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A digestibility enhancing effect of natural food on stomachless fish model (Cyprinus carpio) was verified by fluorogenic substrate assays of enzymatic activities in experimental pond carp gut flush and planktonic food over a full vegetative season. Then compared with size-matched conspecific grown artificially (tank carp) and an advanced omnivore species possessing true stomach (tilapia, Oreochromis niloticus). Results suggested activities of digestive enzymes (except amylolytic) were significantly higher in pond carp (p ≤ 0.05) than in the size-matched tank carp. Even compared to tilapia, pond carp appeared superior (p < 0.05; proteolytic or chitinolytic activities) or comparable (p > 0.05; phosphatase or cellulolytic activities). Amylolytic, chitinolytic, and phosphatases activities in pond carp gut significantly increased (p ≤ 0.01) over season. Several orders-of-magnitude higher enzymatic activities were detected in planktonic natural food than expressed in carp gut. Amino acid markers in planktonic food revealed a higher share of zooplankton (microcrustaceans), but not phytoplankton, synchronized with higher activities of complex polysaccharide-splitting enzymes (cellulolytic and chitinolytic) in fish gut. Periods of clear water phase low in chlorophyll-a and nutrients, but high in certain zooplankton (preferably cladocerans), may create a synergistic digestibility effect in pond carp. We conclude aquatic ecosystem components (natural food, water, microbiota) enhance fishes' hydrolyzing capabilities of C/N/P macromolecules and even their complex polymers such as cellulose, chitin, and maybe phytate (to be validated), to the extent that being stomachless is not an issue. Aquatic nutritional ecologists may consider that laboratory-based understandings of digestibility may underestimate digestion efficiency of free-ranging fish in ponds or lakes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8 + effector T (T EFF ) cells and TIM3 + PD1 + , hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8 + T cells. Conversely, CD8 + T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1 + PD1 + T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8 + T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1 + PD1 + CD8 + T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC., (© 2024. The Author(s).)

Purpose: The CD47 molecule, often referred to as the "do not eat me" signal, is frequently overexpressed in tumor cells. This signaling pathway limits phagocytosis by macrophages. Our objective was to determine CD47 abundance in various soft tissue sarcomas (STS) to investigate whether it could serve as a potential evasion mechanism for tumor cells. Additionally, we aimed to assess the prognostic value of CD47 expression by examining its association with different clinicopathological factors. This study aimed to elucidate the significance of CD47 in the context of emerging anti-tumor targeting approaches., Methods: In this retrospective study, formalin-fixed paraffine-embedded (FFPE) tumor tissues of 55 treatment-naïve patients were evaluated by immunohistochemistry for the abundance of CD47 molecule on tumor cells. The categorization of CD47 positivity was as follows: 0 (no staining of tumor cells), 1 + (less than 1/3 of tumor area positive), 2 + (between 1/3 and 2/3 of tumor area positive), and 3 + (more than 2/3 of tumor area positive for CD47). Next, we compared CD47 abundance between different tumor grades (G1-3). We used Kaplan-Meier survival curves with log-rank test to analyze the differences in survival between patients with different CD47 expression. Moreover, we performed Cox proportional hazards regression model to evaluate the clinical significance of CD47., Results: CD47 is widely prevalent across distinct STS subtypes. More than 80% of high grade undifferentiated pleiomorphic sarcoma (UPS), 70% of myxofibrosarcoma (MFS) and more than 60% of liposarcoma (LPS) samples displayed a pattern of moderate-to-diffuse positivity. This phenomenon remains consistent regardless of the tumor grade. However, there was a tendency for higher CD47 expression levels in the G3 group compared to the combined G1 + G2 groups when all LPS, MFS, and UPS were analyzed together. No significant associations were observed between CD47 abundance, death, and metastatic status. Additionally, high CD47 expression was associated with a statistically significant increase in progression-free survival in the studied cohort of patients., Conclusion: This study highlights the potential of the CD47 molecule as a promising immunotherapeutic target in STS, particularly given its elevated expression levels in diverse sarcoma types. Our data showed a notable trend linking CD47 expression to tumor grade, while also suggesting an interesting correlation between enhanced abundance of CD47 expression and a reduced hazard risk of disease progression. Although these findings shed light on different roles of CD47 in STS, further research is crucial to assess its potential in clinical settings., (© 2024. The Author(s).)

Purpose: The treatment options for metastatic soft tissue sarcomas (STSs) are limited. In most cases, immunotherapy with immune checkpoint inhibitors has not been successful so far. Macrophages dominate the immune landscape of STSs; thus, combinatorial strategies aiming at both tumor-infiltrating lymphocytes and macrophages may represent a particularly relevant treatment approach for metastatic or recurrent STSs., Methods: In this cohort study, 66 patients who underwent surgery for STSs were enrolled. Tumor cells and tumor-infiltrating immune cells were analyzed using flow cytometry and immunohistochemistry. In cell suspensions obtained from surgical resections, human T cells were activated by superparamagnetic polymer beads and cultured at a concentration of 0.3 × 10 6 /µl in the absence or presence of therapeutic monoclonal antibodies (anti-PD-1, anti-CD47, and anti-PD-1 + anti-CD47). Supernatants from cell suspensions were analyzed using multiplex Luminex cytokine bead-based immunoassays., Results: The most profound response to anti-CD47 therapy was observed in an undifferentiated pleiomorphic sarcoma which also displayed high expression of CD47 in the tumor microenvironment. Both anti-PD-1 and anti-CD47 therapies drastically increased the production of pro-inflammatory cytokines in the tumor microenvironment of STSs, but co-administration of both agents did not further increase cytokine secretion. Furthermore, all patient samples treated with a combination of both anti-PD-1 and anti-CD47 antibodies showed a dramatic reduction in cytokine secretion., Conclusion: Our findings suggest that anti-PD-1 and anti-CD47 therapies do not enhance each other, and the combined application of anti-PD-1 and anti-CD47 agents in vitro limits rather than potentiates their efficacy., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Salivary gland carcinomas (SGC) are extremely rare malignancies with only limited treatment options for the metastatic phase of the disease. Treatment with anti-CD47 antibodies could represent a potent therapy for SGCs by promoting the phagocytic clearance of tumor cells through various mechanisms. However, the efficacy of anti-CD47 therapy is largely dependent on the expression of CD47 within the tumor microenvironment (TME)., Materials and Methods: In 43 patients with SGC, we were the first to investigate the CD47 expression in both tumor cells and tumor-infiltrating immune cells (TIIC) in the center and periphery of primary tumors. We also correlated the data with the clinicopathological variables of the patients and offered novel insights into the potential effectiveness of anti-CD47 therapy in SGCs., Results: We observed that the CD47 + tumor cells are outnumbered by CD47 + TIICs in mucoepidermoid carcinoma. In the tumor center, the proportion of CD47 + tumor cells was comparable to the proportion of CD47 + TIICs in most histological subtypes. In low-grade tumors, significantly higher expression of CD47 was observed in TIICs in the periphery of the tumor as compared to the center of the tumor., Conclusion: The reason for a high expression of 'don't eat me' signals in TIICs in the tumor periphery is unclear. However, we hypothesize that in the tumor periphery, upregulation of CD47 in TIICs could be a mechanism to protect newly recruited leukocytes from macrophage-mediated phagocytosis, while also allowing the removal of old or exhausted leukocytes in the tumor center., (© 2022. The Author(s).)

Objectives: With the increasing number of detected lung nodules and the need for morphological verification, the number of CT- controlled biopsies is increasing. The aim of this study was to assess the risks and benefits of these biopsies., Methods: This is a prospective and observational study. We evaluated 101 punctures performed on a group of 90 consecutive patients in the Department of Radiology., Results: In patients with a mean age of 66 years, with mostly accidentally detected lung nodules, we observed complications 38 times. The most common were minor pneumothoraxes or insignificant bleedings. In 6 patients, the complications were more serious, 5 times the pneumothoraxes required chest drainage, once massive hemoptysis was recorded. The lesions were successfully biopsied 78 times, the target was missed 23 times. The diagnosis of lung cancer (LC) was confirmed in 60 patients, 49 LCs were verified by puncture under CT control. 42% (25/60) of patients with LC were diagnosed in TNM stages I and II. 23% (14/60) of patients with LC were treated surgically. The remaining 30 patients most often suffered from lung metastazes (13/30), in 8 of them an inflammatory lung disease was diagnosed. 69 patients underwent bronchoscopy, in only 19% (13/69) it contributed to the diagnosis. In a model "screening like" group of 49 patients with only randomly detected lung deposits, we diagnosed LC in 76% (37/49). 49% (18/37) were in TNM stage I and II, 11 were treated surgically., Conclusions: CT-controlled biopsy of lung lesions is an effective and safe diagnostic method.

Salivary gland carcinomas (SGCs) are extremely morphologically heterogeneous, and treatment options for this disease are limited. Immunotherapy with immune checkpoint inhibitors (ICIs) represents a revolutionary treatment approach. However, SGCs remain largely resistant to this therapy. An increasing body of evidence suggests that resistance to ICI therapy is modulated by the Fas (CD95)-Fas ligand (FasL, CD178) interplay between tumor cells and immune cells. In this study, we examined the Fas-FasL interplay between tumor cells and tumor-infiltrating immune cells (TIICs) in the center and periphery of SGCs from 62 patients. We found that the Fas-expressing tumor cells accumulated in the center of SGC tumors with increasing tumor stage. Furthermore, this accumulation occurred regardless of the presence of TIICs expressing high levels of FasL. On the contrary, a loss of Fas-expressing TIICs with increasing tumor stage was found in the tumor periphery, whereas FasL expression in tumor cells in the tumor periphery correlated with tumor stage. These data suggest that SGC cells are resistant to FasL-induced apoptosis by TIICs but could utilize FasL to eliminate these cells in high-stage tumors to provide resistance to immunotherapy.

The treatment options for patients with advanced salivary gland cancers (SGCs) are limited. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. However, the response to ICI immunotherapy is largely driven by the immune cell signatures within the tumor tissue and the para-tumoral tissue compartments. To date, there are no data on the expression of programed cell death protein-1/programed cell death protein-ligand 1 (PD-1/PD-L1) in SGC, which may enable the implementation of ICI immunotherapy for this disease. Thus, we performed an immunohistochemical analysis of PD-1 and PD-L1 expression in tumor cells and tumor-infiltrating immune cells (TIICs) in the tumor center and periphery of 62 SGC patients. The tumor periphery showed significantly higher expression of PD-L1 in tumor cells than in TIICs. Moreover, peripheral TIICs had significantly higher PD-1 expression than peripheral tumor cells. PD-1-positive tumor cells were detected exclusively in the tumor center of high-grade tumors, and most importantly, the presence of lymph node (LN) metastases and primary tumor stage significantly correlated with the presence of PD-L1-positive tumor cells in the tumor periphery. The PD-1/PD-L1 molecular signatures in SGC are clustered predominantly in the tumor periphery, reflect disease severity, and may predict the response to ICI immunotherapy in SGC patients.

Background: We have performed a head to head comparison of all-oral triplet combination of ixazomib, lenalidomide and dexamethasone (IRD) versus lenalidomide and dexamethasone (RD) in patients with relapsed and refractory multiple myeloma (RRMM) in the routine clinical practice., Methods: A total of 344 patients treated with IRD (N = 127) or RD (N = 217) were selected for analysis from the Czech Registry of Monoclonal Gammopathies (RMG). Descriptive statistics were used to assess patient's characteristics associated with the respective therapy. The primary endpoint was progression free survival (PFS), secondary end points included response rates and overall survival (OS). Survival endpoints were plotted using Kaplan-Meier methodology at 95% Greenwood confidence interval. Univariable and multivariable Cox proportional hazards models were used to evaluate the effect of treatment regimens and the significance of uneven variables. Statistical tests were performed at significance level 0.05., Results: In the whole cohort, median PFS for IRD was 17.5 and for RD was 11.5 months favoring the all-oral triplet, p = 0.005; in patients within relapse 1-3, the median PFS was 23.1 vs 11.6 months, p = 0.001. The hazard ratio for PFS was 0.67 (95% confidence interval [CI] 0.51-0.89, p = 0.006). The PFS advantage translated into improved OS for patients treated with IRD, median 36.6 months vs 26.0 months (p = 0.008). The overall response rate (ORR) was 73.0% in the IRD group vs 66.2% in the RD group with a complete response rate (CR) of 11.1% vs 8.8%, and very good partial response (VGPR) 22.2% vs 13.9%, IRD vs RD respectively. The IRD regimen was most beneficial in patients ≤75 years with ISS I, II, and in the first and second relapse. Patients with the presence of extramedullary disease did not benefit from IRD treatment (median PFS 6.5 months). Both regimens were well tolerated, and the incidence of total as well as grade 3/4 toxicities was comparable., Conclusions: Our analysis confirms the results of the TOURMALINE-MM1 study and shows benefit of all-oral triplet IRD treatment versus RD doublet. It demonstrates that the addition of ixazomib to RD improves key survival endpoints in patients with RRMM in a routine clinical setting.

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

Background: The Memorial Sloan-Kettering Cancer Center (MSKCC) prognostic model has been widely used for the prediction of the outcome of metastatic renal cell carcinoma (mRCC) patients treated with systemic therapies, however, data from large studies are limited. This study aimed at the evaluation of the impact of the MSKCC score on the outcomes in mRCC patients treated with first-line sunitinib, with a focus on the intermediate-risk group., Methods: Clinical data from 2390 mRCC patients were analysed retrospectively. Progression-free survival (PFS), overall survival (OS), and objective response rate (ORR) were analysed according to the MSKCC risk score., Results: ORR, median PFS, and OS for patients with one risk factor were 26.7%, 10.1, and 28.2 months versus 18.7%, 6.2, and 16.2 months, respectively, for those with two risk factors (ORR: p = 0.001, PFS: p < 0.001, OS: p < 0.001). ORR, median PFS, and OS were 33.0%, 17.0, and 44.7 months versus 24.1%, 9.0, and 24.1 months versus 13.4%, 4.5, and 9.5 months in the favourable-, intermediate-, and poor-risk groups, respectively (ORR: p < 0.001, PFS: p < 0.001, OS: p < 0.001)., Conclusions: The results of the present retrospective study demonstrate the suitability of the MSKCC model in mRCC patients treated with first-line sunitinib and suggest different outcomes between patients with one or two risk factors.

Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: To determine the exact incidence of lung cancer, pulmonary emphysema and pleural effusion we decided to carry out an autopsy study., Methods: In this autopsy study carried out over two years, we compared the results of autopsy findings with the clinical data in accompanying records of the deceased., Results: Among the 708 deceased subjects, there were 398 males and 310 females with a median age of 71 years. At autopsy, 55 cases of lung carcinoma (BCA) were found, of which 24 have not been identified during life (44%). Among the deceased with BCA, emphysema was also observed at autopsy in 40% of the cases. Pulmonary emphysema was described macroscopically in 28% of the full set of 708 deceased, whereas the accompanying records of the deceased described this condition in only 12% of the cases. Microscopic changes compatible with emphysema were identified in 54% of the examined lungs. Pleural effusions were described in the accompanying records of 13% of the deceased, while the autopsies showed this condition in 33% of the deceased. BCA was accompanied by effusion in 25% of the cases., Conclusions: The obtained results show that the studied conditions are present in more cases than are reported by clinicians. The study confirms the commonly accepted association between lung cancer and emphysema.

Background: Lung carcinogenesis is a multistep process of accumulation of genetic changes, including loss of heterozygosity (LOH), and precedes phenotypic transformation of the bronchial mucosa. The activity of telomerase, correlating with the hTERT mRNA expression, is detectable in a majority of neoplasms. In this study, the frequency of LOH and hTERT expression in bronchial mucosa of heavy smokers in bronchoscopic biopsies was analyzed., Methods: LOH was examined in 122 bronchial specimens from 81 smokers (67 normal mucosa/bronchitis, 12 squamous metaplasia, 28 dysplasia, 15 bronchogenic carcinoma specimens) by polymerase chain reaction (PCR) and capillary electrophoresis by using 7 fluorescence-labeled markers matching 5 chromosomal regions. hTERT expression was analyzed in 87 specimens (45 normal mucosa/bronchitis, 12 squamous metaplasia, 18 dysplasia, 12 bronchogenic carcinoma specimens) by real-time quantitative reverse-transcription PCR., Results: LOH was detected in at least 1 chromosomal region in 51 of 122 (41.8%) specimens; the incidence in normal bronchial mucosa and preneoplastic lesions was similar (20%-40%); a substantial rise (87%) occurred in carcinomas. The median normalized hTERT(N) values were 6.67 in normal epithelium/chronic bronchitis, 18.38 in squamous metaplasia, 13.31 in epithelial dysplasia, and 75.46 in carcinomas. These results were significantly different (P=.0036). With an increasing number of LOH, the median value of hTERT(N) expression rose, but hTERT was expressed also in tissue samples without any LOH detection., Conclusions: Results indicated that hTERT expression, together with LOH, represent early events in lung carcinogenesis, as both were detected in precancerous lesions and in normal epithelium of heavy smokers.