RNA-sequencing predicts a role of androgen receptor and aldehyde dehydrogenase 1A1 in osteosarcoma lung metastases.

One-third of pediatric patients with osteosarcoma (OS) develop lung metastases (LM), which is the primary predictor of mortality. While current treatments of patients with localized bone disease have been successful in producing 5-year survival rates of 65-70%, patients with LM experience poor survival rates of only 19-30%. Unacceptably, this situation that has remained unchanged for 30 years. Thus, there is an urgent need to elucidate the mechanisms of metastatic spread in OS and to identify targetable molecular pathways that enable more effective treatments for patients with LM. We aimed to identify OS-specific gene alterations using RNA-sequencing of extremity and LM human tissues. Samples of extremity and LM tumors, including 4 matched sets, were obtained from patients with OS. Our data demonstrate aberrant regulation of the androgen receptor (AR) pathway in LM and predicts aldehyde dehydrogenase 1A1 (ALDH1A1) as a downstream target. Identification of AR pathway upregulation in human LM tissue samples may provide a target for novel therapeutics for patients with LM resistant to conventional chemotherapy.

Grade 2, 3 and Dedifferentiated Chondrosarcomas: A Comparative Study of Isocitrate Dehydrogenase-Mutant and Wild-Type Tumors with Implications for Prognosis and Therapy.

BACKGROUND: Grade 2 and 3 and dedifferentiated chondrosarcomas (CS) are frequently associated with isocitrate dehydrogenase (IDH) mutations and often exhibit a poor clinical outcome. Treatment is limited mainly to surgery. Defining IDH status (wild type (WT) and mutant) and the associated transcriptome may prove useful in determining other therapeutic options in these neoplasms. METHODS: Formalin-fixed paraffin-embedded material from 69 primary and recurrent grade 2, 3 and dedifferentiated CS was obtained. DNA sequencing for IDH1 and IDH2 mutations (n = 47) and RNA sequencing via Nextseq 2000 (n = 14) were performed. Differentially expressed genes (DEGs) were identified and used to predict aberrant biological pathways with Ingenuity Pathway Analysis (IPA) software (Qiagen). Gene Set Enrichment Analyses (GSEA) using subsets C3, C5 and C7 were performed. Differentially expressed genes were validated by immunohistochemistry. Outcome analysis was performed using the Wilcoxon test. RESULTS: A set of 69 CS (28 females, 41 males), average age 65, distributed among femur, pelvis, humerus, and chest wall were identified from available clinical material. After further selection based on available IDH status, we evaluated 15 IDH WT and 32 IDH mutant tumors as part of this dataset. Out of 15 IDH WT tumors, 7 involved the chest wall/scapula, while 1 of 32 mutants arose in the scapula. There were far more genes overexpressed in IDH WT tumors compared to IDH mutant tumors. Furthermore, IDH WT and IDH mutant tumors were transcriptomically distinct in the IPA and GSEA, with IDH mutant tumors showing increased activity in methylation pathways and endochondral ossification, while IDH WT tumors showed more activity in normal matrix development pathways. Validation immunohistochemistry demonstrated expression of WT1 and AR in IDH WT tumors, but not in IDH mutants. SATB2 was expressed in IDH mutant tumors and not in WT tumors. Outcome analysis revealed differences in overall survival between mutant and WT tumors (p = 0.04), dedifferentiated mutant and higher-grade (2, 3) mutant tumors (p = 0.03), and dedifferentiated mutant and higher-grade (2, 3) WT tumors (p = 0.03). The longest survival times were observed in patients with higher-grade WT tumors, while patients with dedifferentiated mutant tumors showed the lowest survival. Generally, patients with IDH WT tumors displayed longer survival in both the higher-grade and dedifferentiated groups. CONCLUSIONS: Grade 2, 3 and dedifferentiated chondrosarcomas are further characterized by IDH status, which in turn informs transcriptomic phenotype and overall survival. The transcriptome is distinct depending on IDH status, and implies different treatment targets.

Molecular-derived risk of malignancy and the related positive call rate of indeterminate thyroid cytology diagnoses as quality metrics for individual cytopathologists.

BACKGROUND: Indeterminate thyroid cytopathology diagnoses represent differing degrees of risk that are corroborated by follow-up studies. However, traditional cytologic-histologic correlation may overestimate the risk of malignancy (ROM) because only a subset of cases undergo resection. Alternatively, some molecular tests provide probability of malignancy data to calculate the molecular-derived risk of malignancy (MDROM) and the positive call rate (PCR). The authors investigated MDROMs and PCRs of indeterminate diagnoses for individual cytopathologists as quality metrics. METHODS: This study was approved by the Department of Pathology Quality Improvement Program. Thyroid cytopathology diagnoses and ThyroSeq v3 results were retrieved for each cytopathologist for a 2-year period with at least 3 years of follow-up for the atypia of undetermined significance (AUS), follicular neoplasia (FN), and follicular neoplasia, oncocytic-type (ONC) cytopathologic diagnoses. MDROMs and PCRs were compared with reference ROMs and cytologic-histologic correlation outcomes. RESULTS: The overall MDROMs (and ranges for cytopathologists) for the AUS, FN, and ONC categories were 13.4% (range, 5.8%-20.8%), 28.1% (range, 22.1%-36.7%), and 27.0% (range, 19.5%-41.5%), respectively, and most individual cytopathologists' MDROMs were within reference ROM ranges. However, PCRs more effectively parsed the differences in cytopathologists' ROM performance. Although the overall PCRs were not significantly different across cytopathologists (p = .06), the AUS PCRs were quite different (p = .002). By cytologic-histologic correlation, six of 55 resected cases (10.9%) were falsely negative, and there were no false-positive cases. CONCLUSIONS: MDROMs and PCRs evaluate concordance with reference ROMs and with one another and provide individual feedback, which potentially facilitates quality improvement.

UNSEG: unsupervised segmentation of cells and their nuclei in complex tissue samples.

Multiplexed imaging technologies have made it possible to interrogate complex tumor microenvironments at sub-cellular resolution within their native spatial context. However, proper quantification of this complexity requires the ability to easily and accurately segment cells into their sub-cellular compartments. Within the supervised learning paradigm, deep learning based segmentation methods demonstrating human level performance have emerged. Here we present an unsupervised segmentation (UNSEG) method that achieves deep learning level performance without requiring any training data. UNSEG leverages a Bayesian-like framework and the specificity of nucleus and cell membrane markers to construct an a posteriori probability estimate of each pixel belonging to the nucleus, cell membrane, or background. It uses this estimate to segment each cell into its nuclear and cell-membrane compartments. We show that UNSEG is more internally consistent and better at generalizing to the complexity of tissue samples than current deep learning methods. This allows UNSEG to unambiguously identify the cytoplasmic compartment of a cell, which we employ to demonstrate its use in an example biological scenario. Within the UNSEG framework, we also introduce a new perturbed watershed algorithm capable of stably and accurately segmenting a cell nuclei cluster into individual cell nuclei. Perturbed watershed can also be used as a standalone algorithm that researchers can incorporate within their supervised or unsupervised learning approaches to replace classical watershed. Finally, as part of developing UNSEG, we have generated a high-quality annotated gastrointestinal tissue dataset, which we anticipate will be useful for the broader research community. Segmentation, despite its long antecedents, remains a challenging problem, particularly in the context of tissue samples. UNSEG, an easy-to-use algorithm, provides an unsupervised approach to overcome this bottleneck, and as we discuss, can help improve deep learning based segmentation methods by providing a bridge between unsupervised and supervised learning paradigms.

A thyroid EIF1AX story: how clinical, cytologic, and molecular surveillance led to appropriate management.

INTRODUCTION: Indeterminate thyroid cytology diagnoses are associated with intermediate risks of malignancy. Application of molecular testing (MT) to indeterminate specimens provides additional diagnostic and prognostic information. While a positive or suspicious MT result may prompt surgery, a negative MT result is associated with a low probability of cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear features and approximates that of a benign cytology diagnosis. Furthermore, ThyroSeq v3 MT has a "currently negative" result for findings with the probability of cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear feature that is slightly greater than that for the negative ThyroSeq v3 MT result but less than 10%, suggesting active surveillance. In this report, we discuss a case of a patient for whom clinical, cytologic, and molecular surveillance led to timely surgery and management. CLINICAL DETAILS: A 53-year-old man with a thyroid isthmus nodule had a fine-needle aspiration cytology diagnosis of atypia of undetermined significance and a subsequent ThyroSeq v3 MT, which revealed an EIF1AX mutation and a "currently negative" MT result. Surveillance with additional fine-needle aspiration samples demonstrated concerning genomic alterations (fluctuating EIF1AX allelic frequency and a non-V600E BRAF mutation), culminating in the conversion to a positive MT result 3 years later. Resection revealed an encapsulated noninvasive, oncocytic solid subtype of papillary thyroid carcinoma with increased mitotic activity. CONCLUSION: The case is notable for clinical, cytologic, and molecular surveillance demonstrating sequential pathologic alterations in an indeterminate thyroid nodule with EIF1AX mutation, leading to timely resection of the neoplasm before invasion manifested.

Patient-Derived Spheroid Culture Models Are Better Than Monolayer Models in Chondrosarcoma Research.

Purpose: Chondrosarcoma (CSA) are mesenchymal tissue-derived bone tumors. CSA mainly occurs in older people. CSA has demonstrated resistance to chemotherapy and radiation; complete surgical removal with negative margins is the only treatment option. In the case of metastatic CSA, the chance of survival is meager. Since the conventional two-dimensional cell culture models failed to retain tumor characteristics, developing preclinical models mimicking the disease with the highest fidelity is paramount for personalized treatments. Methods: In this study, we established spherical cultured cells as new models for CSA. First, we demonstrated that CSA cells could form spheroids when cultured in ultra-low attachment plates. Next, tissue samples from CSA patients were collected and processed into primary cells, which were subsequently cultured as primary spheroids. The growth rate of primary spheroids was monitored and the histology of mature spheroids were characterized. These primary spheroids were used in drug susceptibility studies where traditional doxorubicin therapy and our novel disulfiram-copper therapy were tested. Results: Compared with conventional monolayer cultures, spheroids better recapitulated the features of the in vivo tumor in the aspect of the formation of extracellular matrix. In the drug susceptibility study, spheroids demonstrated high resistance to the classic therapies, suggesting that monolayer cultures may give false positive results. Therefore, using spheroids for drug research and development in the CSA field should provide more accurate results. Conclusion: In summary, our study of primary CSA spheroids brought new insight into their chemoresistance and demonstrated its potential for personalized treatment of CSA in clinical medicine.

Postoperative infection and bone sarcoma survival: systematic review of the role of infection in bone sarcoma prognosis.

Background: Osteosarcoma (OS) and chondrosarcoma (CS) are primary bone malignancies whose prognoses have stagnated despite advancements in surgical management, chemotherapy, radiation therapy, and immunotherapy. The role of the immune system in generating anti-cancer physiologic responses is critical to prognosis. Prior studies have explored if immune system activation via infection enhances survival in bone sarcomas without a clear consensus. Methods: This study sought to (I) retrospectively examine the effect of postoperative infection on survival in OS and CS and (II) systematically review the effect of postoperative infection on survival in primary bone malignancies. We performed a retrospective case-control study of 192 patients treated between 1/2000-12/2015 at a single academic sarcoma referral center. Patients with OS or CS undergoing operative resection were included. Eligible patients were grouped by presence of metastasis, and survival was compared between patients with or without postoperative infection. Furthermore, we performed a systematic review following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines investigating the effect of infection on primary bone malignancy survival. Risk of bias assessment was performed utilizing the ROBINS-I (Risk of Bias in Non-randomized Studies-of Interventions) assessment tool. All presented studies included author information, study population, and overall or disease-free survival results. Results: One hundred and four patients were included, with 85 without infection (26 metastatic, 59 non-metastatic) and 19 with infection (10 metastatic, 9 non-metastatic). Five-year survival was greatest in patients without metastasis with a postoperative infection (100%), followed by patients without metastasis who were infection-free (80%). Five-year survival was comparatively lower in patients with metastasis who were infection-free (35%) and lowest in patients with metastasis with a postoperative infection (20%). No significant differences were present (P=0.17) on log-rank analysis. Our systematic review collected six studies exploring the impact of infection on primary bone malignancy survival, with two studies reporting significant findings of infection improving survival. Limitations of this review included risk of bias due to confounding, inconsistency comparing outcomes, and differences in patient populations. Conclusions: This retrospective study and systematic review suggests postoperative infection may play a role in modulating immune response to malignancy. Understanding the synergy between anti-pathogen and anti-cancer responses warrants further investigation as an alternative method of targeted cancer treatment.

Accuracy of definitive rapid onsite evaluation cytopathology diagnoses: Assessment of potentially critical diagnoses as a quality assurance measure.

INTRODUCTION: Intraprocedural rapid onsite evaluation (ROSE) of cytology specimens enhances cytopathology practice. More recently, ROSE diagnoses, like frozen section (FS) diagnoses, have guided immediate clinical decisions. In this study, we evaluated the diagnostic accuracy of definitive ROSE diagnoses in our quality assurance system over a 52-month period. MATERIALS AND METHODS: Cytopathology cases with ROSE from January 2017 to April 2021were retrieved from our laboratory information system. After excluding cases that were deferred or nondiagnostic/unsatisfactory, each definitive ROSE diagnosis (ie, negative for malignant cells or positive for malignant cells) was categorized as having agreement or disagreement with the final diagnosis. For comparison, concordance of FS diagnoses from the same time period were tabulated and compared to those of ROSE diagnoses by using χ2 testing with P < 0.05 considered statistically significant. RESULTS: Of the 1649 ROSE diagnoses, there were 15 disagreements (0.9%) with 1 final moderate interpretive disagreement (0.06%). By comparison, of the 17,469 FS diagnoses, there were 141 disagreements (0.8%) with 49 final moderate or major interpretive disagreements (0.3%). The remaining disagreements were minor. There were no statistically significant differences in the rates of final moderate and major interpretive disagreements. CONCLUSIONS: The final interpretive disagreement rates for definitive ROSE and FS diagnoses were similar in this study. Given the expanding role of ROSE and its use for immediate clinical decisions in some cases, monitoring the accuracy of definitive diagnoses may serve as an initial quality assurance measure.

Differential expression of angiogenesis markers HSP70, HSP90, VEGF and pERK1/2 in both components of dedifferentiated chondrosarcomas.

Dedifferentiated chondrosarcomas (DDCS) are highly malignant bimorphic mesenchymal tumors with poor outcome and limited treatment options. Genes and proteins involved in angiogenesis play an important role in the development of invasion and metastasis. Immunohistochemical stains targeting HSP70, pERK1/2 and VEGFA were applied to a TMA containing 29 DDCS cases representing both tumor components. Higher expression of HSP70 and pERK1/2 was noted in the dedifferentiated component. RNA sequencing performed in 8 paired cases of DDCS comparing well differentiated and dedifferentiated components, showed higher expression of several HSP70 family members and HSP90 in the dedifferentiated component. Furthermore, high mobility group AT-hook 2 (HMAG2) and SET nuclear proto-oncogene demonstrated higher expression in the dedifferentiated component. Thus, the well differentiated and dedifferentiated components of DDCS are different, histologically and transcriptomically. The dedifferentiated component of DDCS shows higher expression of markers that are associated with malignant behavior. Some of these may represent future treatment targets.

Do Patient-derived Spheroid Culture Models Have Relevance in Chondrosarcoma Research?

BACKGROUND: In high-grade chondrosarcoma, 5-year survival is lower than 50%. Therefore, it is important that preclinical models that mimic the disease with the greatest possible fidelity are used to potentially develop new treatments. Accumulating evidence suggests that two-dimensional (2-D) cell culture may not accurately represent the tumor's biology. It has been demonstrated in other cancers that three-dimensional (3-D) cancer cell spheroids may recapitulate tumor biology and response to treatment with greater fidelity than traditional 2-D techniques. To our knowledge, the formation of patient-derived chondrosarcoma spheroids has not been described. QUESTIONS/PURPOSES: (1) Can patient-derived chondrosarcoma spheroids be produced? (2) Do spheroids recapitulate human chondrosarcoma better than 2-D cultures, both morphologically and molecularly? (3) Can chondrosarcoma spheroids provide an accurate model to test novel treatments? METHODS: Experiments to test the feasibility of spheroid formation of chondrosarcoma cells were performed using HT-1080, an established chondrosarcoma cell line, and two patient-derived populations, TP19-S26 and TP19-S115. Cells were cultured in flasks, trypsinized, and seeded into 96-well ultra-low attachment plates with culture media. After spheroids formed, they were monitored daily by bright-field microscopy. Spheroids were fixed using paraformaldehyde and embedded in agarose. After dehydration with isopropanol, paraffin-embedded spheroids were sectioned, and slides were stained with hematoxylin and eosin. To compare differences and similarities in gene expression between 2-D and 3-D chondrosarcoma cultures and primary tumors, and to determine whether these spheroids recapitulated the biology of chondrosarcoma, RNA was extracted from 2-D cultures, spheroids, and tumors. Quantitative polymerase chain reaction was performed to detect chondrosarcoma markers of interest, including vascular endothelial growth factor alpha, hypoxia-inducible factor 1α, COL2A1, and COL10A1. To determine whether 2-D and 3-D cultures responded differently to novel chondrosarcoma treatments, we compared their sensitivities to disulfiram and copper chloride treatment. To test their sensitivity to disulfiram and copper chloride treatment, 10,000 cells were seeded into 96-well plates for 2-D culturing and 3000 cells in each well for 3-D culturing. After treating the cells with disulfiram and copper for 48 hours, we detected cell viability using quantitative presto-blue staining and measured via plate reader. RESULTS: Cell-line and patient-derived spheroids were cultured and monitored over 12 days. Qualitatively, we observed that HT-1080 demonstrated unlimited growth, while TP19-S26 and TP19-S115 contracted during culturing relative to their initial size. Hematoxylin and eosin staining of HT-1080 spheroids revealed that cell-cell attachments were more pronounced at the periphery of the spheroid structure than at the core, while the core was less dense. Spheroids derived from the intermediate-grade chondrosarcoma TP19-S26 were abundant in extracellular matrix, and spheroids derived from the dedifferentiated chondrosarcoma TP19-S115 had a higher cellularity and heterogeneity with spindle cells at the periphery. In the HT-1080 cells, differences in gene expression were appreciated with spheroids demonstrating greater expressions of VEGF-α (1.01 ± 0.16 versus 6.48 ± 0.55; p = 0.003), COL2A1 (1.00 ± 0.10 versus 7.46 ± 2.52; p < 0.001), and COL10A1 (1.01 ± 0.19 versus 22.53 ± 4.91; p < 0.001). Differences in gene expressions were also noted between primary tumors, spheroids, and 2-D cultures in the patient-derived samples TP19-S26 and TP19-S115. TP19-S26 is an intermediate-grade chondrosarcoma. With the numbers we had, we could not detect a difference in VEGF-α and HIF1α gene expression compared with the primary tumor. COL2A1 (1.00 ± 0.14 versus 1.76 ± 0.10 versus 335.66 ± 31.13) and COL10A1 (1.06 ± 0.378 versus 5.98 ± 0.45 versus 138.82 ± 23.4) expressions were both greater in the tumor (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) and 3-D cultures (p (COL2A1) = 0.004; p (COL10A1) < 0.0001) compared with 2-D cultures. We could not demonstrate a difference in VEGF-α and HIF1α expressions in TP19-S115, a dedifferentiated chondrosarcoma, in the tumor compared with 2-D and 3-D cultures. COL2A1 (1.00 ± 0.02 versus 1.86 ± 0.18 versus 2.95 ± 0.56) and COL10A1 (1.00 ± 0.03 versus 5.52 ± 0.66 versus 3.79 ± 0.36) expressions were both greater in spheroids (p (COL2A1) = 0.003; p (COL10A1) < 0.0001) and tumors (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) compared with 2-D cultures. Disulfiram-copper chloride treatment demonstrated high cytotoxicity in HT-1080 and SW-1353 chondrosarcoma cells grown in the 2-D monolayer, but 3-D spheroids were highly resistant to this treatment. CONCLUSION: We provide preliminary findings that it is possible to generate 3-D spheroids from chondrosarcoma cell lines and two human chondrosarcomas (one dedifferentiated chondrosarcoma and one intermediate-grade chondrosarcoma). Chondrosarcoma spheroids derived from human tumors demonstrated morphology more reminiscent of primary tumors than cells grown in 2-D culture. Spheroids displayed similar expressions of cartilage markers as the primary tumor, and we observed a higher expression of collagen markers in the spheroids compared with cells grown in monolayer. Spheroids also demonstrated greater chemotherapy resistance than monolayer cells, but more patient-derived spheroids are needed to further conclude that 3-D cultures may mimic the chemoresistance that chondrosarcomas demonstrate clinically. Additional studies on patient-derived chondrosarcoma spheroids are warranted. CLINICAL RELEVANCE: Chondrosarcomas demonstrate resistance to chemotherapy and radiation, and we believe that if they can be replicated, models such as 3-D spheroids may provide a method to test novel treatments for human chondrosarcoma. Additional comprehensive genomic studies are required to compare 2-D and 3-D models with the primary tumor to determine the most effective way to study this disease in vitro.

Cytomorphology of Mycobacterium avium intracellulare-associated ascites.

Ascites due to Mycobacterium avium intracellulare (MAI) infection is extremely rare and associated with a poor outcome. The cytomorphology of this condition has not been previously reported. We present a unique case of a 45-year-old woman with iatrogenic immunodeficiency who developed MAI-associated chylous ascites. The ascitic fluid cytology showed numerous lymphocytes and foamy histiocytes with abundant intracytoplasmic MAI organisms. The diagnosis was confirmed by tissue biopsy showing MAI mesenteritis. It is important to consider MAI-associated ascites in the differential diagnosis whenever ascitic fluid shows a predominant population of lymphocytes and macrophages, especially in immunocompromised patients.

Design and evaluation of collagen-inspired mineral-hydrogel nanocomposites for bone regeneration.

Bone loss due to trauma and tumors remains a serious clinical concern. Due to limited availability and disease transmission risk with autografts and allografts, calcium phosphate bone fillers and growth factor-based substitute bone grafts are currently used in the clinic. However, substitute grafts lack bone regeneration potential when used without growth factors. When used along with the added growth factors, they lead to unwanted side effects such as uncontrolled bone growth. Collagen-based hydrogel grafts available on the market fail to provide structural guidance to native cells due to high water-solubility and faster degradation. To overcome these limitations, we employed bioinspired material design and fabricated three different hydrogels with structural features similar to native collagen at multiple length-scales. These hydrogels fabricated using polyionic complexation of oppositely charged natural polysaccharides exhibited multi-scale architecture mimicking nanoscale banding pattern, and microscale fibrous structure of native collagen. All three hydrogels promoted biomimetic apatite-like mineral deposition in vitro elucidating crystalline structure on the surface while amorphous calcium phosphate inside the hydrogels resulting in mineral-hydrogel nanocomposites. When evaluated in a non-load bearing critical size mouse calvaria defect model, chitosan - kappa carrageenan mineral-hydrogel nanocomposites enhanced bone regeneration without added growth factors compared to empty defect as well as widely used marketed collagen scaffolds. Histological assessment of the regenerated bone revealed improved healing and tissue remodeling with mineral-hydrogel nanocomposites. Overall, these collagen-inspired mineral-hydrogel nanocomposites showed multi-scale hierarchical structure and can potentially serve as promising bioactive hydrogel to promote bone regeneration. STATEMENT OF SIGNIFICANCE: Hydrogels, especially collagen, are widely used in bone tissue engineering. Collagen fibrils play arguably the most important role during natural bone development. Its multi-scale hierarchical structure to form fibers from fibrils and electrostatic charges enable mineral sequestration, nucleation, and growth. However, bulk collagen hydrogels exhibit limited bone regeneration and are mostly used as carriers for highly potent growth factors such as bone morphogenic protein-2, which increase the risk of uncontrolled bone growth. Thus, there is an unmet clinical need for a collagen-inspired biomaterial that can recreate structural hierarchy, mineral sequestration ability, and stimulate recruitment of host progenitor cells to facilitate bone regeneration. Here, we propose collagen-inspired bioactive mineral-hydrogel nanocomposites as a growth factor-free approach to guide and enhance bone regeneration.

Molecular-derived estimation of risk of malignancy for indeterminate thyroid cytology diagnoses.

INTRODUCTION: One of the key features of the Bethesda System for Reporting Thyroid Cytopathology is the risk of malignancy (ROM), which guides management for each diagnostic category. However, calculation of the ROM can be challenging for indeterminate diagnoses because only a portion of cases will be resected for cytologic-histologic correlation (CHC) analyses. In the present study, we used the probability of cancer information from ThyroSeq, version 3, reports to calculate the molecular-derived (MD) ROM for indeterminate categories. MATERIALS AND METHODS: Cytology cases with indeterminate BSRTC diagnoses and adequate molecular test results were retrieved from our cytopathology laboratory for a 12-month period. The probability of cancer information from the ThyroSeq, version 3, molecular reports were tabulated, and the mean ROM was calculated for each diagnostic category. The MDROM included noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) as a "malignant" outcome because it is considered a surgical disease. RESULTS: A total of 361 cases had adequate material for molecular testing. The diagnostic distribution was as follows: atypia of undetermined significance/follicular lesion of undetermined significance, 271 cases (75.1%), follicular neoplasm/suspicious for a follicular neoplasm, 59 cases (16.3%), and Hürthle cell type/suspicious for a follicular neoplasm, Hürthle cell type, 31 cases (8.6%). The corresponding estimated MDROMs were 14.9%, 32.6%, and 34.4%. A comparison with the CHC data was performed, and the 95% confidence intervals of the MDROMs overlapped well with the 2 endpoint CHC values. CONCLUSIONS: Calculation of the MDROMs provides a new method to approximate the ROMs of indeterminate diagnoses and has the advantage of potentially evaluating all cases, not just those resected. Furthermore, for those using the same platform, interinstitutional comparisons will be possible.

Tumor-to-Tumor Metastasis: Prostate Carcinoma Metastatic to Soft Tissue Sarcoma, a Previously Unreported Event.

Tumor-to-tumor metastasis is an unusual phenomenon wherein one distinct malignancy is present within the substance of another independent tumor. This event is rare, difficult to detect with imaging, and, due to conflicting terminology in the literature, can be challenging to classify. This article reports the first documented case of tumor-to-tumor metastasis involving prostatic adenocarcinoma and myxoid liposarcoma, reviews the available literature for carcinoma metastatic to sarcoma, and discusses the current situation within the context of the established criteria for the classification of combination tumors.

Bottom-Up Self-assembled Hydrogel-Mineral Composites Regenerate Rabbit Ulna Defect without Added Growth Factors.

Hydrogel-based biomaterials have advanced bone tissue engineering approaches in the last decade, through their ability to serve as a carrier for potent growth factor, bone morphogenic protein-2 (BMP-2). However, biophysical properties of hydrogels such as multiscale structural hierarchy and bone extracellular matrix (ECM)-mimetic microarchitecture are underutilized while designing current bone grafts. Incorporation of these properties offers great potential to create a favorable biomimetic microenvironment to harness their regenerative potential. Here, we present our approach to fabricate collagen-inspired bioactive hydrogel scaffolds (referred to as "RegenMatrix") to guide and enhance bone regeneration in a rabbit ulna defect model through the mimicry of multiscale architecture of bone ECM, i.e., native collagen. Specifically, we employed polyelectrolyte complexation to promote bottom-up self-assembly of oppositely charged polysaccharides (chitosan and kappa-carrageenan) at multiple length scales forming fibrils, which further assemble into fibers. The self-assembly and bioinspired scaffold fabrication method resulted in robust cylindrical RegenMatrix with excellent retention of the multiscale architecture and uniform mineral deposition throughout the scaffolds. RegenMatrix, in both nonmineralized and mineralized forms, enhanced bone regeneration in the semiload-bearing ulna defect when compared to the empty defect. RegenMatrix also showed greater histocompatibility without any fibrous tissue formation. Collectively, the RegenMatrix developed in this study has a great potential as a bioactive bone graft without any added growth factors.

Genetic variants and copy number changes in soft tissue leiomyosarcoma detected by targeted amplicon sequencing.

AIMS: Leiomyosarcomas (LMSs) occur in various tissues and harbour potential for metastases. The genomic landscape of LMS is poorly understood. In an effort to improve understanding of the LMS genome, we analysed 11 LMSs of somatic soft tissue including matching tissue of normal phenotype. METHODS: DNA derived from microdissected tumour domains and matching normal tissue underwent amplicon sequencing of 409 tumour suppressors and oncogenes using the Ion Torrent Comprehensive Cancer Panel. RESULTS: Genomic changes were heterogeneous with few recurrent abnormalities detected. Coding variants were identified in genes involved in signal transduction, transcriptional regulation and DNA repair. There were variants in several genes related to angiogenesis and GPR124 variants (TEM5) were confirmed by immunohistochemical analysis of a LMS tissue microarray. Surprisingly, there were shared coding variants in tumour and corresponding normal tissue. CONCLUSIONS: LMSs are a very heterogeneous population lacking recurrent somatic abnormalities. The presence of damaging mutations in normal tissue may reflect either a germline predisposition or field effect rather than tissue contamination. Hopeful therapeutic targets appear to be those related to AKT/MTOR pathway.

Dermatofibrosarcoma protuberans with fibrosarcomatous transformation: our experience, molecular evaluation of selected cases, and short literature review.

Dermatofibrosarcoma protuberans with fibrosarcomatous transformation (DFSP-FS) is a higher grade tumor arising from dermatofibrosarcoma protuberans (DFSP). Recent literature highlights its impact on recurrence rates, metastatic rates, and survival. In this article, we aim to describe our experience with 13 cases of DFSP-FS in terms of pathologic findings, molecular alterations, clinical outcomes, management, and also perform a short recent literature review.

Benign call rate and molecular test result distribution of ThyroSeq v3.

BACKGROUND: The benign call rate (BCR) is the percentage of cytomorphologically indeterminate cases with subsequent benign or negative molecular results. For rule-out tests, the BCR is an important parameter because these molecular "negative" cases may be managed similarly to those with a benign cytology diagnosis. Although earlier versions of ThyroSeq molecular tests were less effective in excluding malignancy, the extensively expanded v3 version with a high negative predictive value is considered to represent a rule-out test. In the current study, the authors evaluated the BCR and the overall molecular result distribution of ThyroSeq v3. METHODS: All indeterminate thyroid fine-needle aspiration cases (except those deemed suspicious for malignancy) with available ThyroSeq v3 results from November 2017 to June 2018 were retrieved from the cytopathology files. Because the ThyroSeq v3 genomic classifier was designed for thyroid follicular-derived lesions, cases in which parathyroid and medullary carcinoma molecular markers were detected and those that were inadequate or limited for molecular testing were excluded from the study. For the remaining cases, the indeterminate diagnoses were correlated with molecular and histologic results. RESULTS: Among the 224 indeterminate cases (except those deemed suspicious for malignancy), the overall ThyroSeq v3 molecular test BCR was 74.1% (166 of 224 cases). The category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) had higher BCRs compared with the other indeterminate diagnostic categories. RAS mutations were the most common positive results. CONCLUSIONS: The high BCR demonstrates that ThyroSeq v3 is potentially effective in sparing the large majority of indeterminate cases with "negative" results from surgery while offering risk assessment for the positive cases and guiding clinical management.

Giant Cell Poor Extra-Articular Diffuse-Type Tenosynovial Giant Cell Tumor With Extensive Desmin Expression: A Potential Diagnostic Pitfall With Cytogenetic Confirmation.

Diffuse-type tenosynovial giant cell tumor can rarely present as an entirely extra-articular mass, which can be misdiagnosed as a sarcoma especially when giant cells are absent, dominated by large dendritic mononuclear cells, and desmin expression is extensive.