Hypoxia Is Associated with Increased Immune Infiltrates and Both Anti-Tumour and Immune Suppressive Signalling in Muscle-Invasive Bladder Cancer.

Hypoxia and a suppressive tumour microenvironment (TME) are both independent negative prognostic factors for muscle-invasive bladder cancer (MIBC) that contribute to treatment resistance. Hypoxia has been shown to induce an immune suppressive TME by recruiting myeloid cells that inhibit anti-tumour T cell responses. Recent transcriptomic analyses show hypoxia increases suppressive and anti-tumour immune signalling and infiltrates in bladder cancer. This study sought to investigate the relationship between hypoxia-inducible factor (HIF)-1 and -2, hypoxia, and immune signalling and infiltrates in MIBC. ChIP-seq was performed to identify HIF1α, HIF2α, and HIF1ß binding in the genome of the MIBC cell line T24 cultured in 1% and 0.1% oxygen for 24 h. Microarray data from four MIBC cell lines (T24, J82, UMUC3, and HT1376) cultured under 1%, 0.2%, and 0.1% oxygen for 24 h were used. Differences in the immune contexture between high- and low-hypoxia tumours were investigated using in silico analyses of two bladder cancer cohorts (BCON and TCGA) filtered to only include MIBC cases. GO and GSEA were used with the R packages "limma" and "fgsea". Immune deconvolution was performed using ImSig and TIMER algorithms. RStudio was used for all analyses. Under hypoxia, HIF1α and HIF2α bound to ~11.5-13.5% and ~4.5-7.5% of immune-related genes, respectively (1-0.1% O2). HIF1α and HIF2α both bound to genes associated with T cell activation and differentiation signalling pathways. HIF1α and HIF2α had distinct roles in immune-related signalling. HIF1 was associated with interferon production specifically, whilst HIF2 was associated with generic cytokine signalling as well as humoral and toll-like receptor immune responses. Neutrophil and myeloid cell signalling was enriched under hypoxia, alongside hallmark pathways associated with Tregs and macrophages. High-hypoxia MIBC tumours had increased expression of both suppressive and anti-tumour immune gene signatures and were associated with increased immune infiltrates. Overall, hypoxia is associated with increased inflammation for both suppressive and anti-tumour-related immune signalling and immune infiltrates, as seen in vitro and in situ using MIBC patient tumours.

Data-independent acquisition mass spectrometry in severe rheumatic heart disease (RHD) identifies a proteomic signature showing ongoing inflammation and effectively classifying RHD cases.

BACKGROUND: Rheumatic heart disease (RHD) remains a major source of morbidity and mortality in developing countries. A deeper insight into the pathogenetic mechanisms underlying RHD could provide opportunities for drug repurposing, guide recommendations for secondary penicillin prophylaxis, and/or inform development of near-patient diagnostics. METHODS: We performed quantitative proteomics using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectrometry (SWATH-MS) to screen protein expression in 215 African patients with severe RHD, and 230 controls. We applied a machine learning (ML) approach to feature selection among the 366 proteins quantifiable in at least 40% of samples, using the Boruta wrapper algorithm. The case-control differences and contribution to Area Under the Receiver Operating Curve (AUC) for each of the 56 proteins identified by the Boruta algorithm were calculated by Logistic Regression adjusted for age, sex and BMI. Biological pathways and functions enriched for proteins were identified using ClueGo pathway analyses. RESULTS: Adiponectin, complement component C7 and fibulin-1, a component of heart valve matrix, were significantly higher in cases when compared with controls. Ficolin-3, a protein with calcium-independent lectin activity that activates the complement pathway, was lower in cases than controls. The top six biomarkers from the Boruta analyses conferred an AUC of 0.90 indicating excellent discriminatory capacity between RHD cases and controls. CONCLUSIONS: These results support the presence of an ongoing inflammatory response in RHD, at a time when severe valve disease has developed, and distant from previous episodes of acute rheumatic fever. This biomarker signature could have potential utility in recognizing different degrees of ongoing inflammation in RHD patients, which may, in turn, be related to prognostic severity.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

MPFL reconstruction corrects patella alta: a cohort study.

PURPOSE: Patella alta is an established risk factor for recurrent lateral patella dislocations. Medial patellofemoral ligament (MPFL) reconstruction has been shown to reduce patella height. Our hypothesis is that isolated MPFL reconstruction corrects patella alta, thereby reducing the risk of recurrent patella dislocation. METHODS: A prospective cohort study of 33 knees which underwent MPFL reconstruction for recurrent patella dislocation. The pre-operative and post-operative patella height and Kujala knee outcome scores, as well as tibial tuberosity-trochlear groove (TT-TG) distance, and the grade of trochlear dysplasia were recorded. RESULTS: The mean age was 21.5 years (16-34 years). There was significant reduction in patella height in all patients (p < 0.001), a 67% normalisation of patella alta (CD ≥ 1.2) (p < 0.004), and improvement in the Kujala scores 57.1 (pre-operatively) to 94.8 (post-operatively, p < 0.0001). The recurrent dislocation rate was 3%. CONCLUSION: MPFL reconstruction alone results in correction in patella alta and improvement in clinical outcomes.

The use of missing values in proteomic data-independent acquisition mass spectrometry to enable disease activity discrimination.

MOTIVATION: Data-independent acquisition mass spectrometry allows for comprehensive peptide detection and relative quantification than standard data-dependent approaches. While less prone to missing values, these still exist. Current approaches for handling the so-called missingness have challenges. We hypothesized that non-random missingness is a useful biological measure and demonstrate the importance of analysing missingness for proteomic discovery within a longitudinal study of disease activity. RESULTS: The magnitude of missingness did not correlate with mean peptide concentration. The magnitude of missingness for each protein strongly correlated between collection time points (baseline, 3 months, 6 months; R = 0.95-0.97, confidence interval = 0.94-0.97) indicating little time-dependent effect. This allowed for the identification of proteins with outlier levels of missingness that differentiate between the patient groups characterized by different patterns of disease activity. The association of these proteins with disease activity was confirmed by machine learning techniques. Our novel approach complements analyses on complete observations and other missing value strategies in biomarker prediction of disease activity. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Application of SWATH mass spectrometry in the identification of circulating proteins does not predict future weight gain in early psychosis.

Weight gain is a common consequence of treatment with antipsychotic drugs in early psychosis, leading to further morbidity and poor treatment adherence. Identifying tools that can predict weight change in early psychosis may contribute to better-individualised treatment and adherence. Recently we showed that proteomic profiling with sequential window acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry (MS) can identify individuals with pre-diabetes more likely to experience weight change in relation to lifestyle change. We investigated whether baseline proteomic profiles predicted weight change over time using data from the BeneMin clinical trial of the anti-inflammatory antibiotic, minocycline, versus placebo. Expression levels for 844 proteins were determined by SWATH proteomics in 83 people (60 men and 23 women). Hierarchical clustering analysis and principal component analysis of baseline proteomics data did not reveal distinct separation between the proteome profiles of participants in different weight change categories. However, individuals with the highest weight loss had higher Positive and Negative Syndrome Scale (PANSS) scores. Our findings imply that mode of treatment i.e. the pharmacological intervention for psychosis may be the determining factor in weight change after diagnosis, rather than predisposing proteomic dynamics.

All-Inside Lateral Meniscal Repair via Anterolateral Portal Increases Risk of Vascular Injury: A Cadaveric Study.

PURPOSE: To compare the distance from the device tip to the neurovascular structures during an all-inside medial and lateral meniscal repair using anteromedial and anterolateral portals in a fresh-frozen cadaveric study. METHODS: Ten fresh-frozen cadaveric knees were studied. The popliteal artery, popliteal vein, and tibial nerve were identified after dissection via a posterior approach. An all-inside meniscal repair device was set to a 20-mm depth limit and inserted into a fixed point in the posterior horn at the meniscocapsular junction. This was performed for medial and lateral menisci via anteromedial and anterolateral arthroscopic portals. The distances between the device tip and the neurovascular structures were measured. We performed t tests to determine statistical significance. RESULTS: The distance between the device and popliteal artery was significantly closer when aimed at the posterior horn of the lateral meniscus via the anterolateral portal (4.7 ± 2.3 mm) versus the anteromedial portal (13.0 ± 8.0 mm, P = .010). The distance to the popliteal vein was closer via the anterolateral portal (6.7 ± 2.9 mm) versus the anteromedial portal (13.9 ± 5.8 mm, P = .004). For medial meniscal repair, the distance to the popliteal artery was significantly closer via the anteromedial portal (12.8 ± 11.3 mm) versus the anterolateral portal (23.8 ± 7.7 mm, P = .022). The distance to the popliteal vein was closer via the anteromedial portal (16.5 ± 11.3 mm) versus the anterolateral portal (28.3 ± 8.2 mm, P = .017). No significant difference was found in the distance to the tibial nerve when aimed at either meniscus via either portal. CONCLUSION: For all-inside meniscal repair, the popliteal vein is at risk and the popliteal artery is at high risk of injury when the posterior horn of the lateral meniscus is repaired via an anterolateral working portal. CLINICAL RELEVANCE: The popliteal artery and vein are at risk of injury when the posterior horn of the lateral meniscus undergoes all-inside repair via the anterolateral portal. Surgeons need to be aware of the risks when performing this repair.

PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline.

Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms, and demonstrate its performance on ground-truth synthetic peptide reference data sets, one previously published small data set, one new larger data set, and also on a previously published phosphoenriched data set where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.

Reliability of magnetic resonance imaging in evaluating meniscal and cartilage injuries in anterior cruciate ligament-deficient knees.

PURPOSE: The accuracy of magnetic resonance (MR) imaging in assessing meniscal and cartilage injuries in anterior cruciate ligament (ACL)-deficient knees as compared to arthroscopy was evaluated in the present study. METHODS: The results of all preoperative MR imaging performed within 3 months prior to the ACL reconstruction were compared against intraoperative arthroscopic findings. A total of 206 patients were identified. The location and type of meniscal injuries as well as the location and grade of the cartilage injuries were studied. The negative predictive value, positive predictive value, sensitivity, specificity and accuracy of MR imaging for these 206 cases were calculated and analysed. RESULTS: In patients with an ACL injury, the highest incidence of concomitant injury was that of medial meniscus tears, 124 (60.2 %), followed by lateral meniscus tears, 105 (51.0 %), and cartilage injuries, 66 (32.0 %). Twenty-three (11.2 %) patients sustained injuries to all of the previously named structures. MR imaging was most accurate in detecting medial meniscus tears (85.9 %). MR imaging for medial meniscus tears also had the highest sensitivity (88.0 %) and positive predictive value (88.7 %), while MR imaging for cartilage injuries had the largest specificity (84.1 %) and negative predictive value (87.1 %). It was least accurate in evaluating lateral meniscus tears (74.3 %). The diagnostic accuracy of medial meniscus imaging is significantly influenced by age and the presence of lateral meniscus tears, while the duration between MR imaging and surgery has greater impact on the likelihood of lateral meniscus and cartilage injuries actually being present during surgery. The majority of meniscus tears missed by MR imaging affected the posterior horn and were complex in nature. Cartilage injuries affecting the medial femoral condyle or medial patella facet were also often missed by MR imaging. CONCLUSION: MR imaging remains a reliable tool for assessing meniscus tears and cartilage defects preoperatively. It is most accurate when evaluating medial meniscus tears. However, MR imaging should be used with discretion especially if there is a high index of suspicion of lateral meniscus tears. LEVEL OF EVIDENCE: IV.

Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.

Computational phosphoproteomics: from identification to localization.

Analysis of the phosphoproteome by MS has become a key technology for the characterization of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable side chain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or "localize" the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards.

Surface histidine mutations for the metal affinity purification of a ß-carbonic anhydrase.

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.

Arthroscopic biological internal bracing with remnant repair for subacute ACL femoral avulsions.

Arthroscopic anterior cruciate ligament (ACL) reconstruction predictably restores sagittal plane knee stability, however its inability to replicate a complex fan-shaped ligament of multiple fascicles, along with deficient restoration of normal rotational knee kinematics, results in failure to reverse a high risk for premature post-traumatic osteoarthritis. Although arthroscopic repair for acute ACL femoral avulsions is proposed to counter these deficiencies, the risk of early failure following non-healing, along with lack of convincing evidence of efficacy has impeded its universal acceptance. Moreover, since ACL repair needs to be performed in the acute phase following injury, it has an increased risk of developing arthrofibrosis, besides precluding any possibility to achieve natural healing of an ACL avulsion with non-operative treatment. The technique of biological internal bracing with remnant repair incorporates the advantages of both reconstruction and repair, and is indicated for patients with persistent ACL deficiency in the subacute phase (6-12 weeks) following an ACL femoral avulsion. This operation essentially involves two steps. The step of biological internal bracing is similar to a conventional ACL reconstruction using a small diameter hamstring graft that is targeted to the centre of the anteromedial ACL bundle on the femur, whereas the tibial socket is located posteriorly within the ACL tibial footprint so as to preserve the anterior fan-shaped morphology of the ACL tibial insertion. The second step involves repairing the remnant ACL tibial stump using one of three techniques. Although technically more complex than an ACL reconstruction, this novel technique provides native anatomy restoration with potential biomechanical and functional advantages, and should be considered for unhealed subacute ACL avulsion injuries.

Do meniscal repairs with meniscus cyst do better than meniscectomy?-a systematic review of meniscal cyst treatment.

Background: This systematic review aims to determine the best modality for the management of meniscal cysts and its associated meniscus tear; whether the meniscal cyst treated via arthroscopy or open methods and whether meniscal debridement or repair achieves better results. Methods: This systematic review was performed using PRISMA guidelines. A literature search of PubMed, EMBASE and Cochrane was carried out in July 2020 using the search terms 'meniscal cyst' and 'treatment'. All clinic studies that included filters for papers in the last 20 years, English language, and meniscal cysts found in humans were included. Studies that contained case reports, were in any language other than English, and with subjects that were not humans were excluded. The methodology quality assessment was performed through the modified Coleman methodology score (CMS). Results: A total of 166 results were obtained from PubMed, Cochrane library and EMBASE. Of them, 12 duplicates were identified across the databases and removed from consideration. Six papers were found relevant from EMBASE in which 1 was eventually included in this paper. In total, 12 papers were used in this study. The weighted mean age of the patients was 35.1 years, with total of 523 meniscal cysts, of which 488 of these cysts are associated with meniscal tears (93.31%). The studies included performed cystectomies and/or decompression of meniscal cysts while some left the meniscal cyst alone and dealt with the meniscal lesion instead. All clinical scores showed significant improvement following surgical procedures. Conclusions: Both arthroscopic and open methods can be used for meniscal cysts treatment. Open cystectomy rather than decompression seemed to confer lower risk of cyst recurrences and complications. It is inconclusive to whether meniscal repair or meniscus debridement influenced recurrence and outcome scores. A recommendation for meniscus repair cannot be made due to insufficient high-quality level I or II trials.

Consideration of growth factors and bio-scaffolds for treatment of combined grade II MCL and ACL injury.

The literature suggests that a Grade II medial collateral ligament (MCL) injury in combination with anterior cruciate ligament (ACL) injury will heal naturally and not compromise patient outcome following ACL reconstruction. Evidence based on bone-patella tendon-bone autograft use is stronger than evidence supporting anatomically placed soft tissue graft use. Current ACL reconstruction practices make greater use of soft tissue grafts, differing fixation methods, and anatomically lower placement on the inner wall of the lateral femoral condyle. Anatomical graft placement aligns the femoral bone tunnel more directly with valgus knee loading forces. Differences in the soft tissue graft-bone tunnel integration and ligamentization timetable following ACL reconstruction also increase concerns regarding residual Grade II MCL laxity and functional deficiency during accelerated functional rehabilitation. MCL dysfunction may increase susceptibility to early ACL graft slippage, elongation, outright failure, and medial femoral condyle lift-off with valgus knee loading. This concept paper discusses the potential role of growth factors and bio-scaffolds for improving Grade II MCL injury healing and mechanical integrity when the injury occurs in combination with an ACL injury that is reconstructed with a soft tissue graft and an anatomical surgical approach.

Red flags for spinal pain in patients diagnosed with spinal infection in Nigeria: A 10-year medical records review.

BACKGROUND: Spinal infection is a diagnostic challenge, the personal and economic consequences of misdiagnosis can be significant resulting in paralysis and instability of the spine and can ultimately be fatal. To aid identification of those at risk of spinal infection, a better understanding of the red flags for spinal infection is needed. OBJECTIVE: To better understand which red flags may help to identify spinal infection. DESIGN: and Methods: A 10-year medical records review of red flags for spinal infection in Nigeria, using a bespoke data extraction tool. Univariable and multivariable logistic regression was used to identify the main independent predictors of spinal pain. RESULTS: 124,913 records were reviewed, 1,645 patients were diagnosed with spinal infection. 79% of patients presented with spinal pain Univariable analysis revealed nine factors (some centres, all age groups above 16 years, co-morbidities, environmental factors, history of TB, radicular pain, pins and needles, numbness and spine tenderness.) were associated with greater odds (OR = 1.77-21.7, p < 0.001), whilst four (some centres, fatigue, fever and myotomal weakness) were associated with lower odds (OR = 0.51-0.59) of spine pain. Six factors were included in the final multivariable model associated with higher odds of spine pain: age groups above 16 years (OR 2.57 to 5.33, p < 0.05), co-morbidity (OR = 1.68, p < 0.05), history of TB (OR = 3.02, p < 0.05), weight loss (OR = 1.75, p < 0.01), radicular pain (OR = 19.88, p < 0.001); spine tenderness (OR = 6.54, p < 0.001). Myotomal weakness (OR = 0.66, p < 0.05) and fatigue (OR = 0.50, p < 0.01) were associated with lower odds of spinal pain in the final model. CONCLUSION: Using data from ten hospitals in Nigeria within a ten-year period, we have produced a shortlist of red flags that can inform clinical decision making about potential spinal infection.

Return to sport and patient satisfaction after arthroscopic Bankart repair: a single-institution experience.

Introduction: Arthroscopic Bankart repair is a widely accepted procedure to treat recurrent shoulder dislocation. This study aims to describe our experience with arthroscopic Bankart repair and its functional outcome. Methods: 107 patients who underwent arthroscopic Bankart repair from 2008 to 2013 were followed up for a minimum of three years and reviewed by an independent observer. 80 consented to being interviewed using the Oxford Shoulder Instability Score (OSIS) and Simple Shoulder Test. Results: 82 shoulders (two bilateral) were studied. Mean age at first dislocation was 19.4 ± 3.4 (12.0-31.0) years. Mean follow-up was 4.4 ± 1.3 (3.0-9.0) years and 2.5 ± 3.0 (0.1-15.4) years elapsed from first dislocation to surgery. 41 (50.0%) patients played overhead or contact sports and 44 (53.7%) played competitive sports before injury; 8 (9.8%) patients reported recurrence of dislocation, which was significantly associated with playing competitive sports before injury (p <0.039), 5 (6.1%) underwent revision surgery and 22 (26.8%) reported residual instability after surgery. 49 (59.8%) patients returned to playing sports, 75 (91.5%) were satisfied with their surgery and 79 (96.3%) were willing to undergo the surgery again. 74 (90.2%) patients had two-year good/excellent OSIS, which was significantly associated with playing competitive sports before injury (p = 0.039), self-reported stability after surgery (p = 0.017), satisfaction with surgery (p = 0.018) and willingness to undergo surgery again (p = 0.024). Conclusion: Arthroscopic Bankart repair yields good functional outcomes and is associated with high patient satisfaction, although not all patients return to sports.

Tumour infiltrating B cells discriminate checkpoint blockade-induced responses.

BACKGROUND: Immune cell-driven anti-cancer activity is paramount for effective responses to checkpoint inhibitors (ICB). However, the contribution of the different immune cell subsets in the circulation and within the tumour is poorly understood. MATERIALS AND METHODS: To elucidate the role of the different cell subsets in anti-tumour responses elicited by ICB, we performed single-cell analysis of the transcriptome and surface proteome of paired pre- and early on-treatment metastatic melanoma tumour biopsies and matched peripheral blood mononuclear cell samples. We next compared the survival of metastatic melanoma patients treated with ICB according to the abundance of pre-treatment tumour-infiltrating B cell clonotypes. RESULTS: We identified cell clusters associated with disease control or progression, defined differential expression of biological pathways likely involved in the immune awakening against the tumour and examined how cell-cell communication patterns between the tumour cell subsets change during treatment. Furthermore, we discovered that B cells (immunoglobulin expression and abundance of B cell clonotypes) discriminate the clinical response after ICB and propose that B cells likely contribute to anti-tumour immunity by antigen presentation through major histocompatibility complex molecules. Finally, we demonstrated that the abundance of tumour-infiltrating B cell clonotypes at baseline identifies two distinct risk groups, a finding that we confirmed in an independent cohort. CONCLUSIONS: Our exploratory translational study provides new insights on the mechanistic role of B cells in anti-melanoma immunity during treatment with ICB. Additionally, we support pre-treatment B cell tumour infiltration as a promising prognostic biomarker to be further validated as a tool for clinical risk stratification.

Safe Femoral Fixation Depth and Orientation for Lateral Extra-Articular Tenodesis in Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Patients who undergo anterior cruciate ligament (ACL) reconstruction (ACLR) can have a persistent postoperative pivot shift. Performing lateral extra-articular tenodesis (LET) concurrently has been proposed to address this; however, LET femoral fixation may interfere with the ACLR femoral tunnel, which could damage the ACL graft or its fixation. PURPOSE: To evaluate the safe maximum implant or tunnel depth for a modified Lemaire LET when combined with ACLR anteromedial portal femoral tunnel drilling and to validate the safe LET drilling angles to avoid conflict with the ACLR femoral tunnel. STUDY DESIGN: Descriptive laboratory study. METHODS: Twelve fresh-frozen cadaveric knees were used. With each knee at 120° of flexion, an ACLR femoral tunnel in the anteromedial bundle position was created arthroscopically via the anteromedial portal using a 5-mm offset guide, a guide wire, and an 8-mm reamer, which was left in situ. A modified Lemaire LET was performed using a 1 cm-wide iliotibial band strip harvested with the distal attachment intact, to be fixed in the femur. The desired LET fixation point was identified with an external aperture 10 mm proximal and 5 mm posterior to the fibular collateral ligament's femoral attachment, and a 2.4-mm guide wire was drilled, aiming at 0°, 10°, 20°, or 30° anteriorly in the axial plane and at 0°, 10°, or 20° proximally in the coronal plane (12 different drilling angle combinations). The relationship between the LET drilling guide wire and the ACLR femoral tunnel reamer was recorded for each combination. When a collision with the femoral tunnel was recorded, the LET wire depth was measured. RESULTS: Collision with the ACLR femoral tunnel occurred at a mean LET wire depth of 23.6 mm (range, 15-33 mm). No correlation existed between LET wire depth and LET drilling orientation (r = 0.066; P = .67). Drilling angle in the axial plane was significantly associated with the occurrence of tunnel conflict (P < .001). However, no such association was detected when comparing the drilling angle in the coronal plane (P = .267). CONCLUSION: Conflict of LET femoral fixation with the ACLR femoral tunnel using anteromedial portal drilling occurred at a mean depth of 23.6 mm but also at a depth as little as 15 mm, which is shorter than most implants. When longer implants or tunnels are used, the orientation should be directed at least 30° anteriorly in the axial plane to minimize the risk of tunnel conflict, bearing in mind the risk of joint violation. CLINICAL RELEVANCE: This study provides important information for surgeons performing LET in combination with ACLR anteromedial portal femoral tunnel drilling regarding safe femoral implant or tunnel length and orientation.

CKAP2L Promotes Non-Small Cell Lung Cancer Progression through Regulation of Transcription Elongation.

Chromosomal instability (CIN) is a driver of clonal diversification and intratumor heterogeneity, providing genetic diversity that contributes to tumor progression. It is estimated that approximately 80% of solid cancers, including non-small cell lung cancer (NSCLC), exhibit features of CIN, which affects tumor growth and response to therapy. However, the molecular mechanisms connecting CIN to tumor progression are still poorly understood. Through an RNAi screen performed on genes involved in CIN and overexpressed in human lung adenocarcinoma samples, we identified the cytoskeleton-associated protein 2-like (CKAP2L) as a potential oncogene that promotes lung cancer proliferation and growth in vitro and in vivo. Mechanistically, CKAP2L directly interacted with RNA Pol II and regulated transcription elongation of key genes involved in spindle assembly checkpoint, chromosome segregation, cell cycle, and E2F signaling. Furthermore, depletion of CKAP2L increased the sensitivity of NSCLC cells to alvocidib, a pan-CDK inhibitor, leading to a significant reduction of cell proliferation and an increase in cell death. Altogether, these findings shed light on the molecular mechanisms through which CKAP2L, a protein involved in CIN, promotes cancer progression and suggest that its inhibition represents a novel therapeutic strategy in NSCLC. SIGNIFICANCE: These findings demonstrate the oncogenic function of CKAP2L through regulation of transcription elongation and suggest that targeting CKAP2L could enhance therapeutic response in patients with NSCLC.