Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Rare multi-fungal sepsis: a case of triple-impact immunoparalysis.

Patients with burn injury and inhalation injury are highly susceptible to infectious complications, including opportunistic pathogens, due to the loss of skin cover and mucosal damage of respiratory tract as well as the disruption of homeostasis. This case report, a 34-year-old man suffered critical burns, provides the first literature description of triple-impact immunoparalysis (critical burns, inhalation injury, and SARS-CoV-2 infection), leading to a lethal multifocal infection caused by several fungi including very rare environmental representatives Metschnikowia pulcherrima and Wickerhamomyces anomalus. The co-infection by these common environmental yeasts in a human is unique and has not yet been described in the literature. Importantly, our patient developed refractory septic shock and died despite targeted antifungal therapy including the most potent current antifungal agent-isavuconazole. It can be assumed that besides immunoparalysis, effectiveness of therapy by isavuconazole was impaired by the large distribution volume in this case. As this is a common situation in intensive care patients, routine monitoring of plasmatic concentration of isavuconazole can be helpful in personalization of the treatment and dose optimization. Whatmore, many fungal species often remain underdiagnosed during infectious complications, which could be prevented by implementation of new methods, such as next-generation sequencing, into clinical practice.

Tazemetostat in the therapy of pediatric INI1-negative malignant rhabdoid tumors.

Rhabdoid tumors are aggressive tumors that may arise in the kidney, soft tissue, central nervous system, or other organs. They are defined by SMARCB1 (INI1) or SMARCA4 alterations. Often, very young children are affected, and the prognosis is dismal. Four patients with primary atypical teratoid rhabdoid tumor (AT/RT, a rhabdoid tumor in the central nervous system) were treated by resection and high dose chemotherapy. Tazemetostat was introduced after completion of chemotherapy. Three patients have achieved an event free survival of 32, 34, and 30 months respectively. One progressed and died. His overall survival was 20 months. One patient was treated for a relapsed atypical teratoid rhabdoid tumor. The treatment combined metronomic therapy, radiotherapy, tazemetostat and immunotherapy. This patient died of disease progression, with an overall survival of 37 months. One patient was treated for a rhabdoid tumor of the ovary. Tazemetostat was given as maintenance after resection, chemotherapy, and radiotherapy, concomitantly with immunotherapy. Her event free survival is 44 months. Only approximately 40% of patients with rhabdoid tumors achieve long-term survival. Nearly all relapses occur within two years from diagnosis. The event free survival of four of the six patients in our cohort has exceeded this timepoint. Tazemetostat has been mostly tested as a single agent in the relapsed setting. We present promising results when applied as maintenance or add on in the first line treatment.

Serum concentrations of proinflammatory biomarker interleukin-6 (IL-6) as a predictor of postoperative complications after elective colorectal surgery.

BACKGROUND: The aim of this prospective study was to evaluate the role of serum IL-6 as a potential predictive biomarker of postoperative complications (POC) in elective colorectal surgery. METHOD: A total of 115 patients underwent colorectal surgery for malignancy. IL-6 was measured on the first and third postoperative days (POD1, POD3), and C-reactive protein (CRP) was measured on the POD3. POC was analysed in subgroups according to Clavien‒Dindo (CD), antibiotic (ATB) treatment, intensive care unit (ICU) and hospital length of stay. The predictive power of variables for evaluated endpoints was analysed using receiver-operating characteristic (ROC) analysis and described by area under the curve (AUC). ROC analysis was adopted for the identification of optimal cut-offs. Histological analysis was performed to verify IL-6 production by the tumour. RESULTS: Out of 115 patients who were analysed, 42% had POC. Patients with POC had significantly higher serum levels of IL-6 on POD1 (p < 0.001) and POD3 (p < 0.001). IL-6 early on POD1 as a predictor of antibiotic treatment, ICU stay and hospital stay (AUC 0.818; 0.811; 0.771) did not significantly differ from the AUC of CRP late on POD3 (0.879; 0.838, 0.752). A cut-off IL-6 value of 113 pg/ml on POD1 and 180.5 pg/ml on POD3 in severe complications (CD > 3a) resulted in 75% and 72% sensitivity, 78.6% and 99% specificity, negative predictive value 96.4% and 97% and positive predictive value 29% and 88.9%. CONCLUSION: The serum level of interleukin-6 can predict severe (CD > 3a) POC early on POD1. On POD3, IL-6 is superior to CRP in terms of high positive predictive power of severe POC. Interestingly, the advantage of IL-6 on POD1 is early prediction of the need for antibiotic treatment, ICU stay and hospital stay, which is comparable to the CRP serum level late on the third POD.

Prognostic Significance of Immune-checkpoint Molecule PD-L1 in Classical Hodgkin Lymphoma: A Clinicopathologic Study of 120 Cases.

BACKGROUND/AIM: Classic Hodgkin lymphoma (cHL) is a common B-cell malignancy. Despite the good prognosis, in some patients the standard chemotherapy and radiotherapy-based approach does not lead to long-term remission, and these patients eventually relapse. Moreover, the primary refractory disease is of major concern regarding prognosis. PATIENTS AND METHODS: We performed a retrospective analysis to evaluate PD-L1 expression in 120 patients with classic Hodgkin lymphoma (cHL). RESULTS: The median follow-up of the entire group of patients was 90 months. After initial therapy, complete remission was achieved in 113 (94.2%) patients. During the follow-up, cHL relapse/refractory disease was reported in 23 (19.2%) cases. A total of five patients died during the follow-up period, all from cHL progression. When determining PD-L1 expression on Hodgkin-Reed-Sternberg (HRS) cells, 37 cases (30.8%) were evaluated as negative, and 83 cases (69.2%) as positive. In the negative PD-L1 group of patients, no cHL relapse/refractory disease was observed during the follow-up period. However, out of 83 patients with positive PD-L1 expression on HRS cells, 23 (28%) showed relapse/refractory cHL. CONCLUSION: A significantly higher relapse rate was observed in PD-L1-positive patients diagnosed with cHL.

Metals and Trace Elements in Calcified Valves in Patients with Acquired Severe Aortic Valve Stenosis: Is There a Connection with the Degeneration Process?

BACKGROUND: Acquired calcified aortic valve stenosis is the most common valve disease in adulthood. In the etiopathogenesis of this complex pathology, the importance of inflammation is mentioned, in which non-infectious influences represented by the biological effects of metal pollutants may participate. The main goal of the study was to determine the concentration of 21 metals and trace elements-aluminium (Al), barium (Ba), cadmium (Cd), calcium (Ca), chrome (Cr), cobalt (Co), copper (Cu), gold (Au), lead (Pb), magnesium (Mg), mercury (Hg), molybdenum (Mo), nickel (Ni), phosphorus (P), selenium (Se), strontium (Sr), sulfur (S), tin (Sn), titanium (Ti), vanadium (V) and zinc (Zn)-in the tissue of calcified aortic valves and to compare them with the concentrations of the same elements in the tissue of healthy aortic valves in the control group. MATERIAL AND METHODS: The study group consisted of 49 patients (25 men, mean age: 74) with acquired, severe, calcified aortic valve stenosis with indicated heart surgery. The control group included 34 deceased (20 men, median age: 53) with no evidence of heart disease. Calcified valves were explanted during cardiac surgery and deep frozen. Similarly, the valves of the control group were removed. All valves were lyophilized and analyzed by inductively coupled plasma mass spectrometry. The concentrations of selected elements were compared by means of standard statistical methods. RESULTS: Calcified aortic valves contained significantly higher (p < 0.05) concentrations of Ba, Ca, Co, Cr, Mg, P, Pb, Se, Sn, Sr and Zn and-in contrast-lower concentrations of Cd, Cu, Mo, S and V than valves of the control group. Significant positive correlations of concentrations between the pairs Ca-P, Cu-S and Se-S and strong negative correlations between the elements Mg-Se, P-S and Ca-S were found in the affected valves. CONCLUSION: Aortic valve calcification is associated with increased tissue accumulation of the majority of the analyzed elements, including metal pollutants. Some exposure factors may increase their accumulation in the valve tissue. A relationship between exposure to environmental burden and the aortic valve calcification process cannot be ruled out. Advances in histochemical and imaging techniques allowing imaging of metal pollutants directly in valve tissue may represent an important future perspective.

Small RNA Sequencing Identifies a Six-MicroRNA Signature Enabling Classification of Brain Metastases According to their Origin.

BACKGROUND/AIM: Brain metastases (BMs) are the most frequent intracranial tumors in adults and one of the greatest challenges for modern oncology. Most are derived from lung, breast, renal cell, and colorectal carcinomas and melanomas. Up to 14% of patients are diagnosed with BMs of unknown primary, which are commonly characterized by an early and aggressive metastatic spread. It is important to discover novel biomarkers for early identification of BM origin, allowing better management of patients with this disease. Our study focused on microRNAs (miRNAs), which are very stable in frozen native and FFPE tissues and have been shown to be sensitive and specific diagnostic biomarkers of cancer. We aimed to identify miRNAs with significantly different expression in the five most frequent groups of BMs and develop a diagnostic classifier capable of sensitive and specific classification of BMs. MATERIALS AND METHODS: Total RNA enriched for miRNAs was isolated using the mirVana miRNA Isolation Kit from 71 fresh-frozen histopathologically confirmed BM tissues originating in 5 cancer types. Sequencing libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the NextSeq 500 platform. MiRNA expression was further validated by RT-qPCR. RESULTS: Differential analysis identified 373 miRNAs with significantly different expression between 5 BM groups (p<0.001). A classifier model was developed based on the expression of 6 miRNAs (hsa-miR-141-3p, hsa-miR-141-5p, hsa-miR-146a-5p, hsa-miR-194-5p, hsa-miR-200b-3p and hsa-miR-365b-5p) with the ability to correctly classify 91.5% of samples. Subsequent validation confirmed both significantly different expression of selected miRNAs in 5 BM groups as well as their diagnostic potential. CONCLUSION: To date, our study is the first to analyze miRNA expression in various types of BMs using small RNA sequencing to develop a diagnostic classifier and, thus, to help stratify BMs of unknown primary. The presented results confirm the importance of studying the dysregulated expression of miRNAs in BMs and the diagnostic potential of the validated 6-miRNA signature.

Case report: Diagnostic challenge: a new multiple sclerosis "relapse" leading to the diagnosis of anaplastic astrocytoma.

Cerebral tumors and multiple sclerosis (MS) can show overlapping clinical and magnetic resonance imaging (MRI) features and even occur concurrently. Due to the emergence of new symptoms, not usually MS related, an MRI was conducted in a 29-year-old woman with relapsing-remitting MS and showed a significant size progression of a parieto-occipital lesion, with mild clinical correlates, such as blurred vision, difficulty in speaking, and headache. Contrast-enhanced MRI and fluorothymidine positron-emission tomography (PET) did not point toward neoplasm, a lesion biopsy, however, showed astrocytoma, which was confirmed as grade III astrocytoma after the radical resection of the tumor. In the case of an atypical lesion, a tumor should be considered in patients with MS. A small fraction of high-grade gliomas show no enhancement on MRI and no hypermetabolism on PET. Biopsy proved to be the essential step in a successful diagnostic workup. To the best of our knowledge, this is the first case of anaplastic astrocytoma with these radiological features reported in a patient with MS.

HLA-E and HLA-F Are Overexpressed in Glioblastoma and HLA-E Increased After Exposure to Ionizing Radiation.

BACKGROUND/AIM: Glioblastoma (GBM) is one of the deadliest human cancers responding very poorly to therapy. Although the central nervous system has been traditionally considered an immunologically privileged site with an enhanced immune response, GBM appears to benefit from this immunosuppressive milieu. Immunomodulatory molecules play an important role in immune tumor-host interactions. Non-classical human leukocyte antigens (HLA) class Ib molecules HLA-E, HLA-F, and HLA-G have been previously described to be involved in protecting semi-allogeneic fetal allografts from the maternal immune response and in transplant tolerance as well as tumoral immune escape. Unfortunately, their role in GBM remains poorly understood. Our study, therefore, aimed to characterize the relationship between the expression of these molecules in GBM on the transcriptional level and clinicopathological and molecular features of GBM as well as the effect of ionizing radiation. MATERIALS AND METHODS: We performed the analysis of HLA-E, HLA-F, and HLA-G mRNA expression in 69 GBM tissue samples and 21 non-tumor brain tissue samples (controls) by reverse transcription polymerase chain reaction. Furthermore, two primary GBM cell cultures had been irradiated to identify the effect of ionizing radiation on the expression of non-classical HLA molecules. RESULTS: Analyses revealed that both HLA-E and HLA-F are significantly up-regulated in GBM samples. Subsequent survival analysis showed a significant association between low expression of HLA-E and shorter survival of GBM patients. The dysregulated expression of both molecules was also observed between patients with methylated and unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Finally, we showed that ionizing radiation increased HLA-E expression level in GBM cells in vitro. CONCLUSION: HLA-E and HLA-F play an important role in GBM biology and could be used as diagnostic biomarkers, and in the case of HLA-E also as a prognostic biomarker.

A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Progressive Tumefactive Demyelination as the Only Result of Extensive Diagnostic Work-Up: A Case Report.

Tumefactive demyelinating lesions belong to the rare variants of multiple sclerosis, posing a diagnostic challenge since it is difficult to distinguish them from a neoplasm or other brain lesions and they require a careful differential diagnosis. This contribution presents the case report of a young female with progressive tumefactive demyelinating brain and spinal cord lesions. An extensive diagnostic process including two brain biopsies and an autopsy did not reveal any explanatory diagnosis other than multiple sclerosis. The patient was treated by various disease-modifying treatments without significant effect and died from ascendent infection via ventriculoperitoneal shunt resulting in Staphylococcus aureus meningitis.

Pancytopenia as the main manifestation of human immunodeficiency virus infection in the AIDS stage The authors present a case.

The authors present a case report of 59-years-old woman examined for pancytopenia recently diagnosed during hospitalization for bilateral interstitial pneumonia without any confirmed etiological agents. Concomitantly, some systemic symptoms like lack of appetite and weight loss were present. Primary hematological disease was ruled out. Positivity of serological screening for HIV-1,2 was rather surprising. Absolute count of CD4+ lymphocytes was 8/μl. Thus, HIV infection was already in stage of AIDS and retrospectively, the interstitial pneumonia has to be judged as AIDS-indicative illness.

Lumbar Interbody Fusion Conducted on a Porcine Model with a Bioresorbable Ceramic/Biopolymer Hybrid Implant Enriched with Hyperstable Fibroblast Growth Factor 2.

Many growth factors have been studied as additives accelerating lumbar fusion rates in different animal models. However, their low hydrolytic and thermal stability both in vitro and in vivo limits their workability and use. In the proposed work, a stabilized vasculogenic and prohealing fibroblast growth factor-2 (FGF2-STAB®) exhibiting a functional half-life in vitro at 37 °C more than 20 days was applied for lumbar fusion in combination with a bioresorbable scaffold on porcine models. An experimental animal study was designed to investigate the intervertebral fusion efficiency and safety of a bioresorbable ceramic/biopolymer hybrid implant enriched with FGF2-STAB® in comparison with a tricortical bone autograft used as a gold standard. Twenty-four experimental pigs underwent L2/3 discectomy with implantation of either the tricortical iliac crest bone autograft or the bioresorbable hybrid implant (BHI) followed by lateral intervertebral fixation. The quality of spinal fusion was assessed by micro-computed tomography (micro-CT), biomechanical testing, and histological examination at both 8 and 16 weeks after the surgery. While 8 weeks after implantation, micro-CT analysis demonstrated similar fusion quality in both groups, in contrast, spines with BHI involving inorganic hydroxyapatite and tricalcium phosphate along with organic collagen, oxidized cellulose, and FGF2- STAB® showed a significant increase in a fusion quality in comparison to the autograft group 16 weeks post-surgery (p = 0.023). Biomechanical testing revealed significantly higher stiffness of spines treated with the bioresorbable hybrid implant group compared to the autograft group (p < 0.05). Whilst histomorphological evaluation showed significant progression of new bone formation in the BHI group besides non-union and fibrocartilage tissue formed in the autograft group. Significant osteoinductive effects of BHI based on bioceramics, collagen, oxidized cellulose, and FGF2-STAB® could improve outcomes in spinal fusion surgery and bone tissue regeneration.

FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

SDHC Methylation Pattern in Patients With Carney Triad.

Carney triad is a multitumor syndrome affecting almost exclusively young women in a nonfamilial setting, which manifests by multifocal gastric gastrointestinal stromal tumors, paragangliomas, and pulmonary chondroma. The Carney triad-associated tumors are characterized by a deficiency of the mitochondrial succinate dehydrogenase enzymatic complex. Recently, it has been observed that the deficiency results from epigenetic silencing of the SDHC gene by its promoter hypermethylation. To elucidate anatomic distribution of SDHC promoter methylation in Carney triad patients and thus to shed some light on the possible natural development of this epigenetic change, both neoplastic and available non-neoplastic tissues of 3 patients with Carney triad were tested for hypermethylation at the SDHC promoter site. SDHC promoter hypermethylation was proven in all tumors studied. Lack of SDHC epigenetic silencing in the non-neoplastic lymphoid and duodenal tissue (ie, tissues not involved in the development of Carney triad-associated tumors) together with the finding of SDHC promoter hypermethylation in the non-neoplastic gastric wall favors the hypothesis of postzygotic somatic mosaicism as the biological background of Carney triad; it also offers an explanation of the multifocality of gastrointestinal stromal tumors of the stomach occurring in this scenario as well. However, the precise mechanism responsible for the peculiar organ-specific distribution of Carney triad-associated tumors is still unknown.

miR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors.

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40-NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by BCR activity.

Assessment of Tumor Mutational Burden in Pediatric Tumors by Real-Life Whole-Exome Sequencing and In Silico Simulation of Targeted Gene Panels: How the Choice of Method Could Affect the Clinical Decision?

BACKGROUND: Tumor mutational burden (TMB) is an emerging genomic biomarker in cancer that has been associated with improved response to immune checkpoint inhibitors (ICIs) in adult cancers. It was described that variability in TMB assessment is introduced by different laboratory techniques and various settings of bioinformatic pipelines. In pediatric oncology, no study has been published describing this variability so far. METHODS: In our study, we performed whole exome sequencing (WES, both germline and somatic) and calculated TMB in 106 patients with high-risk/recurrent pediatric solid tumors of 28 distinct cancer types. Subsequently, we used WES data for TMB calculation using an in silico approach simulating two The Food and Drug Administration (FDA)-approved/authorized comprehensive genomic panels for cancer. RESULTS: We describe a strong correlation between WES-based and panel-based TMBs; however, we show that this high correlation is significantly affected by inclusion of only a few hypermutated cases. In the series of nine cases, we determined TMB in two sequentially collected tumor tissue specimens and observed an increase in TMB along with tumor progression. Furthermore, we evaluated the extent to which potential ICI indication could be affected by variability in techniques and bioinformatic pipelines used for TMB assessment. We confirmed that this technological variability could significantly affect ICI indication in pediatric cancer patients; however, this significance decreases with the increasing cut-off values. CONCLUSIONS: For the first time in pediatric oncology, we assessed the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels and confirmed a significant technological variability to be introduced by different laboratory techniques and various settings of bioinformatic pipelines.

N-Glycan profiling of lung adenocarcinoma in patients at different stages of disease.

Lung adenocarcinoma (LAC) is the most common form of lung cancer that increases in non-smokers at younger age. Altered protein glycosylation is one of the hallmarks of malignancy, its role in cancer progression is still poorly understood. In this study, we report mass spectrometric (MS) analysis of N-glycans released from fresh or defrosted tissue specimens from 24 patients with LAC. Comparison of cancerous versus adjacent healthy tissues revealed substantial differences in N-glycan profiles associated with disease. The significant increase in paucimannose and high-mannose glycans with 6-9 mannose residues and decline in the sialylated complex biantenary core fucosylated glycan with composition NeuAcGal2GlcNAc2Man3GlcNAc2Fuc were general features of tumors. In addition, 42 new N-glycan compositions were detected in cancerous tissues. The prominent changes in advanced disease stages were mostly observed in core fucosylated N-glycans with additional fucose (Fuc) residue/s and enhanced branching with non-galactosylated N-acetyl-glucosamine (GlcNAc) units. Both of these monosaccharide types were linked preferably on the 6-antenna. Importantly, as compared with noncancerous tissues, a number of these significant changes were clearly detectable early on in stage I. Application of N-glycan data obtained from tissues was next assessed and validated for evaluation of small sized biopsies obtained via bronchoscopy. In summary, observed alterations and data of newly detected N-glycans expand knowledge about the glycosylation in LAC and may contribute to research in more tailored therapies. Moreover, the results demonstrate effectiveness of the presented approach for utility in rapid discrimination of cancerous from healthy lung tissues.

Mantle cell lymphoma diagnosed from radical prostatectomy for prostate adenocarcinoma: a case report.

We present a case of 64 - year - old man with mantle cell lymphoma (MCL) diagnosed in radical prostatectomy for adenocarcinoma. Lymphoma of the prostate, either primary or secondary, is a rare entity. The prognosis of lymphoma involving the prostate is usually poor. The most common form of leukemia/lymphoma involving the prostate is that of chronic lymphocytic leukemia/lymphoma (CLL/SLL). Mantle cell lymphoma of the prostate is an exceedingly rare entity. The association between MCL and urologic cancer was previously described, however the etiologic connection between these two conditions remains unknown. The pathologists should pay attention to all lymphoid infiltrates in the prostate, especially in cases when these are dense.

Phospho-Protein Arrays as Effective Tools for Screening Possible Targets for Kinase Inhibitors and Their Use in Precision Pediatric Oncology.

The specific targeting of signal transduction by low-molecular-weight inhibitors or monoclonal antibodies represents a very promising personalized treatment strategy in pediatric oncology. In this study, we present the successful and clinically relevant use of commercially available phospho-protein arrays for analyses of the phosphorylation profiles of a broad spectrum of receptor tyrosine kinases and their downstream signaling proteins in tumor tissue samples. Although these arrays were made for research purposes on human biological samples, they have already been used by several authors to profile various tumor types. Our study performed a systematic analysis of the advantages and pitfalls of the use of this method for personalized clinical medicine. In certain clinical cases and their series, we demonstrated the important aspects of data processing and evaluation, the use of phospho-protein arrays for single sample and serial sample analyses, and the validation of obtained results by immunohistochemistry, as well as the possibilities of this method for the hierarchical clustering of pediatric solid tumors. Our results clearly show that phospho-protein arrays are apparently useful for the clinical consideration of druggable molecular targets within a specific tumor. Thus, their potential validation for diagnostic purposes may substantially improve the personalized approach in the treatment of relapsed or refractory solid tumors.