Production of a Low-Cost, Off-the-Shelf, Decellularized Cartilage Xenograft for Tissue Regeneration.

BACKGROUND: Reconstruction of cartilaginous deformities is a well-established surgical challenge with high levels of unpredictability and complication. Because of the morbidity associated with autologous cartilage grafting, combined with its limited supply and the significant expense of commercially decellularized allografts, increasing efforts have sought to produce an acellular, nonimmunogenic cartilage xenograft. We have developed and validated a novel protocol for high throughput decellularization of ovine costal cartilage with immediate translational potential for preclinical investigation of novel strategies for cartilaginous reconstruction. METHODS: Floating ribs were isolated from freshly slaughtered rack of lamb and after cleaning, the ribs were either minced into 2-mm cubes or zested into 1-mm flakes. Tissue was then decellularized via a protocol consisting of 4 freeze/thaw cycles, digestion with trypsin, incubation in hyperosmolar and hypoosmolar salt solutions, with incubation in 1% Tween following both the hyperosmolar and hypoosmolar steps, a 48-hour incubation in nucleases, DNA elution via EDTA, and 2 terminal sterilization steps. Protocol success was evaluated via histologic analysis with hematoxylin and eosin, DAPI, and safranin-O staining, as well as DNA quantification. RESULTS: Histologic analysis of the decellularized tissue revealed a significant reduction in nuclei as evidenced by hematoxylin and eosin and DAPI staining (P < 0.01). Safranin-O staining demonstrated a depletion of glycosaminoglycan content in the decellularized cartilage but with preservation of tissue architecture. Unprocessed lamb cartilage contained 421 ± 60 ng DNA/mg of lyophilized tissue, whereas decellularized zested and minced costal cartilage contained 27 ± 2 ng DNA/mg lyophilized tissue (P < 0.0001) and 24 ± 2.3 ng DNA/mg lyophilized tissue (p < 0.0001), respectively, well below the threshold of 50 ng accepted as evidence of suitable decellularization. In comparison, commercial allograft cartilage contained 17 ± 5 ng DNA/mg of lyophilized tissue. CONCLUSIONS: We have developed a novel protocol for the decellularization of xenogeneic cartilage graft. This structurally stable, low immunogenicity decellularized cartilage can be produced at low cost in large quantities for use in preclinical investigation.

Off-the-Shelf Nipple Engineering: Neonipple Formation via Implantation of Scaffolded Decellularized Ovine Xenograft.

BACKGROUND: Nipple reconstruction is widely regarded as the final step in postmastectomy breast reconstruction. While grafts, local flaps, or combination approaches have been used in nipple reconstruction, none has been able to achieve reliable long-term projection preservation. In response, we have sought to bioengineer neonipples in situ via the implantation of processed, decellularized cartilage xenografts placed within 3-dimensional-printed polylactic acid (PLA) scaffolds. MATERIALS AND METHODS: External nipple scaffolds were designed in-house and 3-dimensional-printed with PLA filament. Decellularized ovine xenograft infill was prepared and processed by mincing or zesting. All nipple scaffolds were placed subcutaneously on the dorsa of Sprague-Dawley rats and explanted after 1, 3, and 6 months for analysis. RESULTS: Explanted nipple scaffolds demonstrated gross maintenance of scaffold shape, diameter, and projection with accompanying increases in tissue volume. Histologic analyses revealed preservation of native cartilage architecture after 6 months without evidence of degradation. Analysis of formed tissue within the scaffolds revealed a progressive invasion of fibrovascular tissue with identifiable vascular channels and adipose tissue after 6 months in vivo. Confined compression testing revealed equilibrium moduli of both minced and zested samples that were within the expected range of previously reported human nipple tissue, while these data revealed no differences in the mechanical properties of the neotissue between time points or processing techniques. CONCLUSIONS: These preliminary data support potential use of decellularized allograft to foster healthy tissue ingrowth within a PLA scaffold, thereby offering a novel solution to current limitations in nipple reconstruction.

Revealing the Invisible Emotion Work of Caregivers: A Photovoice Exploration of Informal Care Provided by Family Caregivers for Post-9/11 Veterans With Traumatic Brain Injuries.

OBJECTIVE: Describe the different forms of emotion work performed by family caregivers of veterans living with a traumatic brain injury (TBI). DESIGN: Collaborators were provided cameras to take photographs illustrating their experiences as family caregivers. The meaning behind caregiver photographs was solicited using photoelicitation interviews and coded. SETTING: Homes of veterans or other informal settings in 2 regions of the United States served by the Southeast Louisiana Veterans Health Care System and the Veterans Affairs Portland Health Care System. PARTICIPANTS: Twenty-six family caregivers of post-9/11 era veterans with TBI. RESULTS: Caregivers described performing different types of intangible, and largely invisible, work centered on emotion management. Emotion work primarily involved creating a new normal, keeping things calm, and suppressing their own emotional experiences to "put on a brave face." Although having derived a sense of satisfaction and identity from their role, caregivers acknowledged that emotion work was challenging and sometimes stressful. The Photovoice method allowed caregivers to express through metaphor experiences that otherwise would have been hard to articulate and share with others. CONCLUSION: Findings signal a need for healthcare systems and providers to acknowledge emotion work as a potential source of stress and to provide multifaceted support for veterans and family caregivers.

Molecular Profiling of Clear Cell Ovarian Cancers: Identifying Potential Treatment Targets for Clinical Trials.

BACKGROUND: Advanced stage/recurrent clear cell ovarian cancers (CCOCs) are characterized by a low response to chemotherapy and a poor prognosis. There is growing interest in investigating novel/molecular targeted therapies in patients with CCOC in histotype-specific trials. However, CCOCs are not a uniform entity and comprise a number of molecular subtypes and it is unlikely that a single approach to treatment will be appropriate for all patients. The aim of this study was to analyze the results of a multiplatform profiling panel in CCOCs to identify potential therapeutic targets. PATIENTS AND METHODS: Tumor profiling was performed on 521 CCOCs. They were grouped into pure (n = 422) and mixed (n = 99) CCOC for analysis. Testing included a combination of DNA sequencing (including next-generation sequencing) using a 46-gene panel, immunohistochemistry, fluorescent or chromogenic in situ hybridization, and RNA fragment analysis. RESULTS: The most common findings were in the PIK3CA/Akt/mTOR pathway, with 61% of all CCOCs showing a molecular alteration in one of these pathway components. Next-generation sequencing revealed PIK3CA mutations in 50% of pure CCOCs. Significant differences were observed between pure and mixed CCOCs with respect to hormone receptor expression (9% vs 34.7% for ER, 13.45 vs 26.4% for PR), cMET (24.1% vs 11.6%), PD-1 tumor infiltrating lymphocytes (48.1% vs 100%), expression of PD-L1 (7.4% vs 25%), and TOPO1 (41% vs 27.1%) on immunohistochemistry, whereas next-generation sequencing revealed significant differences in mutation frequency in PIK3CA (50% vs 18.5%), TP53 (18.1% vs 57.7%), KRAS (12.4% vs 3.7%), and cMET (1.9% vs 11.1%). CONCLUSIONS: This large study confirms that the PIK3CA/Akt/mTOR pathway is commonly altered in CCOCs, and highlights the significant differences between pure and mixed CCOCs. Clear cell ovarian cancers are molecularly heterogeneous and there are a number of potential therapeutic targets which could be tested in clinical trials.

Customizable, biocompatible implants for dorsal nasal augmentation: An in vivo pilot study of eight polylactic acid scaffold designs.

Augmentation of the nasal dorsum often requires implantation of structural material. Existing methods include autologous, cadaveric or alloplastic materials and injectable hydrogels. Each of these options is associated with considerable limitations. There is an ongoing need for precise and versatile implants that produce long-lasting craniofacial augmentation. Four separate polylactic acid (PLA) dorsal nasal implant designs were 3D-printed. Two implants had internal PLA rebar of differing porosities and two were designed as "shells" of differing porosities. Shell designs were implanted without infill or with either minced or zested processed decellularized ovine cartilage infill to serve as a "biologic rebar", yielding eight total treatment groups. Scaffolds were implanted heterotopically on rat dorsa (N = 4 implants per rat) for explant after 3, 6, and 12 months followed by volumetric, histopathologic, and biomechanical analysis. Low porosity implants with either minced cartilage or PLA rebar infill had superior volume retention across all timepoints. Overall, histopathologic and immunohistochemical analysis showed a resolving inflammatory response with an M1/M2 ratio consistently favoring tissue regeneration over the study course. However, xenograft cartilage showed areas of degradation and pro-inflammatory infiltrate contributing to volume and contour loss over time. Biomechanical analysis revealed all constructs had equilibrium and instantaneous moduli higher than human septal cartilage controls. Biocompatible, degradable polymer implants can induce healthy neotissue ingrowth resulting in guided soft tissue augmentation and offer a simple, customizable and clinically-translatable alternative to existing craniofacial soft tissue augmentation materials. PLA-only implants may be superior to combination PLA and xenograft implants due to contour irregularities associated with cartilage degradation.

Neurophysiological correlates of motor and working memory performance following subthalamic nucleus stimulation.

Subthalamic nucleus (STN) deep brain stimulation (DBS) has become an accepted treatment for the motor manifestations of Parkinson disease (PD). The beneficial motor effects of STN DBS are likely due to modulation of BG output to frontal cortical regions associated with motor control, but the underlying neurophysiology of STN DBS effects, especially at the level of the cortex, is not well understood. In this study, we examined the effects of STN DBS on motor disability and visual working memory, a cognitive process supported by pFC. We tested 10 PD participants off medications, ON and OFF stimulation, along with 20 normal controls on a visual working memory task while simultaneously recording cortical EEG. In the OFF state, PD patients had poor motor function, were slower and less accurate in performing the working memory task, and had greater amplitudes and shorter latencies of the N200 ERP response. DBS improved clinical motor function, reduced N200 amplitudes, and increased N200 latencies but had little effect on working memory performance. We conclude that STN DBS normalizes neurophysiological activity in fronto striatal circuits and this may independently affect motor and cognitive function.

Identification of HRAS mutations and absence of GNAQ or GNA11 mutations in deep penetrating nevi.

HRAS is mutated in ∼15% of Spitz nevi, and GNAQ or GNA11 is mutated in blue nevi (46-83% and ∼7% respectively). Epithelioid blue nevi and deep penetrating nevi show features of both blue nevi (intradermal location, pigmentation) and Spitz nevi (epithelioid morphology). Epithelioid blue nevi and deep penetrating nevi can also show overlapping features with melanoma, posing a diagnostic challenge. Although epithelioid blue nevi are considered blue nevic variants, no GNAQ or GNA11 mutations have been reported. Classification of deep penetrating nevi as blue nevic variants has also been proposed, however, no GNAQ or GNA11 mutations have been reported and none have been tested for HRAS mutations. To better characterize these tumors, we performed mutational analysis for GNAQ, GNA11, and HRAS, with blue nevi and Spitz nevi as controls. Within deep penetrating nevi, none demonstrated GNAQ or GNA11 mutations (0/38). However, 6% revealed HRAS mutation (2/32). Twenty percent of epithelioid blue nevi contained a GNAQ mutation (2/10), while none displayed GNA11 or HRAS mutation. Eighty-seven percent of blue nevi contained a GNAQ mutation (26/30), 4% a GNA11 mutation (1/28), and none an HRAS mutation. Within Spitz nevi, none demonstrated GNAQ or GNA11 mutations (0/30). Seventeen percent contained an HRAS mutation (5/30). All GNAQ and GNA11 mutations were p.Q209L (c.626A>T) point mutations, except 2 GNAQ mutations, which contained novel c.625_626CA>TT double mutations. Four HRAS mutations were in exon 2, and three in exon 3. This is the first study to identify HRAS mutations in deep penetrating nevi. The presence of HRAS mutations and absence of GNAQ or GNA11 mutations in deep penetrating nevi suggests classification of these unusual nevi within the Spitz nevus category of melanocytic tumors, rather than the blue nevus category.

Perfuse and Reuse: A Low-Cost Three-Dimensional-Printed Perfusion Bioreactor for Tissue Engineering.

This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow centimeter-scale vascularized or otherwise canalized tissue constructs to be maintained in weeks long static and/or perfusion culture at an exceptionally low cost, with intermittent live imaging and media sampling capabilities. The perfusion system includes a reusable polydimethylsiloxane (PDMS) lid generated from a three-dimensional (3D)-printed poly-lactic acid (PLA) mold and several lengths of perfusion tubing. The coverslip tissue culture chamber includes PDMS components built with 3D-printed PLA molds, as well as 3D-printed PLA frames and glass coverslips that house perfusable hydrogel constructs. As proof of concept, we fabricated a vascularized hydrogel construct, which was subjected to static and perfusion tissue culture, as well as flow studies using fluorescent beads and widefield fluorescent microscopy. This system can be readily reproduced, promoting the advancement of tissue engineering and regenerative medicine research.

High burden of clonal hematopoiesis in first responders exposed to the World Trade Center disaster.

The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64-6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population.

Twice-Daily Nitrofurantoin Administration Following Short-term Transurethral Catheterization After Pelvic Reconstructive Surgery: A Randomized Clinical Trial.

OBJECTIVE: The objective of this study was to evaluate if twice-daily nitrofurantoin for 5 days after discontinuation of transurethral catheterization decreases the rate of urinary tract infection (UTI) in women with postoperative urinary retention (POUR) after pelvic reconstructive surgery. METHODS: This was a double-blind, placebo-controlled, randomized trial conducted across 2 clinical sites between October 2017 and April 2019. Women with acute POUR after pelvic reconstructive surgery were included and randomized to nitrofurantoin (100 mg) or placebo twice-daily for 5 days. The primary outcome was clinically suspected UTI (defined as dysuria, frequency, and irritation in the absence of vaginal discharge) and/or culture-proven UTI (defined as greater than 105 colony forming units of a single organism) within 30 days of surgery. Secondary outcomes included evaluation of adverse events related to study medication and medication adherence. RESULTS: Data from 164 participants were eligible for intention-to-treat analysis (nitrofurantoin, n = 82; placebo, n = 82). There were no significant demographic or intraoperative differences except for body mass index and race. Median duration of catheterization was 3 days (interquartile range, 2-5 days, P = 0.12). Fifteen women in the nitrofurantoin group and 14 women in the placebo group experienced UTI within 30 days (18.3% vs 17.1%; P = 0.84; odds ratio, 1.09; 95% confidence interval, 0.49-2.43). There were no study medication allergies; however, nausea was the most common intolerance. Most women in each group completed the study drug treatment (91.5% vs 86.4%, P = 0.30). CONCLUSIONS: Nitrofurantoin prophylaxis after transurethral catheter removal did not reduce the risk of UTI in women with acute POUR after pelvic reconstructive surgery.

Facilitated self-assembly of a prevascularized dermal/epidermal collagen scaffold.

Introduction: Resurfacing complex full thickness wounds requires free tissue transfer which creates donor site morbidity. We describe a method to fabricate a skin flap equivalent with a hierarchical microvascular network. Materials & methods: We fabricated a flap of skin-like tissue containing a hierarchical vascular network by sacrificing Pluronic® F127 macrofibers and interwoven microfibers within collagen encapsulating human pericytes and fibroblasts. Channels were seeded with smooth muscle and endothelial cells. Constructs were topically seeded with keratinocytes. Results: After 28 days in culture, multiphoton microscopy revealed a hierarchical interconnected network of macro- and micro-vessels; larger vessels (>100 µm) were lined with a monolayer endothelial neointima and a subendothelial smooth muscle neomedia. Neoangiogenic sprouts formed in the collagen protodermis and pericytes self-assembled around both fabricated vessels and neoangiogenic sprouts. Conclusion: We fabricated a prevascularized scaffold containing a hierarchical 3D network of interconnected macro- and microchannels within a collagen protodermis subjacent to an overlying protoepidermis with the potential for recipient microvascular anastomosis.

Microstructured hydrogel scaffolds containing differential density interfaces promote rapid cellular invasion and vascularization.

INTRODUCTION: Insufficient vascularization of currently available clinical biomaterials has limited their application to optimal wound beds. We designed a hydrogel scaffold with a unique internal microstructure of differential collagen densities to induce cellular invasion and neovascularization. METHODS: Microsphere scaffolds (MSS) were fabricated by encasing 1% (w/v) type 1 collagen microspheres 50-150 µm in diameter in 0.3% collagen bulk. 1% and 0.3% monophase collagen scaffolds and Integra® disks served as controls. Mechanical characterization as well as in vitro and in vivo invasion assays were performed. Cell number and depth of invasion were analyzed using Imaris™. Cell identity was assessed immunohistochemically. RESULTS: In vitro, MSS exhibited significantly greater average depth of cellular invasion than Integra® and monophase collagen controls. MSS also demonstrated significantly higher cell counts than controls. In vivo, MSS revealed significantly more cellular invasion spanning the entire scaffold depth at 14 days than Integra®. CD31+ expressing luminal structures suggestive of neovasculature were seen within MSS at 7 days and were more prevalent after 14 days. Multiphoton microscopy of MSS demonstrated erythrocytes within luminal structures after 14 days. CONCLUSION: By harnessing simple architectural cues to induce cellular migration, MSS holds great potential for clinical translation as the next generation dermal replacement product. STATEMENT OF SIGNIFICANCE: Large skin wounds require tissue engineered dermal substitutes in order to promote healing. Currently available dermal replacement products do not always adequately incorporate into the body, especially in complex wounds, due to poor neovascularization. In this paper, we present a hydrogel with an innovative microarchitecture that is composed of dense type I collagen microspheres suspended in a less-dense collagen bulk. We show that cell invasion into the scaffold is driven solely by mechanical cues inherent within this differential density interface, and that this induces robust vascular cell invasion both in vitro and in a rodent model. Our hydrogel performs favorably compared to the current clinical gold standard, Integra®. We believe this hydrogel scaffold may be the first of the next generation of dermal replacement products.

Stem cell mutations can be detected in myeloma patients years before onset of secondary leukemias.

Therapy-related acute myeloid leukemia and myelodysplastic syndromes (t-AML/t-MDS) are secondary hematologic malignancies associated with poor prognosis, warranting insights into their predisposing conditions and cells of origin. We identified patients with myeloma who developed t-AML/t-MDS and analyzed their stem and progenitor cells collected years before the onset of secondary disease. We demonstrate that aberrant stem cells with high CD123 expression can be detected long before the onset of overt leukemia. Rigorous sorting, followed by targeted sequencing, resulted in ultradeep functional depth of sequencing and revealed preexisting mutant hematopoietic stem cell (HSC) clones, mainly harboring TP53 mutations, that became the dominant population at the time of leukemic presentation. Taken together, these data show that HSCs can act as reservoirs for leukemia-initiating cells many years before the onset of myeloid leukemia.

PD-L1 Status in Refractory Lymphomas.

Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones (SP142 and SP263, Ventana). Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (≥5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.

Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast.

Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases having matched primary and metastatic tumors) were profiled using gene sequencing, gene copy number analysis, whole genome expression, and protein expression. Whole genome expression analysis demonstrated consistent over-expression of genes involved in angiogenesis including VEGFA, Angiopoietin-2, VCAM1, PDGFRA, and PTTG1. EGFR protein overexpression was observed in 26/27 (96%) of cases with amplification of the EGFR gene in 8/24 (33%) cases. Two EGFR mutations were identified including EGFRvIII and a presumed pathogenic V774M mutation, respectively. The most common pathogenic mutations included TP53 (50%) and PIK3CA (15%). Cases with matched primary and metastatic tumors harbored identical mutations in both sites (PIK3CA/KRAS and RB1 gene mutations, respectively). Tumor expression of PD-L1 immunoregulatory protein was observed in 3/22 (14%) of cases. Overexpression of molecular biomarkers of increased angiogenesis, EGFR and immune checkpoints provides novel targeted therapy options in malignant phyllodes tumors of the breast.

Multi-platform molecular profiling of a large cohort of glioblastomas reveals potential therapeutic strategies.

Glioblastomas (GBM) are the most aggressive and prevalent form of gliomas with abysmal prognosis and limited treatment options. We analyzed clinically relevant molecular aberrations suggestive of response to therapies in 1035 GBM tumors. Our analysis revealed mutations in 39 genes of 48 tested. IHC revealed expression of PD-L1 in 19% and PD-1 in 46%. MGMT-methylation was seen in 43%, EGFRvIII in 19% and 1p19q co-deletion in 2%. TP53 mutation was associated with concurrent mutations, while IDH1 mutation was associated with MGMT-methylation and TP53 mutation and was mutually exclusive of EGFRvIII mutation. Distinct biomarker profiles were seen in GBM compared with WHO grade III astrocytoma, suggesting different biology and potentially different treatment approaches. Analysis of 17 metachronous paired tumors showed frequent biomarker changes, including MGMT-methylation and EGFR aberrations, indicating the need for a re-biopsy for tumor profiling to direct subsequent therapy. MGMT-methylation, PR and TOPO1 appeared as significant prognostic markers in sub-cohorts of GBM defined by age. The current study represents the largest biomarker study on clinical GBM tumors using multiple technologies to detect gene mutation, amplification, protein expression and promoter methylation. These data will inform planning for future personalized biomarker-based clinical trials and identifying effective treatments based on tumor biomarkers.

Stimulation of topoisomerase II-mediated DNA cleavage by benzene metabolites.

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic and quinone-based metabolites, especially 1,4-benzoquinone. Since the DNA damage induced by 1,4-benzoquinone is consistent with that of topoisomerase II-targeted drugs, it has been proposed that the compound initiates specific types of leukemia by acting as a topoisomerase II poison. This hypothesis, however, was not supported by initial in vitro studies. While 1,4-benzoquinone inhibited topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage were not observed. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of benzene metabolites (including 1,4-benzoquinone, 1,4-hydroquinone, catechol, 1,2,4-benzenetriol, 2,2'-biphenol, and 4,4'-biphenol) on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. Other metabolites displayed considerably less activity. DNA cleavage enhancement by 1,4-benzoquinone was unseen in previous studies due to the presence of reducing agents and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. Unlike anticancer drugs such as etoposide that interact with topoisomerase IIalpha in a noncovalent manner, the actions of 1,4-benzoquinone appear to involve a covalent attachment to the enzyme. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. These findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of some benzene-induced leukemias.

Molecular profiling of infiltrating urothelial carcinoma of bladder and nonbladder origin.

BACKGROUND: Infiltrating UC represents the second most common genitourinary malignancy. Advanced UC has a poor prognosis and new treatments are needed. Molecular profiling of UC might identify biomarkers associated with targeted therapies or chemotherapeutics, providing physicians with new treatment options. MATERIALS AND METHODS: Five hundred thirty-seven cases of locally advanced or metastatic UC of the bladder, 74 nonbladder, and 55 nonurothelial bladder cancers were profiled using mutation analysis, in situ hybridization, and immunohistochemistry assays for biomarkers predictive of therapy response. RESULTS: Molecular profiling of UC showed high overexpression of topoisomerase 2α, common phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha and/or phosophatase and tensin homolog (PTEN) alterations in nonbladder (27%) and bladder UC (21%), and rare gene mutations across subtypes. Compared with nonbladder, bladder UC consistently exhibited more frequent abnormal protein expression, including HER2 (10% vs. 3%; P = .04), tyrosine protein c-Kit receptor kinases (11% vs. 5%), c-Met proto-oncogene, receptor tyrosine kinases (25% vs. 8%), androgen receptor (16% vs. 6%), O(6)-methylguanine-methyltransferase (63% vs. 43%), ribonucleotide reductase M1 (32% vs. 11%), Serum protein acidic and rich in cysteine (SPARC) (69% vs. 33%), and topoisomerase 1 (63% vs. 39%). Bladder UC also exhibited increased amplification of HER2 (12% vs. 2%; P = .06). CONCLUSION: Comprehensive molecular profiling of UC identified a large number of biomarkers aberrations that might direct treatment in conventional chemotherapies and targeted therapies, not currently recommended in this population. As a group, bladder UC exhibited higher levels of actionable biomarkers, suggesting that UC from different primary sites and non-UC are driven by different molecular pathways. These differences could have clinical implications resulting in different treatment regimens depending on the site of origin of UC.

Mutations in the Kinase Domain of the HER2/ERBB2 Gene Identified in a Wide Variety of Human Cancers.

The HER2 (official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non-small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2 mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2 mutations identified as the sole driver mutation. HER2 mutations coexisted with HER2 gene amplification and overexpression and with mutations in other functionally important genes. HER2 mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted.

Disseminated histiocytoses biomarkers beyond BRAFV600E: frequent expression of PD-L1.

The histiocytoses are rare tumors characterized by the primary accumulation and tissue infiltration of histiocytes and dendritic cells. Identification of the activating BRAFV600E mutation in Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH) cases provided the basis for the treatment with BRAF and/or MEK inhibitors, but additional treatment options are needed. Twenty-four cases of neoplastic histiocytic diseases [11 extrapulmonary LCH, 4 ECD, 4 extranodal Rosai-Dorfman disease (RDD), 3 follicular dendritic cell sarcoma (FDCS), 1 histiocytic sarcoma (HS) and 1 blastic plasmacytoid dendritic cell neoplasm (BPDCN)] were analyzed using immunohistochemical and mutational analysis in search of biomarkers for targeted therapy. BRAF V600E mutations were detected in 4/11 LCH and 4/4 ECD cases. A pathogenic PTEN gene mutation and loss of PTEN protein expression were identified in the case of HS. Increased expression of PD-L1 (≥2+/≥5%) was seen in 3/4 ECD, 7/8 LCH, 3/3 FDCS and 1/1 HS, with overall 81% concordance between 2 antibodies used in the study (SP142 vs. MAB1561 clone). These results show for the first time significant expression of the PD-L1 immune checkpoint protein in these disorders, which may provide rationale for addition of immune check-point inhibitors in treatment of disseminated and/or refractory histiocytoses.