Chronic Myeloid Leukemia, Version 2.2024, NCCN Clinical Practice Guidelines in Oncology.

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome resulting from a reciprocal translocation between chromosomes 9 and 22 [t9;22] that gives rise to a BCR::ABL1 fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase in developed countries. Tyrosine kinase inhibitor (TKI) therapy is a highly effective treatment option for patients with chronic phase-CML. The primary goal of TKI therapy in patients with chronic phase-CML is to prevent disease progression to accelerated phase-CML or blast phase-CML. Discontinuation of TKI therapy with careful monitoring is feasible in selected patients. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with chronic phase-CML.

Generation of antigen-specific mature T cells from RAG1-/-RAG2-/-B2M-/- stem cells by engineering their microenvironment.

Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1-/-RAG2-/-B2M-/- human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.

Strength of CAR signaling determines T cell versus ILC differentiation from pluripotent stem cells.

Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.

Evaluation of a modified radiographic union scale for tibial fractures scoring system in staged bilateral tibial plateau leveling osteotomy procedures and comparison of first and second side radiographic bone healing.

OBJECTIVE: To evaluate the inter- and intra-rater reliability of the tibial plateau leveling osteotomy (TPLO) modified radiographic union scale for tibial fractures (mRUST), a semiquantitative scoring system, as compared with the subjective evaluation of radiographic union for staged TPLOs. STUDY DESIGN: Retrospective study. ANIMALS: Forty-eight dogs, 96 stifles. METHODS: Medical records were reviewed for dogs with bilateral cranial cruciate ligament injuries diagnosed at presentation that underwent staged bilateral TPLOs within 6 months and had both immediate and recheck postoperative radiographs. Radiographs were anonymized, reviewers were blinded, radiographic union was evaluated subjectively, and TPLO mRUST scores were assigned. RESULTS: The subjective evaluation's intra-rater reliability was 0.72 (Kappa 95% CI 0.65-0.79) and inter-rater reliability was 0.33 (Kappa 95% CI 0.28-0.39). The TPLO mRUST scoring system intra-rater reliability was 0.73 (95% CI 0.68-0.78) and inter-rater reliability was 0.56 (95% CI 0.41-0.68). There was no difference in the degree of bone healing quantified by the TPLO mRUST scoring system (95% CI - 0.1-1.2, P = .09) or subjective evaluation (P = .48) between the first and second side TPLOs. The TPLO mRUST scores were positively correlated with subjective healing (r = 0.94, 95% CI 0.92-0.96, P < .0001, and for scores ≥10/12, 99%, 244/246) were subjectively assigned as radiographically healed. CONCLUSION: The TPLO mRUST scoring system improved inter-rater reliability compared to subjective evaluation of radiographic union. CLINICAL SIGNIFICANCE: The TPLO mRUST scoring system should be considered as a semiquantitative supplemental tool for evaluating radiographic union.

Generation of Artificial Thymic Organoids from Human and Murine Hematopoietic Stem and Progenitor Cells.

The generation of T cells is a complex, carefully orchestrated process that occurs in the thymus. The ability to mimic T cell differentiation in vitro has opened up avenues to better understand different stages of thymopoiesis but has also enabled the in vitro production of mature T cells suitable for immunotherapy. Among existing protocols, the artificial thymic organoid (ATO) system has been shown to be the most efficient at producing mature conventional T cells. In this serum-free model, human or murine hematopoietic stem and progenitor cells (HSPCs) are combined with a murine stromal cell line expressing a Notch ligand in a 3D cell aggregate. In ATOs, although only simple medium changes are required throughout the cultures, HSPCs differentiate into T cells with kinetics and phenotypes similar to those of endogenous thymopoiesis. This article describes protocols for the generation of ATOs from human and murine HSPCs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expansion and preparation of MS5-hDLL4 or MS5-mDLL4 cells Basic Protocol 2: Isolation of human hematopoietic stem and progenitor cells (HSPCs; CD34+ cells) Support Protocol 1: Transduction of human HSPCs (CD34+ cells) Basic Protocol 3: Production of thymic progenitors and mature T cells from human HSPCs in artificial thymic organoids (ATOs) Support Protocol 2: Phenotype analysis of human ATO cells by flow cytometry Basic Protocol 4: Isolation of murine HSPCs (Lin- Sca1+ cKit+; LSK) and hematopoietic stem cells (LSK CD150+ CD48-) Basic Protocol 5: Production of thymic progenitors and mature T cells from murine HSPCs in ATOs Support Protocol 3: Phenotype analysis of murine ATO cells by flow cytometry Alternate Protocol: Generation of ATOs from single HSPCs.

Humeral Head Morphology Influences Outcomes of Arthroscopic Interposition Glenoid Patch Allograft for Glenohumeral Arthritis.

PURPOSE: To determine short- to midterm patient-reported outcomes of arthroscopic soft-tissue interposition arthroplasty using acellular dermal allograft with a minimum follow-up of 1 year and to assess outcomes in patients with and without flattening of the humeral head. METHODS: Patients with a diagnosis of primary glenohumeral arthritis who underwent arthroscopic soft-tissue interposition arthroplasty with an acellular dermal allograft from July 2010 to November 2019 were retrospectively enrolled. Inclusion criteria were a primary diagnosis of glenohumeral arthritis and Outerbridge 4 full-thickness cartilage loss of ≥50% of the glenoid articular surface. Patients underwent arthroscopic debridement, microfracture, and biological arthroscopic soft-tissue interposition arthroplasty with an acellular dermal matrix. Postoperative outcomes included American Shoulder and Elbow Surgeon (ASES) score, Single Assessment Numeric Evaluation (SANE) score, Penn Shoulder Score (PSS), numeric rating scale (NRS) pain score, analgesic use, and conversion to total shoulder arthroplasty (TSA). Results were stratified according to humeral head morphology on preoperative radiographs. RESULTS: A total of 25 patients were included, with a mean age of 56.0 years (range 19.2 to 74.8) and a mean follow-up of 3.36 years (range 1.03 to 8.98). The mean postoperative ASES score was 64.1 (range 11.7 to 100.0), SANE score was 62% (range 5% to 100%), and PSS was 61.2 (range 10.6 to 97.9). Additionally, 56% of patients rated their shoulder function as improved or much improved, and 36% of patients converted to TSA at a mean of 2.35 years. Patients with and without humeral flattening had similar postoperative ASES scores (P = .44), SANE scores (P = .90), PSS (P = .73), and conversions to TSA (P = .83). Patients with humeral flattening were more likely to have shoulder pain at night (83.3% versus 28.6%, P = .02). CONCLUSION: Arthroscopic soft-tissue interposition arthroplasty with an acellular dermal allograft resulted in satisfactory short- to mid-term postoperative outcomes for younger patients with glenohumeral arthritis but demonstrated a TSA conversion rate of 36%. Patients with humeral head flattening also had satisfactory shoulder function but were more likely to experience shoulder pain at night. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Selective and noncovalent targeting of RAS mutants for inhibition and degradation.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells.

We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαß and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1-/- marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.

The Clinical Spectrum of PTEN Hamartoma Tumor Syndrome: Exploring the Value of Thyroid Surveillance.

INTRODUCTION: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) comprises a collection of clinical features characterized by constitutional variants in PTEN. Several guidelines recommend thyroid screening, beginning at the pediatric age at the time of PHTS diagnosis; however, the benefits of early surveillance has not been well defined. METHODS: We conducted a retrospective investigation of patients followed up at the Children's Hospital of Philadelphia with a diagnosis of PHTS between January 2003 and June 2019. In total, 81 patients younger than 19 years were identified. RESULTS: The most common clinical feature at presentation was macrocephaly (85.1%), followed by impaired development (42.0%), skin/oral lesions (30.9%), and autism spectrum disorder (27.2%). A total of 58 of 81 patients underwent thyroid surveillance, with 30 patients (51.7%) found to have a nodule(s). Ultimately, 16 patients underwent thyroidectomy, with 7.4% (6/81) diagnosed with thyroid cancer. All thyroid cancer patients were older than 10 years at diagnosis, and all displayed low-invasive behavior. Of the patients younger than 10 years at the time of thyroid ultrasound (US) surveillance, 71.4% (15/21) had a normal US. The remaining 6 patients had thyroid nodules, including 4 undergoing thyroid surgery with benign histology. DISCUSSION/CONCLUSION: Patients with macrocephaly, impaired cognitive development and thyroid nodules, and/or early-onset gastrointestinal polyps should undergo constitutional testing for PHTS. There does not appear to be a clinical advantage to initiating thyroid US surveillance before 10 years of age. In PHTS patients with a normal physical examination, thyroid US surveillance can be delayed until 10 years of age.

Clinical Utility of Intraoperative Parathyroid Hormone Measurement in Children and Adolescents Undergoing Total Thyroidectomy.

Background: Hypoparathyroidism is one of the most common complications for patients undergoing total thyroidectomy. Our study's primary objective was to assess if intraoperative PTH levels correlate with parathyroid gland function recovery time in pediatric patients following total thyroidectomy. Methods: Retrospective review of pediatric patients who underwent thyroid surgery at CHOP for demographics and laboratory test values (calcium, phosphorus, and parathyroid hormone). We defined Time of Recovery (TOR) as the time difference from first intra-operative parathyroid hormone level (ioPTH) timepoint until normalization of PTH (> 10 pg/mL) post-thyroidectomy. Calcium and vitamin D supplements were weaned following normalization of calcium and phosphorous levels postoperatively. Patients were excluded if they lacked three intraoperative PTH timepoints or were missing postoperative follow-up PTH data. Results: 65 patients (54 female), median age 15 (range 5-23 years), underwent thyroid surgery and met study inclusion criteria. The correlations of 2nd and 3rd ioPTHs with TOR were statistically significant (p < 0.05): the lower the ioPTH, the greater the recovery time. Stratifying patients into high-risk (2nd ioPTH ≤ 10 pg/mL), moderate-risk (2nd ioPTH between 10 and 20 pg/mL), and low-risk (2nd ioPTH ≥ 20 pg/mL) tertiles, the TOR decreased by orders of magnitudes from an average of 43.13 ± 76.00 to 6.10 ± 17.44 to 1.85 ± 6.20 days. These differences were statistically significant (p < 0.05). Conclusions: Our study results confirm the usefulness of intraoperative PTH levels to predict pediatric patient recovery post-surgery and provides useful anticipatory guidance to optimize timing and frequency of postoperative laboratory surveillance.

Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells.

The ability to generate T cells from pluripotent stem cells (PSCs) has the potential to transform autologous T cell immunotherapy by facilitating universal, off-the-shelf cell products. However, differentiation of human PSCs into mature, conventional T cells has been challenging with existing methods. We report that a continuous 3D organoid system induced an orderly sequence of commitment and differentiation from PSC-derived embryonic mesoderm through hematopoietic specification and efficient terminal differentiation to naive CD3+CD8αß+ and CD3+CD4+ conventional T cells with a diverse T cell receptor (TCR) repertoire. Introduction of an MHC class I-restricted TCR in PSCs produced naive, antigen-specific CD8αß+ T cells that lacked endogenous TCR expression and showed anti-tumor efficacy in vitro and in vivo. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naive CD8+ T cells. The PSC-artificial thymic organoid (ATO) system presented here is an efficient platform for generating functional, mature T cells from human PSCs.

Facile target validation in an animal model with intracellularly expressed monobodies.

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

Azygos continuation of the caudal vena cava with segmental aneurysm, lung lobe torsion and pulmonary thromboembolism in a dog.

This case highlights the management and diagnostic evaluation of a dog with two individually rare conditions (lung lobe torsion and vena cava aneurysm) that ultimately resulted in fatal pulmonary thromboembolism.

DNA mismatch repair deficiency and hereditary syndromes in Latino patients with colorectal cancer.

BACKGROUND: The landscape of hereditary syndromes and clinicopathologic characteristics among US Latino/Hispanic individuals with colorectal cancer (CRC) remains poorly understood. METHODS: A total of 265 patients with CRC who were enrolled in the Hispanic Colorectal Cancer Study were included in the current study. Information regarding CRC risk factors was elicited through interviews, and treatment and survival data were abstracted from clinical charts. Tumor studies and germline genetic testing results were collected from medical records or performed using standard molecular methods. RESULTS: The mean age of the patients at the time of diagnosis was 53.7 years (standard deviation, 10.3 years), and 48.3% were female. Overall, 21.2% of patients reported a first-degree or second-degree relative with CRC; 3.4% met Amsterdam I/II criteria. With respect to Bethesda guidelines, 38.5% of patients met at least 1 criterion. Of the 161 individuals who had immunohistochemistry and/or microsatellite instability testing performed, 21 (13.0%) had mismatch repair (MMR)-deficient (dMMR) tumors. dMMR tumors were associated with female sex (61.9%), earlier age at the time of diagnosis (50.4 ± 12.4 years), proximal location (61.9%), and first-degree (23.8%) or second-degree (9.5%) family history of CRC. Among individuals with dMMR tumors, 13 (61.9%) had a germline MMR mutation (MutL homolog 1 [MLH1] in 6 patients; MutS homolog 2 [MSH2] in 4 patients; MutS homolog 6 [MHS6] in 2 patients; and PMS1 homolog 2, mismatch repair system component [PMS2] in 1 patient). The authors identified 2 additional MLH1 mutation carriers by genetic testing who had not received immunohistochemistry/microsatellite instability testing. In total, 5.7% of the entire cohort were confirmed to have Lynch syndrome. In addition, 6 individuals (2.3%) had a polyposis phenotype. CONCLUSIONS: The percentage of dMMR tumors noted among Latino individuals (13%) is similar to estimates in non-Hispanic white individuals. In the current study, the majority of individuals with dMMR tumors were confirmed to have Lynch syndrome. Cancer 2017. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. Cancer 2017;123:3732-3743. © 2017 American Cancer Society.

Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

Imaging characteristics of intrathoracic histiocytic sarcoma in dogs.

In this retrospective study, two observers independently reviewed thoracic imaging studies of 39 dogs with confirmed histiocytic sarcoma. The most common findings were intrathoracic lymphadenopathy, identified by the first and second observers in 82.1% and 87.2% of dogs, respectively, and pulmonary masses (74.4% and 82.1%). Right middle lung lobe masses were significantly more common than masses in any other lung lobe (P < 0.0013), with the majority having a ventral distribution. Sternal and tracheobronchial lymphadenopathy were significantly more common than cranial mediastinal lymphadenopathy (P-values of 0.0002 and 0.012, respectively). Interobserver agreement regarding distribution of lymphadenopathy and pulmonary masses was good (kappa = 0.64 and 0.75, respectively). Other findings included pulmonary nodules, pleural effusion, and abnormal pulmonary patterns. In patients with CT examinations, the majority of masses were mildly to moderately enhancing and heterogeneous, poorly marginated, and bronchocentric. Lymphadenopathy and pulmonary masses are the most common intrathoracic findings in dogs with histiocytic sarcoma, and the strong predilection for the ventral aspect of the right middle lung lube may help to differentiate it from other types of neoplasia.

Molecular characterization of the human NANOG protein.

NANOG is a key transcriptional regulator of pluripotent stem cell (PSC) self-renewal. NANOG occupies promoters that are active and others that are repressed during self-renewal; however, the mechanisms by which NANOG regulates transcriptional repression and activation are unknown. We hypothesized that individual protein domains of NANOG control its interactions with both the promoters and its coregulators. We performed a detailed characterization of the functional domains in the human (h) NANOG protein, using a panel of deletion-mutant and point-mutant constructs. We determined that six amino acids in the homeodomain ((136)YKQVKT(141)) are sufficient for the nuclear localization of hNANOG. We also determined that the tryptophan-rich region (W) of hNANOG contains a CRM1-independent signal for nuclear export, suggesting a possible cellular shuttling behavior that has not been reported for hNANOG. We also show that at least four tryptophans are required for nuclear export. We also determined that similar to murine (m) NANOG, the W region of hNANOG contains a homodimerization domain. Finally, in vitro transactivation analyses identified distinct regions that enhance or diminish activity at gene promoters that are active during self-renewal. Specifically, the N-terminal region interferes with transcription and removal of this region that produced a "super-active" hNANOG with enhanced transcriptional activity. We also confirmed that the transcriptional activator in hNANOG is contained in the C-terminal region, similar to murine NANOG. In summary, this study has characterized the structure and function of hNANOG protein leading to an increased understanding of the mechanism by which hNANOG regulates both transcriptional activation and repression during PSC self-renewal.