous cancer types. Their ability to elicit humoral and cellular immune responses have rendered them promising targets for cancer immunotherapy, but they have never been studied in a large and well-characterised cohort of soft tissue sarcomas (STS). (2) Methods: On a protein level, we examined PRAME, NY-ESO-1, and SSX2 expression in tumour tissues of 249 high-risk STS using immunohistochemistry. We correlated expression levels with clinicopathological parameters including tumour-infiltrating lymphocyte (TIL) counts, grading, and long-term survival. (3) Results: Expression of PRAME, NY-ESO-1, and SSX2 was observed in 25 (10%), 19 (8%), and 11 (4%) of 249 specimens with distinct patterns for histo-subtypes. Expression of PRAME was associated with shorter patient survival (p = 0.005) and higher grade (G2 vs. G3, p = 0.001), while NY-ESO-1 expression was correlated with more favourable survival (p = 0.037) and lower grade (G2 vs. G3, p = 0.029). Both PRAME and NY-ESO-1 expression were more frequent in STS with low TIL counts. In multivariate analysis, high PRAME and low SSX2 expression levels as well as metastatic disease and non-radical resections were independent predictors of shorter overall survival. (4) Conclusions: CTAs PRAME, NY-ESO-1, and SSX2 show distinct expression patterns in different STS subtypes. These results demonstrate their prognostic relevance and may guide future immunotherapeutic approaches in STS.

Structured reporting has the potential to reduce reporting times of dual-energy x-ray absorptiometry exams.

BACKGROUND: In recent years, structured reporting has been shown to be beneficial with regard to report completeness and clinical decision-making as compared to free-text reports (FTR). However, the impact of structured reporting on reporting efficiency has not been thoroughly evaluted yet. The aim of this study was to compare reporting times and report quality of structured reports (SR) to conventional free-text reports of dual-energy x-ray absorptiometry exams (DXA). METHODS: FTRs and SRs of DXA were retrospectively generated by 2 radiology residents and 2 final-year medical students. Time was measured from the first view of the exam until the report was saved. A random sample of DXA reports was selected and sent to 2 referring physicians for further evaluation of report quality. RESULTS: A total of 104 DXA reports (both FTRs and SRs) were generated and 48 randomly selected reports were evaluated by referring physicians. Reporting times were shorter for SRs in both radiology residents and medical students with median reporting times of 2.7 min (residents: 2.7, medical students: 2.7) for SRs and 6.1 min (residents: 5.0, medical students: 7.5) for FTRs. Information extraction was perceived to be significantly easier from SRs vs FTRs (P <  0.001). SRs were rated to answer the clinical question significantly better than FTRs (P <  0.007). Overall report quality was rated significantly higher for SRs compared to FTRs (P <  0.001) with 96% of SRs vs 79% of FTRs receiving high or very high-quality ratings. All readers except for one resident preferred structured reporting over free-text reporting and both referring clinicians preferred SRs over FTRs for DXA. CONCLUSIONS: Template-based structured reporting of DXA might lead to shorter reporting times and increased report quality.

Ifosfamide, Carboplatin, and Etoposide (ICE) in Combination with Regional Hyperthermia as Salvage Therapy in Patients with Locally Advanced Nonmetastatic and Metastatic Soft-Tissue Sarcoma.

Patients with localized relapse of soft-tissue sarcoma (STS) after anthracycline-based chemotherapy have a dismal prognosis, particularly when surgery is not possible. To facilitate resection and improve long-term tumor control, we applied an intensified perioperative treatment consisting of ICE (ifosfamide 6 g/m2, carboplatin 400 mg/m2, and etoposide 600 mg/m2) in combination with regional hyperthermia (RHT) to maximize local control. Here, we retrospectively evaluate the safety and efficacy of this strategy. Patients aged ≥18 years with locally advanced high-risk STS, either with or without metastasis, treated with ICE + RHT after the failure of first-line anthracycline-based chemotherapy were included in this analysis. Radiographic response, toxicity, progression-free survival (PFS), and overall survival (OS) were assessed. Between 1996 and 2018, 213 sarcoma patients received ICE at our centre. Of these, 110 patients met the selection criteria (progressive disease, suitable high-grade STS histology, anthracycline pretreatment, RHT treatment) for this analysis. Fifty-four patients had locally advanced disease without metastases (LA-STS), and 56 patients had additional metastatic disease (M-STS). Disease control was achieved in 59% of LA-STS patients and in 47% of M-STS patients. For LA-STS, 21% of the patients achieved radiographic response, facilitating resection in 4 patients (7%), compared with 11% of the M-STS patients, facilitating resection in 5 patients (9%). PFS was significantly longer in LA-STS than in M-STS (10 vs. 4 months, p < 0.0001). Median OS was 26 months in LA-STS and 12 months in M-STS. Disease control was the only independent prognostic factor for improved OS in multivariate analysis. Toxicity was high with neutropenic fever occurring in 25% of the patients and three therapy-related deaths (3%). ICE + RHT demonstrated activity in high-risk STS and facilitated resection in selected patients after anthracycline failure. Disease control was associated with improved OS. Based on the observed toxicities, the dose should be reduced to 75%.

A comparative view on the expression patterns of PD-L1 and PD-1 in soft tissue sarcomas.

Soft tissue sarcomas (STSs) are heterogeneous cancers associated with poor prognosis due to high rates of local recurrence and metastasis. The programmed death receptor ligand 1 (PD-L1) is expressed in several cancers. PD-L1 interacts with its receptor, PD-1, on the surface of tumor-infiltrating lymphocytes (TILs), thereby attenuating anti-cancer immune response. Immune checkpoint inhibitors targeting this interaction have been established as effective anti-cancer drugs. However, studies on the PD-L1 and PD-1 expression status in STS are commonly limited by small sample size, analysis of single STS subtypes, or lack of combinatorial marker assessment. To overcome these limitations, we evaluated the expression patterns of intratumoral PD-L1, the number of TILs, their PD-1 expression, and associations with clinicopathological parameters in a large and comprehensive cohort of 225 samples comprising six STS subtypes. We found that nearly all STS subtypes showed PD-L1 expression on the tumor cells, albeit with a broad range of positivity across subtypes (50% angiosarcomas to 3% synovial sarcomas). Co-expression and correlation analyses uncovered that PD-L1 expression was associated with more PD-1-positive TILs (P < 0.001), higher tumor grading (P = 0.016), and worse patients' 5-year overall survival (P = 0.028). The results were in line with several publications on single STS subtypes, especially when comparing findings for STS with low and high mutational burden. In sum, the substantial portion of PD-L1 positivity, the co-occurrence of PD-1-positive TILs, and the association of PD-L1 with unfavorable clinical outcome provide rationales for immune checkpoint inhibition in patients with PD-L1-positive STS.

Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

Hallmarks of hyperthermia in driving the future of clinical hyperthermia as targeted therapy: translation into clinical application.

Regional hyperthermia is described as a targeted therapy and the definitions of six hallmarks of hyperthermia are proposed, representing the pleiotropic effect of this therapeutic modality to counteract tumour growth and progression. We recommend the considerations of these hallmarks in the design of clinical trials involving regional hyperthermia as targeted therapy. Randomised clinical studies using loco-regional hyperthermia as an adjuvant to radiotherapy or to chemotherapy for locally advanced tumours demonstrate the benefit of the combination compared to either of the standard treatments alone for tumour response, disease control, and patient survival outcome. These impressive results were obtained from proof-of-concept trials for superficial or deep-seated malignancies in unselected patients. None of these trials was designed as tailored approaches for the treatment of specified targets or to select potentially more sensitive subpopulations of patients using eligibility criteria. Based upon clinical examples of targeted chemotherapy, some guidelines are described for the successful development of targeted therapeutic combinations. We also retrospectively analyse the stepwise process of generating an ongoing new clinical trial using hyperthermia as targeted therapy to evade DNA repair in combination with a DNA damaging anticancer agent to implement this new vision.

Neoadjuvant or adjuvant sirolimus for malignant metastatic or locally advanced perivascular epithelioid cell tumors: two case reports.

Perivascular epithelioid cell tumors (PEComas) are very rare mesenchymal tumors, characterized by the presence of perivascular epithelioid cells. Despite their often benign nature, malignant variants with a locally aggressive growth pattern and even distant metastases are known. We describe two cases of malignant PEComas. The first patient had an extensive peritoneal spread and a history of multiple resections, and received the mechanistic target of rapamycin inhibitor sirolimus in a postoperative setting as maintenance therapy. The second patient presented with locally advanced disease in the iliac fossa and was treated with sirolimus in a neoadjuvant setting and achieved complete remission. Both patients have been under treatment for 18 and 52 months, respectively, and are currently in complete remission. These two cases indicate that mechanistic target of rapamycin inhibition for malignant PEComas could be a safe and successful treatment strategy in a neoadjuvant setting with an acceptable toxicity profile.

VEGFR2 predicts decreased patients survival in soft tissue sarcomas.

AIMS: Tyrosine kinases are promising targets for personalized medicine, and new drugs are currently in phase 2 and phase 3 clinical trials. However, expression analysis of tyrosine kinases as predictive biomarkers is still not a standard approach. Furthermore, only limited studies have investigated the expression of tyrosine kinase receptors on the protein level. In this study, we analysed a well-characterised group of soft tissue sarcomas for different tyrosine kinase receptors and correlated the results with clinicopathological parameters, including survival. METHODS: 275 soft tissue sarcomas of our Sarcoma center at the Ludwig-Maximilians-University (LMU) were reinvestigated and categorized according to the current WHO classification system. The tumor collective included undifferentiated pleomorphic sarcomas (n=81), leiomyosarcomas (n=50), synovial sarcomas (n=27), liposarcomas (n=51), angiosarcomas (n=43) and other soft tissue sarcomas (n=23). RESULTS: On protein levels, high expression of VEGFR1 was detected immunohistochemically in 61%, VEGFR2 (KDR) in 11%, VEGFR3 in 64%, PDGFRA in 42% and PDGFRB in 73%. High expression of VEGFR1-3 and PDGFRB was significantly correlated with higher grading (G2 vs G3, p<0.05), and high VEGFR2 was significantly correlated with decreased patients' survival (p<0.001). CONCLUSIONS: Tyrosine kinase receptors showed a distinct expression pattern in soft tissue sarcomas. High expression of VEGFR2 (KDR) is significantly associated with decreased patients' survival. High VEGFR 1-3 and PDGFRB are significantly correlated with higher tumor grading. Protein signatures might be evaluated before targeted therapy to give a rationale for an eligible personalized therapy.

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis.

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27-mediated through both the intrinsic and the extrinsic apoptotic pathways-at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.

High IGF2 and FGFR3 are associated with tumour progression in undifferentiated pleomorphic sarcomas, but EGFR and FGFR3 mutations are a rare event.

AIM: Pleomorphic undifferentiated sarcomas (formerly known as malignant fibrous histiocytomas) are recognised by the actual WHO classification as an undifferentiated, unclassifiable category of pleomorphic sarcomas which show no definable line of differentiation and are still a diagnosis of exclusion. Therefore, diagnostic, prognostic and therapeutic options of these tumours are urgently needed. METHODS: Three hundred and twenty-seven spindle cell tumours of a German consultation and reference centre of soft tissue tumours consisting of 200 undifferentiated pleomorphic sarcomas (UPS), 45 low-grade sarcomas (10 low-grade fibromyxoid sarcomas, 32 low-grade myofibroblastic sarcomas and three myxoinflammatory fibroblastic sarcomas) and 82 tumours of the fasciitis family were revisited. The specimens were analysed immunohistochemically with distinct markers including tyrosine kinases and expression correlated with clinicopathological parameters. Additionally, mutational analysis was performed on specimens with high expression of EGFR and FGFR3. RESULTS: At the protein level high IGF2 expression was observed in 86 %, FGFR3 (69 %), PDGFRA (62 %), PDGFRB (39 %), FGFR1 (8 %), EGFR (5 %), KDR/VEGFR2 (3 %), ALK (0 %) and high Ki67 (63 %) in UPS. High expressions of IGF2 and FGFR3 are significantly correlated with a higher grading (p = 0.023 and p = 0.016, respectively) and a high Ki67 index (p = 0.017 and p = 0.001, respectively). No mutations were found in the hot spots of tumour specimens with a high expression of EGFR gene (exons 18-21) and FGFR3 gene (exons 7, 10 and 15). CONCLUSIONS: High expressions of IGF2 and FGFR3 are significantly associated with tumour progression, grading and Ki67 and might classify a subgroup of undifferentiated pleomorphic sarcoma. These markers might guide targeted therapies in these neoplasms.

Loss of p16(INK4a) is associated with reduced patient survival in soft tissue tumours, and indicates a senescence barrier.

AIMS: p16(INK4a) is an important factor in carcinogenesis, and its expression is linked to oncogene-induced senescence. Very recently it was shown that upregulation and downregulation of p16 indicates a senescence barrier in the serrated route of colorectal cancer. However, in soft tissue sarcoma (STS), the senescence mechanism is still not understood. In this study, we analysed a well characterised cohort of STS for p16(INK4a) expression and correlated the results with clinicopathological parameters including survival. METHODS: Tissue microarrays (TMA) of 183 soft tissue and bone tumours were analysed immunohistochemically. Furthermore, mRNA expression of p16(INK4a) was evaluated in four sarcoma cell lines, and a demethylation test was performed by treatment with 5-aza-2'-deoxycytide. RESULTS: On protein level, expression of p16(INK4a) was observed in undifferentiated pleomorphic sarcoma (UPS) in 69.1%, leiomyosarcoma in 85.7%, synovial sarcoma in 77.8%, liposarcoma in 88.9%, angiosarcoma in 60.9% and MPNST in 22.2%. Loss of p16(INK4a) was observed in high grade sarcomas and showed a significant correlation with reduced patient survival (p=0.032). On DNA level, one out of four sarcoma cell lines exhibited a methylated p16(INK4a) promoter analysed by methylation-specific PCR. p16(INK4a) mRNA and protein expression was restored after demethylation using 5-aza-2'-deoxycytide. CONCLUSIONS: Upregulation of p16(INK4a) might be associated with the induction of senescence and indicates a senescence barrier. Downregulation of p16(INK4a) is found in malignant progression, and is significantly correlated with reduced patient survival. Downregulation of p16(INK4a) may be explained by DNA-hypermethylation in sarcoma cells.

RAR/RXR and PPAR/RXR signaling in neurological and psychiatric diseases.

Retinoids are important signals in brain development. They regulate gene transcription by binding to retinoic acid receptors (RAR) and, as was discovered recently, a peroxisome proliferator-activated receptor (PPAR). Traditional ligands of PPAR are best known for their functions in lipid metabolism and inflammation. RAR and PPAR are ligand-activated transcription factors, which share members of the retinoid X receptor (RXR) family as heterodimeric partners. Both signal transduction pathways have recently been implicated in the progression of neurodegenerative and psychiatric diseases. Since inflammatory processes contribute to various neurodegenerative diseases, the anti-inflammatory activity of retinoids and PPARgamma agonists recommends them as potential therapeutic targets. In addition, genetic linkage studies, transgenic mouse models and experiments with vitamin A deprivation provide evidence that retinoic acid signaling is directly involved in physiology and pathology of motoneurons, of the basal ganglia and of cognitive functions. The activation of PPAR/RXR and RAR/RXR transcription factors has therefore been proposed as a therapeutic strategy in disorders of the central nervous system.

Anti-inflammatory effect of retinoic acid on prostaglandin synthesis in cultured cortical astrocytes.

Prostanoids are important mediators of inflammation and pain signaling. Although it is now well accepted that astrocytes participate in inflammatory reactions in the CNS, the molecular regulation of this activity is still largely unknown. Specifically, the regulation of prostanoid synthesis by this type of glia remains to be resolved. Recent evidence suggests that the transcriptional regulator retinoic acid (RA) is involved in regulation of the immune response. We have investigated the expression pattern of the enzymes that catalyze prostanoid and leukotriene synthesis in cultured cortical astrocytes, their stimulation by lipopolysaccharides (LPS) and their regulation by RA. The data indicate that astrocytes are an important source of prostaglandins (PGs) and that RA reduces their inflammatory biosynthesis. LPS treatment induced the expression of enzymes for the production of arachidonic acid and PGs but caused down-regulation of a PG degrading enzyme and of leukotriene synthesizing enzymes that compete with PG synthesis. Consequently, the secretion of the PGE(2) was highly increased after LPS exposure. RA counteracted the inflammatory regulation of cyclooxygenase (COX)-2 mRNA and protein in astrocytes and thereby reduced the synthesis of PGE(2) by approximately 60%. In the absence of LPS, RA enhanced the expression of COX-1 mRNA. In conclusion, RA might be effective in suppressing inflammatory processes in the brain by inhibiting PG synthesis.

Expression of enzymes involved in the prostanoid metabolism by cortical astrocytes after LPS-induced inflammation.

Neuroinflammatory processes are a common epiphenomenon for a number of neurological and neurodegenerative diseases. Besides microglia, astrocytes are implicated in brain inflammation in response to harmful stimuli and pathological processes. Bacterial endotoxins can induce the synthesis and release of proinflammatory mediators, i.e., cytokines and chemokines, by astroglia. In this study, we have investigated the effect of lipopolysaccharide (LPS) treatment on the expression of enzymes of prostanoid synthesis and degradation in cultured mouse cortical astrocytes using an Affymetrix Gene Chip array, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and an enzyme-immunosorbent assay. LPS treatment induced an upregulation of enzymes responsible for prostaglandin E2 synthesis, a downregulation of enzymes that catalyzes prostaglandin E2 (PGE2) degradation and production of proinflammatory leukotrienes. Changes in enzyme expression were accompanied by a highly significant increase in extracellular PGE2. Our data demonstrate that astrocytes are directly involved in the complex regulation of proinflammatory prostanoids in the CNS under pathological processes, thus being of potential interest as targets for therapeutical interventions. Further studies are required to unravel the different roles and interactions between astroglia and other cells of the brain-intrinsic innate immune system during inflammation.

Retinoic acid enhances Erk phosphorylation in the chick retina.

The transcriptional activator retinoic acid (RA) is a regulator of neural development and regeneration. Synergistic effects with brain-derived neurotrophic factor suggested that RA influences neurotrophin signaling. To test this hypothesis RA was administered systemically to E17 chick embryos, and retinas were prepared 12h and 24h later to measure mRNA or protein expression. While there was no significant influence on activation of Akt, CREB and STAT-3, RA-treatment caused elevated levels of Erk-phosphorylation, a kinase involved in Trk signaling. A small but significant increase in the expression of TrkB mRNA and protein was observed but no significant change in TrkA, TrkC and p75 expression.