Modern multiligament knee injury surgical reconstruction techniques can achieve excellent knee function and patient satisfaction, with low complication rates.

PURPOSE: To report on the recovery of strength and functional capacity symmetry following multiligament knee surgical reconstruction (MLKR), as well as the capacity of athletes to return to sport. METHODS: This prospective cohort study recruited 47 patients undergoing MLKR between February 2018 and July 2021. Forty patients had full outcome assessment postoperatively at 6, 12 and 24 months and were included in the analysis, 75% were knee dislocation one injuries and 60% were injured playing sport. Patient-reported outcome measures (PROMs) assessed included the International Knee Documentation Committee score, the Knee Outcome Survey, the Lysholm Knee Score and the Tegner Activity Scale (TAS). Patient satisfaction was also assessed. Objective assessment included assessment of active knee flexion and extension range of motion (ROM), the single (single horizontal hop for distance [SHD]) and triple (triple horizontal hop for distance [THD]) hop tests for distance and peak isokinetic knee flexor/extensor torque. RESULTS: All PROMs significantly improved (p < 0.001) from presurgery to 24 months postsurgery. At 24 months, 70% of patients were satisfied with their sports participation. Active knee flexion (p < 0.0001) and extension (p < 0.0001) ROM significantly improved over time, as did the limb symmetry indices (LSIs) for the SHD (p < 0.0001), THD (p < 0.0001), peak knee extensor (p < 0.0001) and flexor (p = 0.012) torque. While LSIs for the SHD, THD and knee flexor strength tended to plateau by 12 months, knee extensor strength continued to improve from 12 to 24 months. CONCLUSIONS: The majority of patients undergoing modern MLKR surgical techniques and rehabilitation can achieve excellent knee function, with low complication rates. LEVEL OF EVIDENCE: Level IV.

Combined quadriceps medial patellofemoral ligament reconstruction and osteochondral fixation offers good patient-reported outcomes and low rates of recurrent instability for osteochondral defects secondary to acute patella dislocation.

PURPOSE: Chondral injuries secondary to traumatic patella dislocation are common, and a subgroup of these are significant defects with fragments amenable to fixation. There is a paucity of published evidence assessing patients managed with combined acute patellofemoral stabilisation and osteochondral fixation. The purpose of this study is to report the outcomes of patients with osteochondral injuries secondary to acute traumatic patella dislocation treated with combined early fragment fixation and MPFL reconstruction using a quadriceps tendon turndown technique which has distinct advantages for this cohort, including preventing chondral overloading and non-violation of the patella bone. METHODS: Patients who underwent combined quadriceps tendon MPFL reconstruction and osteochondral fixation were included. Patient demographics, defect characteristics, complications and reoperations were evaluated. Patients were assessed with Lysholm, Kujala, KOOS-PF scores and satisfaction scale at follow up. Pre-operative MRI was assessed for presence of radiological risk factors for patella dislocation and post-operative MRI was used to assess cartilage quality with MOCART 2.0 score. RESULTS: A total of 19 patients (63.2% female) were included. The mean age was 17.4 ± 4.8 years and patients were followed up at a mean 15.8 ± 5.1 months post-surgery. The mean defect size was 2.4 cm2 ± 1.3 cm2, with the most common defect location being the patella (13/19; 68.4%) followed by the lateral femoral condyle (5/19; 26.3%). At final follow up, the overall mean Lysholm, Kujala, and KOOS-PF scores were 84.9 ± 11.1, 89.7 ± 5.8 and 80.6 ± 13.6, respectively. Seventeen patients (89.5%) were satisfied with their outcome. The mean MOCART 2.0 score at final follow-up was 72.5. One patient required medial capsular plication with removal of a loose chondral body and microfracture and 3 knees required minor reoperations. CONCLUSION: Combined acute osteochondral fragment fixation and MPFL reconstruction using a quadriceps tendon graft offers good radiological and patient-reported outcomes with high satisfaction and low rates of recurrent patella dislocation. To our knowledge, this is currently the largest series of its kind in the literature and the results of this study provide a rationale for a combined approach using a quadriceps tendon graft for this cohort. LEVEL OF EVIDENCE: Level IV.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

Dissecting CLL through high-dimensional single-cell technologies.

We now have the potential to undertake detailed analysis of the inner workings of thousands of cancer cells, one cell at a time, through the emergence of a range of techniques that probe the genome, transcriptome, and proteome combined with the development of bioinformatics pipelines that enable their interpretation. This provides an unprecedented opportunity to better understand the heterogeneity of chronic lymphocytic leukemia and how mutations, activation states, and protein expression at the single-cell level have an impact on disease course, response to treatment, and outcomes. Herein, we review the emerging application of these new techniques to chronic lymphocytic leukemia and examine the insights already attained through this transformative technology.

No clinical difference in 10-year outcomes between standard and minimal graft debridement techniques in patients undergoing anterior cruciate ligament reconstruction using autologous hamstrings: a randomized controlled trial.

PURPOSE: Delayed ligamentization following anterior cruciate ligament reconstruction (ACLR) may result in reduced graft stiffness and strength, and an increased risk of secondary re-tear. Remnant sparing ACLR may accelerate ligamentization and proprioceptive function, theoretically reducing re-injury risk. This study sought to investigate 10-year graft failure rates and patient perceived knee functioning in those undergoing ACLR with remnant preservation (RP), versus remnant debridement (RD). METHODS: A prospective RCT allocated 49 patients to ACLR with a hamstrings autograft together with a RD (n = 25) or RP (n = 24) procedure, of which 86% were clinically evaluated at 10 years (22 RD, 22 RP). A detailed chart review and patient phone consultation was undertaken with all patients at 10 years to evaluate the incidence (and timing) of subsequent re-tear and/or contralateral ACL tear, as well as other knee injuries/surgeries, the patient's ability to perform full work/sport duties and their perceived knee function using a numerical rating scale (NRS). RESULTS: No significant differences existed between groups in descriptive variables. There were 2 graft ruptures (10.0%) in the RP group and 3 (13.6%) in the RD group, with an earlier mean time to graft failure in the RD group (RD 7.7 ± 4.5 months, RP 49.5 ± 17.7 months), albeit the size of this sub-sample was too small for statistical comparison. There was a significantly higher number of patients requiring ≥ 1 additional ipsilateral knee surgery in the RP group (RP = 10, RD = 4, p = 0.048). At 10 years, there were no significant group differences in the percentage of patients returning to unrestricted activity, with 16 (72.7%) and 15 (75.0%) patients in the RD and RP ACLR groups, respectively, unrestricted in work/sport duties. There were no significant group differences in the functional NRS ratings. CONCLUSIONS: No long term clinical benefit of RP ACLR could be determined by this study with similar re-tear incidence and perceived knee function. A statistically higher number of re-operations were observed in RP ACLR patients and, while re-tears were observed later after RP versus RD ACLR, the study was underpowered to detect statistical significance. LEVEL OF EVIDENCE: Level II (prospective randomized controlled trial).

Autologous stem cell transplantation outcomes in elderly patients with B cell Non-Hodgkin Lymphoma.

The precise role of autologous haematopoietic stem cell transplantation (ASCT) remains unclear in patients over 60 years of age. There is potential for increased procedural morbidity and mortality, and differences in disease biology that could impact outcomes. We performed a retrospective single-centre review of 81 elderly B-cell Non-Hodgkin Lymphoma patients undergoing ASCT. Five-year overall survival (OS) and progression-free survival (PFS) was 54·7% and 49·1% respectively. Non-relapse mortality (NRM) at 100 days and 1 year was 1·3% and 2·5%, suggesting no major excess compared to younger cohorts. OS and PFS were significantly worse in those over 65 years compared to those aged 60-64 (47·6% vs. 57·7%, P = 0·0437, and 27·6% vs. 57·7%, P = 0·0052 at 5 years). This resulted largely from an increased relapse risk (RR) (53·8% vs. 30·1%, P = 0·0511) rather than excess NRM, and age remained independently significant for PFS on multivariate analyses [Hazard ratio 2·56 (1·35-4·84, P = 0·0052) for PFS and 1·89 (0·99-3·61, P = 0·054) for OS]. Our data adds to the growing body of evidence demonstrating that ASCT can be an effective treatment strategy with an acceptable safety profile in selected elderly patients. Further evaluation of its overall benefit is warranted, however, in those over 65 years of age, as RR appears to be considerably higher.

Serial MRI and clinical assessment of cyclops lesions.

PURPOSE: 'Clinical cyclops syndrome' is associated with pain and a palpable 'clunk' at terminal extension with the loss of full extension. The aims of this prospective controlled study were: (1) to assess whether the minimal debridement of the ACL stump and notch is associated with an increased incidence of clinical cyclops lesions, (2) to look at the incidence and natural history of 'MRI cyclops' lesions using serial MRI's and (3) to assess whether 'MRI cyclops' lesions are associated with the loss of extension. METHODS: Forty-eight patients were randomized for ACL reconstruction into standard (23) and minimal debridement (24) techniques. One patient was excluded from the study. All patients underwent MR scanning postoperatively at 2, 6 and 12 months, together with the clinical assessment using a KT-1000 arthrometer and International Knee Documentation Committee evaluation. All observations were made by investigators blinded to the surgical technique. RESULTS: There was no statistical difference in the incidence of cyclops lesions between the two groups (n.s.). The overall incidence of cyclops lesions was 46.8% (22 of 47). The natural history is variable with some getting larger, smaller or remaining static in size. Of patients with cyclops lesions, 17 patients (77%) had cyclops lesions in the setting of full extension. Five patients (23%) had loss of extension at 12 months with no MRI cyclops detected at 2 months. CONCLUSIONS: The natural history is variable; although once present, the majority of cyclops remain static or regress in size. The onset of cyclops lesions is usually between 6- and 12-month post-ACL reconstruction. Minimal debridement does not lead to an increased incidence of clinical cyclops lesions. The authors conclude that loss of extension is multi-factorial, and there is a discrepancy between what we term 'MRI cyclops' and true 'clinical cyclops'. LEVEL OF EVIDENCE: Case-control study, Level II.

A case report and the literature review: volar dislocation of the DRUJ and stabilisation using mini-suture anchors.

Volar dislocation of the distal radio-ulnar joint is a rare clinical entity in the orthopaedic literature, and there is little to describe the patho-anatomy, investigative or treatment modalities. We describe the case of an irreducible locked volar dislocation with novel magnetic resonance imaging findings of an ulnar impaction fracture with cartilaginous injury and successful surgical stabilisation using a suture anchor technique.

DIISCO: A Bayesian framework for inferring dynamic intercellular interactions from time-series single-cell data.

Characterizing cell-cell communication and tracking its variability over time is essential for understanding the coordination of biological processes mediating normal development, progression of disease, or responses to perturbations such as therapies. Existing tools lack the ability to capture time-dependent intercellular interactions, such as those influenced by therapy, and primarily rely on existing databases compiled from limited contexts. We present DIISCO, a Bayesian framework for characterizing the temporal dynamics of cellular interactions using single-cell RNA-sequencing data from multiple time points. Our method uses structured Gaussian process regression to unveil time-resolved interactions among diverse cell types according to their co-evolution and incorporates prior knowledge of receptor-ligand complexes. We show the interpretability of DIISCO in simulated data and new data collected from CAR-T cells co-cultured with lymphoma cells, demonstrating its potential to uncover dynamic cell-cell crosstalk.

The immune microenvironment shapes transcriptional and genetic heterogeneity in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor-microenvironment interactions, and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared with PB and in immunoglobulin heavy-chain variable (IGHV) region unmutated compared with mutated cases. Single-cell RNA sequencing (scRNA-seq) distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival (TFS) and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing (WES) of paired PB and LN samples showed subclonal expansion in LN in approximately half of the patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives the clinical outcome. Clonal trajectories are shaped by the LN milieu, where T-cell immunity may contribute to suppressing clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.

Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia.

In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukemia (R/R B-ALL), CD19-targeting chimeric antigen receptor (CAR)-T cells often induce durable remissions, which requires the persistence of CAR-T cells. In this study, we systematically analyzed CD19 CAR-T cells of 10 children with R/R B-ALL enrolled in the CARPALL trial via high-throughput single-cell gene expression and T cell receptor sequencing of infusion products and serial blood and bone marrow samples up to 5 years after infusion. We show that long-lived CAR-T cells developed a CD4/CD8 double-negative phenotype with an exhausted-like memory state and distinct transcriptional signature. This persistence signature was dominant among circulating CAR-T cells in all children with a long-lived treatment response for which sequencing data were sufficient (4/4, 100%). The signature was also present across T cell subsets and clonotypes, indicating that persisting CAR-T cells converge transcriptionally. This persistence signature was also detected in two adult patients with chronic lymphocytic leukemia with decade-long remissions who received a different CD19 CAR-T cell product. Examination of single T cell transcriptomes from a wide range of healthy and diseased tissues across children and adults indicated that the persistence signature may be specific to long-lived CAR-T cells. These findings raise the possibility that a universal transcriptional signature of clinically effective, persistent CD19 CAR-T cells exists.

ZNF683 marks a CD8+ T cell population associated with anti-tumor immunity following anti-PD-1 therapy for Richter syndrome.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.

cyCombine allows for robust integration of single-cell cytometry datasets within and across technologies.

Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. However, in many cases the full potential of co-analyses is not reached due to technical variance between data from different experimental batches. Here, we present cyCombine, a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques, such as CITE-seq, flow cytometry, and mass cytometry. We demonstrate that cyCombine maintains the biological variance and the structure of the data, while minimizing the technical variance between datasets. cyCombine does not require technical replicates across datasets, and computation time scales linearly with the number of cells, allowing for integration of massive datasets. Robust, accurate, and scalable integration of cytometry data enables integration of multiple datasets for primary data analyses and the validation of results using public datasets.