SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

Elevated levels of MMP12 sourced from macrophages are associated with poor prognosis in urothelial bladder cancer.

BACKGROUND: Urothelial bladder cancer is most frequently diagnosed at the non-muscle-invasive stage (NMIBC). However, recurrences and interventions for intermediate and high-risk NMIBC patients impact the quality of life. Biomarkers for patient stratification could help to avoid unnecessary interventions whilst indicating aggressive measures when required. METHODS: In this study, immuno-oncology focused, multiplexed proximity extension assays were utilised to analyse plasma (n = 90) and urine (n = 40) samples from 90 newly-diagnosed and treatment-naïve bladder cancer patients. Public single-cell RNA-sequencing and microarray data from patient tumour tissues and murine OH-BBN-induced urothelial carcinomas were also explored to further corroborate the proteomic findings. RESULTS: Plasma from muscle-invasive, urothelial bladder cancer patients displayed higher levels of MMP7 (p = 0.028) and CCL23 (p = 0.03) compared to NMIBC patients, whereas urine displayed higher levels of CD27 (p = 0.044) and CD40 (p = 0.04) in the NMIBC group by two-sided Wilcoxon rank-sum tests. Random forest survival and multivariable regression analyses identified increased MMP12 plasma levels as an independent marker (p < 0.001) associated with shorter overall survival (HR = 1.8, p < 0.001, 95% CI:1.3-2.5); this finding was validated in an independent patient OLINK cohort, but could not be established using a transcriptomic microarray dataset. Single-cell transcriptomics analyses indicated tumour-infiltrating macrophages as a putative source of MMP12. CONCLUSIONS: The measurable levels of tumour-localised, immune-cell-derived MMP12 in blood suggest MMP12 as an important biomarker that could complement histopathology-based risk stratification. As MMP12 stems from infiltrating immune cells rather than the tumor cells themselves, analyses performed on tissue biopsy material risk a biased selection of biomarkers produced by the tumour, while ignoring the surrounding microenvironment.

Robust humoral and cellular immune responses and low risk for reinfection at least 8 months following asymptomatic to mild COVID-19.

BACKGROUND: Emerging data support detectable immune responses for months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, but it is not yet established to what degree and for how long protection against reinfection lasts. METHODS: We investigated SARS-CoV-2-specific humoral and cellular immune responses more than 8 months post-asymptomatic, mild and severe infection in a cohort of 1884 healthcare workers (HCW) and 51 hospitalized COVID-19 patients. Possible protection against SARS-CoV-2 reinfection was analyzed by a weekly 3-month polymerase chain reaction (PCR) screening of 252 HCW that had seroconverted 7 months prior to start of screening and 48 HCW that had remained seronegative at multiple time points. RESULTS: All COVID-19 patients and 96% (355/370) of HCW who were anti-spike IgG positive at inclusion remained anti-spike IgG positive at the 8-month follow-up. Circulating SARS-CoV-2-specific memory T cell responses were detected in 88% (45/51) of COVID-19 patients and in 63% (233/370) of seropositive HCW. The cumulative incidence of PCR-confirmed SARS-CoV-2 infection was 1% (3/252) among anti-spike IgG positive HCW (0.13 cases per 100 weeks at risk) compared to 23% (11/48) among anti-spike IgG negative HCW (2.78 cases per 100 weeks at risk), resulting in a protective effect of 95.2% (95% CI 81.9%-99.1%). CONCLUSIONS: The vast majority of anti-spike IgG positive individuals remain anti-spike IgG positive for at least 8 months regardless of initial COVID-19 disease severity. The presence of anti-spike IgG antibodies is associated with a substantially reduced risk of reinfection up to 9 months following asymptomatic to mild COVID-19.

Potential of hybrid ¹8F-fluorocholine PET/MRI for prostate cancer imaging.

PURPOSE: To report the first results of hybrid (18)F-fluorocholine PET/MRI imaging for the detection of prostate cancer. METHODS: This analysis included 26 consecutive patients scheduled for prostate PET/MRI before radical prostatectomy. The examinations were performed on a hybrid whole-body PET/MRI scanner. The MR acquisitions which included T2-weighted, diffusion-weighted and dynamic contrast-enhanced sequences were followed during the same session by whole-body PET scans. Parametric maps were constructed to measure normalized T2-weighted intensity (nT2), apparent diffusion coefficient (ADC), volume transfer constant (K (trans)), extravascular extracellular volume fraction (v e) and standardized uptake values (SUV). With pathology as the gold standard, ROC curves were calculated using logistic regression for each parameter and for the best combination with and without PET to obtain a MR model versus a PETMR model. RESULTS: Of the 26 patients initially selected, 3 were excluded due to absence of an endorectal coil (2 patients) or prosthesis artefacts (1 patient). In the whole prostate, the area under the curve (AUC) for SUVmax, ADC, nT2, K (trans) and v e were 0.762, 0.756, 0.685, 0.611 and 0.529 with a best threshold at 3.044 for SUVmax and 1.075 × 10(-3) mm(2)/s for ADC. The anatomical distinction between the transition zone and the peripheral zone showed the potential of the adjunctive use of PET. In the peripheral zone, the AUC of 0.893 for the PETMR model was significantly greater (p = 0.0402) than the AUC of 0.84 for the MR model only. In the whole prostate, no relevant correlation was observed between ADC and SUVmax. The SUVmax was not affected by the Gleason score. CONCLUSION: The performance of a hybrid whole-body (18)F-fluorocholine PET/MRI scan in the same session combined with a prostatic MR examination did not interfere with the diagnostic accuracy of the MR sequences. The registration of the PET data and the T2 anatomical MR sequence data allowed precise localization of hypermetabolic foci in the prostate. While in the transition zone the adenomatous hyperplasia interfered with cancer detection by PET, the quantitative analysis tool performed well for cancer detection in the peripheral zone.

Correction: Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.

[This corrects the article DOI: 10.1371/journal.pone.0253178.].

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BACKGROUND: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. METHODS: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFNγ) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. RESULTS: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFNγ and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFNγ in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. CONCLUSION: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.

Plasma Proteomic Analysis in Non-Small Cell Lung Cancer Patients Treated with PD-1/PD-L1 Blockade.

Checkpoint inhibitors have been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients demonstrate a durable clinical response. PD-L1 scoring is currently the only biomarker measure routinely used to select patients for immunotherapy, but its predictive accuracy is modest. The aim of our study was to evaluate a proteomic assay for the analysis of patient plasma in the context of immunotherapy. Pretreatment plasma samples from 43 NSCLC patients who received anti-PD-(L)1 therapy were analyzed using a proximity extension assay (PEA) to quantify 92 different immune oncology-related proteins. The plasma protein levels were associated with clinical and histopathological parameters, as well as therapy response and survival. Unsupervised hierarchical cluster analysis revealed two patient groups with distinct protein profiles associated with high and low immune protein levels, designated as "hot" and "cold". Further supervised cluster analysis based on T-cell activation markers showed that higher levels of T-cell activation markers were associated with longer progression-free survival (PFS) (p < 0.01). The analysis of single proteins revealed that high plasma levels of CXCL9 and CXCL10 and low ADA levels were associated with better response and prolonged PFS (p < 0.05). Moreover, in an explorative response prediction model, the combination of protein markers (CXCL9, CXCL10, IL-15, CASP8, and ADA) resulted in higher accuracy in predicting response than tumor PD-L1 expression or each protein assayed individually. Our findings demonstrate a proof of concept for the use of multiplex plasma protein levels as a tool for anti-PD-(L)1 response prediction in NSCLC. Additionally, we identified protein signatures that could predict the response to anti-PD-(L)1 therapy.

Fed-batch production assessment of a tetravalent bispecific antibody: A case study on piggyBac stably transfected HEK293 cells.

The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 × 106 viable cells/mL with over 90 % viability 12 days post-inoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody.

Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.

Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.

An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses.

Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.

Reproducibility of Lobar Perfusion and Ventilation Quantification Using SPECT/CT Segmentation Software in Lung Cancer Patients.

Planar perfusion scintigraphy with 99mTc-labeled macroaggregated albumin is often used for pretherapy quantification of regional lung perfusion in lung cancer patients, particularly those with poor respiratory function. However, subdividing lung parenchyma into rectangular regions of interest, as done on planar images, is a poor reflection of true lobar anatomy. New tridimensional methods using SPECT and SPECT/CT have been introduced, including semiautomatic lung segmentation software. The present study evaluated inter- and intraobserver agreement on quantification using SPECT/CT software and compared the results for regional lung contribution obtained with SPECT/CT and planar scintigraphy. Methods: Thirty lung cancer patients underwent ventilation-perfusion scintigraphy with 99mTc-macroaggregated albumin and 99mTc-Technegas. The regional lung contribution to perfusion and ventilation was measured on both planar scintigraphy and SPECT/CT using semiautomatic lung segmentation software by 2 observers. Interobserver and intraobserver agreement for the SPECT/CT software was assessed using the intraclass correlation coefficient, Bland-Altman plots, and absolute differences in measurements. Measurements from planar and tridimensional methods were compared using the paired-sample t test and mean absolute differences. Results: Intraclass correlation coefficients were in the excellent range (above 0.9) for both interobserver and intraobserver agreement using the SPECT/CT software. Bland-Altman analyses showed very narrow limits of agreement. Absolute differences were below 2.0% in 96% of both interobserver and intraobserver measurements. There was a statistically significant difference between planar and SPECT/CT methods (P < 0.001) for quantification of perfusion and ventilation for all right lung lobes, with a maximal mean absolute difference of 20.7% for the right middle lobe. There was no statistically significant difference in quantification of perfusion and ventilation for the left lung lobes using either method; however, absolute differences reached 12.0%. The total right and left lung contributions were similar for the two methods, with a mean difference of 1.2% for perfusion and 2.0% for ventilation. Conclusion: Quantification of regional lung perfusion and ventilation using SPECT/CT-based lung segmentation software is highly reproducible. This tridimensional method yields statistically significant differences in measurements for right lung lobes when compared with planar scintigraphy. We recommend that SPECT/CT-based quantification be used for all lung cancer patients undergoing pretherapy evaluation of regional lung function.

Phase 1-2 Study of Dual-Energy Computed Tomography for Assessment of Pulmonary Function in Radiation Therapy Planning.

PURPOSE: To quantify lung function according to a dual-energy computed tomography (DECT)-derived iodine map in patients treated with radiation therapy for lung cancer, and to assess the dosimetric impact of its integration in radiation therapy planning. METHODS AND MATERIALS: Patients treated with stereotactic ablative radiation therapy for early-stage or intensity modulated radiation therapy for locally advanced lung cancer were prospectively enrolled in this study. A DECT in treatment position was obtained at time of treatment planning. The relative contribution of each voxel to the total lung function was based on iodine distribution. The composition of each voxel was determined on the basis of a 2-material decomposition. The DECT-derived lobar function was compared with single photon emission computed tomography/computed tomography (SPECT/CT). A functional map was integrated in the treatment planning system using 6 subvolumes of increasing iodine distribution levels. Percent lung volume receiving 5 Gy (V5), V20, and mean dose (MLD) to whole lungs (anatomic) versus functional lungs were compared. RESULTS: Twenty-five patients with lung cancer, including 18 patients treated with stereotactic ablative radiation therapy and 7 patients with intensity modulated radiation therapy (locally advanced), were included. Eighty-four percent had chronic obstructive pulmonary disease. Median (range) forced expiratory volume in 1 second was 62% of predicted (29%-113%), and median diffusing capacity of the lung for carbon monoxide was 56% (39%-91%). There was a strong linear correlation between DECT- and SPECT/CT-derived lobar function (Pearson coefficient correlation r=0.89, P<.00001). Mean (range) differences in V5, V20, and MLD between anatomic and functional lung volumes were 16% (0%-48%, P=.03), 5% (1%-15%, P=.12), and 15% (1%-43%, P=.047), respectively. CONCLUSIONS: Lobar function derived from a DECT iodine map correlates well with SPECT/CT, and its integration in lung treatment planning is associated with significant differences in V5 and MLD to functional lungs. Future work will involve integration of the weighted functional volume in the treatment planning system, along with integration of an iodine map for functional lung-sparing IMRT.

The use of gallium-67 scintigraphy in the diagnosis of acute interstitial nephritis.

BACKGROUND: Gallium-67 scintigraphy has been suggested as a noninvasive method to diagnose acute interstitial nephritis (AIN). However, its diagnostic performance and usefulness remain controversial. METHODS: We retrospectively reviewed the charts of 76 patients who underwent gallium-67 scintigraphy for a suspicion of AIN. Patients were classified based on kidney biopsy and/or clinical probability of AIN. Gallium-67 scintigraphy results were reinterpreted blindly using both posterior planar and single photon emission computed tomography (SPECT) imaging. Intensity of radioisotope uptake in the kidney was graded from 0 to 5. RESULTS: The diagnosis of AIN was confirmed in 23 patients and excluded in 44. Nine patients with an uncertain diagnosis were excluded from subsequent analysis. A gallium-67 kidney uptake cutoff of 1 gave a negative predictive value of 100%, whereas a cutoff of 5 had an excellent specificity and positive predictive value for the diagnosis of AIN. When using a cutoff of 3, which had previously been used in the literature, we obtained a sensitivity of 61% and a specificity of 75% with posterior planar imaging. The results of both SPECT and posterior planar imaging modalities were comparable. CONCLUSIONS: Gallium-67 scintigraphy may be of interest in patients with a clinical suspicion of AIN, especially in those who are unable to undergo kidney biopsy. However, results need to be interpreted with caution and depend on the intensity of gallium-67 kidney uptake.