Utility of Clinical Next Generation Sequencing Tests in KIT/PDGFRA/SDH Wild-Type Gastrointestinal Stromal Tumors.

Objective: The vast majority of gastrointestinal stromal tumors (GISTs) are driven by activating mutations in KIT, PDGFRA, or components of the succinate dehydrogenase (SDH) complex (SDHA, SDHB, SDHC, and SDHD genes). A small fraction of GISTs lack alterations in KIT, PDGFRA, and SDH. We aimed to further characterize the clinical and genomic characteristics of these so-called "triple-negative" GISTs. Methods: We extracted clinical and genomic data from patients seen at MD Anderson Cancer Center with a diagnosis of GIST and available clinical next generation sequencing data to identify "triple-negative" patients. Results: Of the 20 patients identified, 11 (55.0%) had gastric, 8 (40.0%) had small intestinal, and 1 (5.0%) had rectal primary sites. In total, 18 patients (90.0%) eventually developed recurrent or metastatic disease, and 8 of these presented with de novo metastatic disease. For the 13 patients with evaluable response to imatinib (e.g., neoadjuvant treatment or for recurrent/metastatic disease), the median PFS with imatinib was 4.4 months (range 0.5-191.8 months). Outcomes varied widely, as some patients rapidly developed progressive disease while others had more indolent disease. Regarding potential genomic drivers, four patients were found to have alterations in the RAS/RAF/MAPK pathway: two with a BRAF V600E mutation and two with NF1 loss-of-function (LOF) mutations (one deletion and one splice site mutation). In addition, we identified two with TP53 LOF mutations, one with NTRK3 fusion (ETV6-NTRK3), one with PTEN deletion, one with FGFR1 gain-of-function (GOF) mutation (K654E), one with CHEK2 LOF mutation (T367fs*), one with Aurora kinase A fusion (AURKA-CSTF1), and one with FANCA deletion. Patients had better responses with molecularly targeted therapies than with imatinib. Conclusions: Triple-negative GISTs comprise a diverse cohort with different driver mutations. Compared to KIT/PDGFRA-mutant GIST, limited benefit was observed with imatinib in triple-negative GIST. In depth molecular profiling can be helpful in identifying driver mutations and guiding therapy.

The Future of Targeted Therapy for Leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive subtype of soft tissue sarcoma that arises from smooth muscle cells, most commonly in the uterus and retroperitoneum. LMS is a heterogeneous disease with diverse clinical and molecular characteristics that have yet to be fully understood. Molecular profiling has uncovered possible targets amenable to treatment, though this has yet to translate into approved targeted therapies in LMS. This review will explore historic and recent findings from molecular profiling, highlight promising avenues of current investigation, and suggest possible future strategies to move toward the goal of molecularly matched treatment of LMS. We focus on targeting the DNA damage response, the macrophage-rich micro-environment, the PI3K/mTOR pathway, epigenetic regulators, and telomere biology.

Clinical Outcomes Among Immunotherapy-Treated Patients With Primary Cardiac Soft Tissue Sarcomas: A Multicenter Retrospective Study.

Background: Primary cardiac soft tissue sarcomas (CSTS) affect young adults, with dismal outcomes. Objectives: The aim of this study was to investigate the clinical outcomes of patients with CSTS receiving immune checkpoint inhibitors (ICIs). Methods: A retrospective, multi-institutional cohort study was conducted among patients with CSTS between 2015 and 2022. The patients were treated with ICI-based regimens. The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). Objective response rates were determined according to Response Evaluation Criteria in Solid Tumors version 1.1. Treatment-related adverse events were graded per the Common Terminology Criteria for Adverse Events version 5.0. Results: Among 24 patients with CSTS, 17 (70.8%) were White, and 13 (54.2%) were male. Eight patients (33.3%) had angiosarcoma. At the time of ICI treatment, 18 patients (75.0%) had metastatic CSTS, and 4 (16.7%) had locally advanced disease. ICIs were administered as the first-line therapy in 6 patients (25.0%) and as the second-line therapy or beyond in 18 patients (75.0%). For the 18 patients with available response data, objective response rate was 11.1% (n = 2 of 18). The median PFS and median OS in advanced and metastatic CSTS (n = 22) were 5.7 months (95% CI: 2.8-13.3 months) and 14.9 months (95% CI: 5.7-23.7 months), respectively. The median PFS and OS were significantly shorter in patients with cardiac angiosarcomas than in those with nonangiosarcoma CSTS: median PFS was 1.7 vs 11 months, respectively (P < 0.0001), and median OS was 3.0 vs 24.0 months, respectively (P = 0.008). Any grade treatment-related adverse events occurred exclusively in the 15 patients with nonangiosarcoma CSTS (n = 7 [46.7%]), of which 6 (40.0%) were grade ≥3. Conclusions: Although ICIs demonstrate modest activity in CSTS, durable benefit was observed in a subset of patients with nonangiosarcoma, albeit with higher toxicity.

Pleural fluid characteristics of patients with COVID-19 infection.

INTRODUCTION: Pleural effusions are known to occur in many cases of COVID-19. Data on typical characteristics of COVID-19-associated pleural effusions are limited. The goal of this project was to characterize the pleural fluid from patients with COVID-19. METHODS: We retrospectively collected electronic medical record data from adults hospitalized at a large metropolitan hospital system with COVID-19 infection who had a pleural effusion and a thoracentesis performed. We assessed pleural fluid characteristics and applied Light's criteria. RESULTS: We identified 128 effusions from 106 unique patients; 45.4% of the effusions had fluid/serum protein ratio greater than 0.5, 33.9% had fluid/serum lactate dehydrogenase (LDH) greater than 0.6, and 56.2% had fluid LDH greater than 2/3 of the serum upper limit of normal. Altogether, 68.5% of effusions met at least one of these three characteristics and therefore were exudative by Light's criteria. The white blood cell (WBC) differential was predominantly lymphocytic (mean 42.8%) or neutrophilic (mean 28.7%); monocytes (mean 12.7%) and eosinophils (mean 2.5%) were less common. CONCLUSION: We demonstrate that 68.5% of pleural effusions in patients with COVID-19 infection were exudative and hypothesize that COVID-19-associated pleural effusions are likely to be exudative with WBC differential more likely to be predominantly lymphocytic.

Clinico-Genomic Profiling of Conventional and Dedifferentiated Chondrosarcomas Reveals TP53 Mutation to Be Associated with Worse Outcomes.

PURPOSE: Chondrosarcomas are the most common primary bone tumor in adults. Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are prevalent. We aimed to assess the clinico-genomic properties of IDH mutant versus IDH wild-type (WT) chondrosarcomas as well as alterations in other genes. EXPERIMENTAL DESIGN: We included 93 patients with conventional and dedifferentiated chondrosarcoma for which there were available clinical next-generation sequencing data. Clinical and genomic data were extracted and compared between IDH mutant and IDH WT chondrosarcomas and between TP53 mutant and TP53 WT chondrosarcomas. RESULTS: IDH1 and IDH2 mutations are prevalent in chondrosarcoma (50.5%), more common in chondrosarcomas arising in the extremities, associated with higher age at diagnosis, and more common in dedifferentiated chondrosarcomas compared with grades 1-3 conventional chondrosarcoma. There was no difference in survival based on IDH mutation in univariate and multivariate analyses. TP53 mutation was the next most prevalent (41.9%) and is associated with worse overall survival and metastasis-free survival in both univariate and multivariate analyses. TP53 mutation was also associated with higher risk of recurrence following curative-intent surgery and worse survival among patients that presented with de novo metastatic disease. CONCLUSIONS: IDH mutations are prevalent in chondrosarcoma though were not associated with survival outcomes in this cohort. TP53 mutations were the next most common alteration and were associated with worse outcomes.

The Landscape of Alterations from 1407 Ultra-Rare Sarcomas from the AACR GENIE Database: Clinical Implications.

PURPOSE: Ultra-rare sarcomas (URS) comprise a group of orphan diseases with an incidence of ≤1/1,000,000 people per year. We aimed to assess clinically actionable genomic alterations in URS. EXPERIMENTAL DESIGN: Data were extracted from the GENIE database using cBioPortal. OncoKB was used to assess for clinical actionability of mutations. Tumor mutational burden (TMB) was inferred from clinical sequencing data. RESULTS: Soft tissue (ST) URS made up 23.5% of ST sarcoma cases, and bone URS made up 16.5% of bone sarcoma cases. The most commonly mutated gene in all four groups was TP53. The most common fusions involved EWSR1. The most common copy-number variations included deletions of CDKN2A and CDKN2B and amplifications of MDM2 and CDK4. TMB was generally low across all four categories of sarcoma, though there was considerable heterogeneity, with 3.8% of ST URS and 0.55% of bone URS having high TMB. We find Level 1 alterations (FDA-recognized biomarker predictive of response to an FDA-approved drug) in 10.0% of ST URS compared with 7.1% of ST non-URS, 1.1% of bone URS, and 4.5% of bone non-URS. Level 1-3 alterations (also include alterations for which there are standard-of-care drugs or clinical evidence supporting a drug) were seen in 27.8% of ST URS, 25.2% of ST non-URS, 20.9% of bone URS, and 17.4% of bone non-URS. CONCLUSIONS: Clinically actionable genomic alterations are seen in a substantial fraction of URS. Clinical sequencing in advanced URS has the potential to guide the treatment of a significant portion of patients with URS.

Misaligned Chromosomes are a Major Source of Chromosomal Instability in Breast Cancer.

Chromosomal instability (CIN), the persistent reshuffling of chromosomes during mitosis, is a hallmark of human cancers that contributes to tumor heterogeneity and has been implicated in driving metastasis and altering responses to therapy. Though multiple mechanisms can produce CIN, lagging chromosomes generated from abnormal merotelic attachments are the major cause of CIN in a variety of cell lines, and are expected to predominate in cancer. Here, we quantify CIN in breast cancer using a tumor microarray, matched primary and metastatic samples, and patient-derived organoids from primary breast cancer. Surprisingly, misaligned chromosomes are more common than lagging chromosomes and represent a major source of CIN in primary and metastatic tumors. This feature of breast cancers is conserved in a majority of breast cancer cell lines. Importantly, though a portion of misaligned chromosomes align before anaphase onset, the fraction that remain represents the largest source of CIN in these cells. Metastatic breast cancers exhibit higher rates of CIN than matched primary cancers, primarily due to increases in misaligned chromosomes. Whether CIN causes immune activation or evasion is controversial. We find that misaligned chromosomes result in immune-activating micronuclei substantially less frequently than lagging and bridge chromosomes and that breast cancers with greater frequencies of lagging chromosomes and chromosome bridges recruit more stromal tumor-infiltrating lymphocytes. These data indicate misaligned chromosomes represent a major mechanism of CIN in breast cancer and provide support for differential immunostimulatory effects of specific types of CIN. Significance: We surveyed the single-cell landscape of mitotic defects that generate CIN in primary and metastatic breast cancer and relevant models. Misaligned chromosomes predominate, and are less immunostimulatory than other chromosome segregation errors.

PLK4 is upregulated in prostate cancer and its inhibition reduces centrosome amplification and causes senescence.

BACKGROUND: Identification of novel molecular target(s) is important for designing newer mechanistically driven approaches for the treatment of prostate cancer (PCa), which is one of the main causes of morbidity and mortality in men. In this study, we determined the role of polo-like kinase 4 (PLK4), which regulates centriole duplication and centrosome amplification (CA), in PCa. MATERIALS AND METHODS: Employing human PCa tissue microarrays, we assessed the prevalence of CA, correlated with Gleason score, and estimated major causes of CA in PCa (cell doubling vs. centriole overduplication) by staining for mother/mature centrioles. We also assessed PLK4 expression and correlated it with CA in human PCa tissues and cell lines. Further, we determined the effects of PLK4 inhibition in human PCa cells. RESULTS: Compared to benign prostate, human PCa demonstrated significantly higher CA, which was also positively correlated with the Gleason score. Further, most cases of CA were found to arise by centriole overduplication rather than cell doubling events (e.g., cytokinesis failure) in PCa. In addition, PLK4 was overexpressed in human PCa cell lines and tumors. Moreover, PLK4 inhibitors CFI-400945 and centrinone-B inhibited cell growth, viability, and colony formation of both androgen-responsive and androgen-independent PCa cell lines. PLK4 inhibition also induced cell cycle arrest and senescence in human PCa cells. CONCLUSIONS: CA is prevalent in PCa and arises predominantly by centriole overduplication as opposed to cell doubling events. Loss of centrioles is cellular stress that can promote senescence and suggests that PLK4 inhibition may be a viable therapeutic strategy in PCa.

Undifferentiated and Unresectable Sarcoma With NTRK3-Fusion in a Pediatric Patient Treated With Larotrectinib and Proton Beam Radiotherapy.

A 6-year-old female presenting with an abdominal mass was found to have an unresectable undifferentiated sarcoma. The tumor did not respond to multiagent chemotherapy. However, molecular testing identified an NTRK3-fusion, and treatment was changed to larotrectinib monotherapy. Following 6 months of therapy, the patient achieved a very good partial response with 96% reduction in tumor size. She underwent proton beam radiation therapy with continued larotrectinib therapy and achieved a complete response. This case report shows that an NTRK fusion positive undifferentiated sarcoma can be safely treated with larotrectinib and radiation therapy and highlights the importance of early molecular testing.

Centriole and Golgi microtubule nucleation are dispensable for the migration of human neutrophil-like cells.

Neutrophils migrate in response to chemoattractants to mediate host defense. Chemoattractants drive rapid intracellular cytoskeletal rearrangements including the radiation of microtubules from the microtubule-organizing center (MTOC) toward the rear of polarized neutrophils. Microtubules regulate neutrophil polarity and motility, but little is known about the specific role of MTOCs. To characterize the role of MTOCs on neutrophil motility, we depleted centrioles in a well-established neutrophil-like cell line. Surprisingly, both chemical and genetic centriole depletion increased neutrophil speed and chemotactic motility, suggesting an inhibitory role for centrioles during directed migration. We also found that depletion of both centrioles and GM130-mediated Golgi microtubule nucleation did not impair neutrophil directed migration. Taken together, our findings demonstrate an inhibitory role for centrioles and a resilient MTOC system in motile human neutrophil-like cells.

Optimization of oxidative stress for mesenchymal stromal/stem cell engraftment, function and longevity.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that possess great potential as a cellular therapeutic based on their ability to differentiate to different lineages and to modulate immune responses. However, their potential is limited by their low tissue abundance, and thus the need for robust ex vivo expansion prior to their application. This creates its own issues, namely replicative senescence, which could lead to reduced MSC functionality and negatively impact their engraftment. Ex vivo expansion and MSC aging are associated with greater oxidative stress. Therefore, there is great need to identify strategies to reduce oxidative stress in MSCs. This review summarizes the achievements made to date in addressing oxidative stress in MSCs and speculates about interesting avenues of future investigation to solve this critical problem.

High nuclear TPX2 expression correlates with TP53 mutation and poor clinical behavior in a large breast cancer cohort, but is not an independent predictor of chromosomal instability.

BACKGROUND: Targeting Protein for Xenopus Kinesin Like Protein 2 (TPX2) is a microtubule associated protein that functions in mitotic spindle assembly. TPX2 also localizes to the nucleus where it functions in DNA damage repair during S-phase. We and others have previously shown that TPX2 RNA levels are strongly associated with chromosomal instability (CIN) in breast and other cancers, and TPX2 RNA levels have been demonstrated to correlate with aggressive behavior and poor clinical outcome across a range of solid malignancies, including breast cancer. METHODS: We perform TPX2 IHC on a cohort of 253 primary breast cancers and adopt a clinically amenable scoring system to separate tumors into low, intermediate, or high TPX2 expression. We then correlate TPX2 expression against diverse pathologic parameters and important measures of clinical outcome, including disease-specific and overall survival. We link TPX2 expression to TP53 mutation and evaluate whether TPX2 is an independent predictor of chromosomal instability (CIN). RESULTS: We find that TPX2 nuclear expression strongly correlates with high grade morphology, elevated clinical stage, negative ER and PR status, and both disease-specific and overall survival. We also show that increased TPX2 nuclear expression correlates with elevated ploidy, supernumerary centrosomes, and TP53 mutation. TPX2 nuclear expression correlates with CIN via univariate analyses but is not independently predictive when compared to ploidy, Ki67, TP53 mutational status, centrosome number, and patient age. CONCLUSIONS: Our findings demonstrate a strong correlation between TPX2 nuclear expression and aggressive tumor behavior, and show that TPX2 overexpression frequently occurs in the setting of TP53 mutation and elevated ploidy. However, TPX2 expression is not an independent predictor of CIN where it fails to outperform existing clinical and pathologic metrics.

Analysis of the "centrosome-ome" identifies MCPH1 deletion as a cause of centrosome amplification in human cancer.

The centrosome is the microtubule organizing center of human cells and facilitates a myriad of cellular functions including organization of the mitotic spindle to ensure faithful chromosome segregation during mitosis, cell polarization and migration, and primary cilia formation. A numerical increase in centrosomes, or centrosome amplification (CA), is common in cancer and correlates with more aggressive clinical features and worse patient outcomes. However, the causes of CA in human cancer are unclear. Many previous studies have identified mechanisms of CA in cellulo, such as overexpression of PLK4, but it is unclear how often these are the primary mechanism in human disease. To identify a primary cause of CA, we analyzed The Cancer Genome Atlas (TCGA) genomic and transcriptomic data for genes encoding the 367 proteins that localize to the centrosome (the "centrosome-ome"). We identified the following candidates for primary causes of CA: gain-of-function alterations of CEP19, CEP72, CTNNB1, PTK2, NDRG1, SPATC1, TBCCD1; and loss-of-function alterations of CEP76, MCPH1, NEURL4, and NPM1. In cellulo analysis of these candidates revealed that loss of MCPH1/microcephalin caused the most robust increase in centriole number. MCPH1 deep gene deletions are seen in 5-15% of human cancers, depending on the anatomic site of the tumor. Mechanistic experiments demonstrated that loss of MCPH1 caused a CDK2-dependent increase in STIL levels at the centrosome to drive CA. We conclude that loss of MCPH1 is common in human cancer and is likely to be a cause of CA.

Centrosome amplification is a frequent event in circulating tumor cells from subjects with metastatic breast cancer.

Centrosome amplification (CA) is a common phenomenon in cancer, promotes genomic stability and cancer evolution, and has been reported to promote metastasis. CA promotes a stochastic gain/loss of chromosomes during cell division, known as chromosomal instability (CIN). However, it is unclear whether CA is present in circulating tumor cells (CTCs), the seeds for metastasis. Here, we surveyed CA in CTCs from human subjects with metastatic breast cancer. CTCs were captured by CD45 exclusion and selection of EpCAM-positive cells using an exclusion-based sample preparation technology platform known as VERSA (versatile exclusion-based rare sample analysis). Centriole amplification (centrin foci> 4) is the definitive assay for CA. However, determination of centrin foci is technically challenging and incompatible with automated analysis. To test if the more technically accessible centrosome marker pericentrin could serve as a surrogate for centriole amplification in CTCs, cells were stained with pericentrin and centrin antibodies to evaluate CA. This assay was first validated using breast cancer cell lines and a nontransformed epithelial cell line model of inducible CA, then translated to CTCs. Pericentrin area and pericentrin area x intensity correlate well with centrin foci, validating pericentrin as a surrogate marker of CA. CA is found in CTCs from 75% of subjects, with variability in the percentage and extent of CA in individual circulating cells in a given subject, similar to the variability previously seen in primary tumors and cell lines. In summary, we created, validated, and implemented a novel method to assess CA in CTCs from subjects with metastatic breast cancer. Such an assay will be useful for longitudinal monitoring of CA in cancer patients and in prospective clinical trials for assessing the impact of CA on response to therapy.

MRSA Septicemia With Septic Arthritis and Prostatic, Intraretinal, Periapical, and Lung Abscesses.

INTRODUCTION: Methicillin-resistant staphylococcus aureus (MRSA) bacteremia is a life-threatening illness and a major global health care problem. It can cause metastatic and complicated infections. CASE PRESENTATION: A 58-year-old man with uncontrolled type 2 diabetes mellitus presented with altered mental status after a fall. He was found to have a hip fracture, diabetic ketoacidosis, and MRSA bacteremia. This was complicated by septic knee arthritis, prostatic abscess, intraretinal abscess, periapical abscesses, and pulmonary abscesses. He was treated with intravenous vancomycin and oral linezolid and eventually recovered. DISCUSSION: Severe metastatic MRSA infection was likely due, in part, to the patient's uncontrolled diabetes, as he has no underlying immunodeficiency and was HIV negative. Prostatic abscesses are a relatively rare occurrence that typically develop in immunocompromised patients. CONCLUSION: This case is an interesting confluence of sequelae of MRSA bacteremia and reinforces the necessity for clinicians to be diligent when evaluating a patient with a suspected prostatic abscess.

Centrosome Amplification in Cancer Disrupts Autophagy and Sensitizes to Autophagy Inhibition.

Centrosome amplification (CA), or a numerical increase in centrosomes, is common in human cancers, particularly those with high-risk features. We have discovered that cells with CA have an increased burden of autophagy, a catabolic process whereby autophagosomes engulf damaged organelles and proteins and deliver these contents to the lysosome for degradation and subsequent recycling. Cells with CA demonstrate an accumulation of autophagosomes. We evaluated the alternative hypotheses that CA alters autophagy by modulating microtubule networks and impairing trafficking versus altering lysosome clustering and organization versus chromosome missegregation-induced proteotoxic stress. Using LC3 reporter assays and autophagosome tracking experiments, we demonstrate that CA causes an accumulation of autophagosomes by interfering with autophagosome trafficking. To establish whether this was a druggable weakness, we tested autophagy inhibitors in our cell models of CA. Cells with CA are sensitized to chemical and genetic autophagy inhibition. Taken together, our results suggest that autophagy is disrupted by CA and sensitizes cells to inhibition of autophagy. These findings suggest a novel precision medicine strategy, whereby CA increases reliance on autophagy and serves as a biomarker for autophagy inhibitors in high-risk cancers. IMPLICATIONS: Our study suggests that CA could be used as a predictive biomarker for treatment with autophagy inhibitors.

Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131).

The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.