Adenoviral Vector Expressing Murine Angiostatin Inhibits a Model of Breast Cancer Metastatic Growth in the Lungs of Mice

Angiostatin, an internal fragment of plasminogen, has been shown to inhibit the process of angiogenesis or neovascularization. In this study, we have expressed the cDNA for murine angiostatin under the control of the human cytomegalovirus promoter from a human type-5 adenovirus and shown that this vector produces a protein which retains biological activity. Angiostatin expression was determined by Northern blot analysis and Western immunoblotting. Ad-angiostatin, but not a control vector Ad-dl70, significantly reduced the viability of infected human umbilical cord vein endothelial cells (HUVEC) in vitro. In an in vivo model of basic fibroblast growth factor-induced angiogenesis, Ad-angiostatin (1 x 10(9) pfu) could inhibit endothelial cell migration and the formation of capillaries within a Matrigel plug which had been implanted for one week subcutaneously into C57BL/6 mice. Endothelial cells in these plugs had an altered, rounded, phenotype with dark picnotic nuclei indicative of apoptosis, which was confirmed using transmission electron microscopy. In contrast, endothelial cells from bFGF alone or in combination with the control vector-treated plugs retained the long spindle shape characteristic of endothelial cells. Intranasal delivery of Ad-angiostatin into the lungs of FVB/n mice demonstrated comparable cellular infiltration in the recovered bronchoalveolar lavage fluid with no signs of abnormal pathology as compared to PBS or control vector-treated animals. In a pulmonary metastatic breast cancer model, the delivery of Ad-angiostatin (1 x 10(9) pfu) to the lung significantly delayed tumor growth as measured by the number of visible surface tumor nodules. This study has demonstrated that the specific targeting of tumors to inhibit angiogenesis using an adenovirus expressing angiostatin, may deliver localized concentrations of protein having a greater impact on inhibition of tumor growth.     

Metastatic Breast Tumour Regression Following Treatment by a Gene-Modified Vaccinia Virus Expressing MUC1 and IL-2

MUC1 is expressed by glandular epithelial cells. It isoverexpressed in the majority of breast tumours, making it apotential target for immune therapy. The objectives of thepresent study were to evaluate the anti-tumour activity andtolerance of repeated administration of TG1031 (an attenuatedrecombinant vaccinia virus containing sequences coding for humanMUC1 and the immune stimulatory cytokine IL-2) in patients withMUC1-positive metastatic breast cancer. This was an open-label,randomised study comparing two dose levels, 5 × 10E6and 5 × 10E7pfu, with 14 patients in each arm.The treatment was administered intramuscularly every 3 weeks forthe first 4 doses and every 6 weeks thereafter, untilprogression. Two patients had a partial tumour regression(> 50%), and 15 patients had stable disease as their bestoverall response until at least the 5th injection. Partialregression lasted for 11 months in one patient and for 12 monthsin the second patient who then underwent surgical resection ofher hepatic metastases. The most frequent adverse events includedinflammation at injection site: 7 patients, itching or pain atinjection site: 5 patients, and moderate fever: 6 patients. Oneresponding patient developed antinuclear, anti-DNA, and increasedanti-TPO antibodies after the fifth injection, and which resolvedat the end of treatment. The treatment regimes were welltolerated with a low toxicity profile. Although clinical efficacyremains limited, this study demonstrates the potential use ofMUC1-based immune therapy in breast cancer.     

Alterations in Copy Number of c-erbB-2 and c-myc Proto-oncogenes in Advanced Stage of Human Breast Cancer

In our previous study, amplification of c- erb B-2 and c- myc proto-oncogenes in DNA of human breast cancer occurred in 16% and 4% of cases, respectively, and increased copy number of these genes is suggested to be associated with aggressive primary tumors. We examined change in the copy number of c-erb B 2 and c-myc proto-oncogenes between primary and multiple metastatic tumors in 10 patients with breast cancer, who underwent breast surgery and were later autopsied, by using DNAs isolated from formalin-fixed paraffin-embedded tissues and the dot blot-hybridization method. In primary tumors, amplification of c-erbB-2 and c-myc was detected in three and two cases, whereas at the stage with systemic metastasis, it was detected in four and three cases, respectively. In all four cases with amplified c-erbB-2 gene and in one of the three with amplified c-myc gene, the copy number was clearly increased in the metastatic tumors in comparison with the primary. Microscopically, more than five mitotic figures per high power field were detected in metastatic tumors of five cases including three with amplified c-erbB-2, but in only two primary. These results suggested that the aggressive nature of breast cancer is frequently enhanced in accordance with cancer metastasis. Acta Pathol Jpn 41: 19–23, 1991.     

Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

FANCD2, a pivotal protein in the Fanconi anemia and BRCA pathway/network, is monoubiquitylated in the nucleus in response to DNA damage. This study examines the subcellular location and relationship with prognostic factors and patient survival of FANCD2 in breast cancer. Antibodies to FANCD2 were used to immunocytochemically stain 16 benign and 20 malignant breast specimens as well as 314 primary breast carcinomas to assess its association with subcellular compartment and prognostic factors using Fisher’s Exact test or with patient survival over 20 years using Wilcoxon-Gehan statistics. Immunoreactive FANCD2 was found in the nucleus and cytoplasm of all 16 benign tissues, but nuclear staining was lost from a significant 19/20 malignant carcinomas (P < 0.0001). Antibodies to FANCD2 stained the cytoplasm of 196 primary carcinomas, leaving 118 as negatively stained. Negative cytoplasmic staining was significantly associated with positive staining for the metastasis-inducing proteins S100A4, S100P, osteopontin, and AGR2 (P ≤ 0.002). Survival of patients with FANCD2-negative carcinomas was significantly worse (P < 0.0001) than those with positively stained carcinomas, and only 4% were alive at the census date. Multivariate regression analysis identified negative staining for cytoplasmic FANCD2 as the most significant indicator of patient death (P = 0.001). Thus FANCD2’s cytoplasmic loss in the primary carcinomas may allow the selection of cells overexpressing proteins that can induce metastases before surgery.Previous reports have shown that the expression of four proteins that can induce metastasis in experimental rats are highly significantly correlated with each other and separately with early patient death in human breast cancer. These four proteins are S100A4, osteopontin (OPN), AGR2, and S100P.^1,2,3,4 If their expression is coordinated, then markers of the underlying mechanism should be even more highly correlated with patient demise. One possible coordination mechanism is the generation of an unstable genome by failure of a DNA repair pathway or some other protective mechanism. The majority of familial breast cancer is associated with individuals heterozygous for the BRCA1 or BRCA2 genes that encode proteins important for the repair of DNA double strand breaks and interstrand crosslinks by homologous recombination.^5,6 Biallelic inactivation of BRCA2 results in one form of the cancer-prone syndrome Fanconi anemia (complementation group FA-D1) and the BRCA2/FANCD1 protein operates with 12 other Fanconi anemia (FA) proteins and BRCA1 in a multifaceted response to DNA damage known as the FA/BRCA tumor suppressor pathway/network.^7,8,9,10 Eight of the 13 proteins, together with two FA-associated-proteins, participate in a nuclear core-complex that is required for the monoubiquitylation of FANCD2 and FANCI.⁸ FANCD2 is pivotal in the FA pathway translocating to chromatin and on monoubiquitylation, co-localizing with BRCA1, BRCA2 and the recombinase RAD51 in DNA damage inducible foci.^{11,12,13,14,15} FANCD2 is primarily regarded as being active within the nucleus and specifically in chromatin, although a cytoplasmic function has recently been proposed.¹⁶ The FA proteins BRCA2/FANCD1 and FANCN/PALB2 are believed to function downstream of, or in parallel with, FANCD2 monoubiquitylation and FANCN and FANCJ/BRIP1, like BRCA2, are now considered to be breast cancer susceptibility genes.^17,18 While FANCD2 directly interacts with BRCA2,¹⁹ and is found in complex with BRCA2 in mammalian cells,^13,20 there is no direct evidence for changes in FANCD2 in the development of familial breast cancer. However, FancD2 knockout mice exhibit an increased incidence of epithelial cancers including hepatic, ovarian, and mammary tumors,²¹ reduced FANCD2 expression is associated with familial ovarian cancer²² and several FA core-complex genes have now been implicated with several types of sporadic cancer, including ovarian and pancreatic cancer.²³ This suggests that a failure to express or post-translationally modify FANCD2 may play a role in the development of sporadic breast cancer. In contrast, a previous report in human breast cancer over a relatively short 6-year follow-up has suggested that high nuclear FANCD2 expression is correlated with poor patient survival.²⁴ To resolve this apparent contradiction we now show that breast carcinomas contain mainly cytoplasmic FANCD2 and that its loss is strongly correlated with the expression of the four metastasis-inducing proteins and particularly with premature death of patients with sporadic breast cancer over a much longer follow-up period of 20 years.     

Spotlight on Trastuzumab in the Management of HER2-Positive Metastatic and Early-Stage Breast Cancer

Trastuzumab (Herceptin) is a humanized monoclonal antibody used in the treatment of breast cancer that overexpresses human epidermal growth factor receptor 2 (HER2), which is associated with clinically aggressive disease and a poor prognosis.The addition of intravenous trastuzumab to first-line chemotherapy improved the time to disease progression, objective response rate, duration of response, and overall survival in randomized, multicenter trials in women with HER2-positive metastatic breast cancer. As such, trastuzumab has become the standard of care in this setting, despite its high acquisition cost and potential for cardiac events, and is licensed for use in combination with paclitaxel (Europe and the US) or docetaxel (Europe). In addition, trastuzumab monotherapy is approved for use in patients with HER2-positive metastatic breast cancer who have previously received chemotherapy for their metastatic disease. Recent data from large phase III trials with trastuzumab in the adjuvant setting revealed significant improvements in disease-free and overall survival. Thus, trastuzumab is also rapidly becoming a standard component of adjuvant therapy for patients with HER2-positive early-stage breast cancer.     

Sox2、EpCAM在三阴乳腺癌及其癌症转移灶中的表达与临床意义

目的 检测Sox2、EpCAM在三阴乳腺癌及其癌症转移灶中的表达并探讨其临床意义.方法 选择2015年1月至2016年12月我院收治的30例发生癌症转移的三阴乳腺癌患者,作为观察组.并选取同期的非三阴乳腺癌患者30例作为对照组,对观察组和对照组癌症原发灶及转移灶进行 Sox2、EpCAM的表达分析.结果 观察组30例三阴乳腺癌中,TNM分期越高,Sox2的表达越高,Ⅲ期阳性率显著高于Ⅰ期和Ⅱ期,差异有统计学意义(P<0.05),而EpCAM的表达差异不具有统计学意义(P>0.05);组织学分级越高,Sox2、EpCAM的表达也越高,Ⅲ级阳性率显著高于Ⅰ级和Ⅱ级,差异有统计学意义(P<0.05).Sox2在观察组转移灶的阳性表达率(93.3%)高于原发灶(70.0%),且显著高于对照组(43.3%),差异具有统计学意义(P<0.05);EpCAM 在观察组转移灶中的阳性表达率(86.7%)与原发灶(80.0%)相比,差异无统计学意义(P>0.05),与对照组(53.3%)相比,差异具有统计学意义(P<0.05).结论 在三阴乳腺癌中SOX2、EpCAM蛋白均呈高表达,可作为临床检测三阴乳腺癌的一个参考指标,且在转移灶中显著表达,也可作为癌症转移的辅助诊断指标.     

Establishment of an Orthotopic Mouse Non-Muscle Invasive Bladder Cancer Model Expressing the Mammalian Target of Rapamycin Signaling Pathway

We established an orthotopic non-muscle invasive bladder cancer (NMIBC) mouse model expressing the mammalian target of the rapamycin (mTOR) signaling pathway. After intravesical instillation of KU-7-lucs (day 0), animals were subsequently monitored by bioluminescence imaging (BLI) on days 4, 7, 14, and 21, and performed histopathological examination. We also validated the orthotopic mouse model expressing the mTOR signaling pathway immunohistochemically. In vitro BLI photon density was correlated with KU-7-luc cell number (r² = 0.97, P < 0.01) and in vivo BLI photon densities increased steadily with time after intravesical instillation. The tumor take rate was 84.2%, formed initially on day 4 and remained NMIBC up to day 21. T1 photon densities were significantly higher than Ta (P < 0.01), and histological tumor volume was positively correlated with BLI photon density (r² = 0.87, P < 0.01). The mTOR signaling pathway-related proteins were expressed in the bladder, and were correlated with the western blot results. Our results suggest successful establishment of an orthotopic mouse NMIBC model expressing the mTOR signaling pathway using KU-7-luc cells. This model is expected to be helpful to evaluate preclinical testing of intravesical therapy based on the mTOR signaling pathway against NMIBC.     

Establishment of Patient-Derived Gastric Cancer Organoid Model From Tissue Obtained by Endoscopic Biopsies

Cancer organoids are three-dimensional mini-organ analogues derived from cancer tissues and have been proposed as models capable of simulating the structure and function of human organs and tissues in vitro. We sought to establish gastric cancer patient-derived organoids (PDOs) from tissues obtained by endoscopic biopsies. Gastric cancer-PDOs were successfully established and cultured from cancer tissues with gastric adenocarcinoma by endoscopic biopsies. To confirm that gastric cancer-PDOs were derived from cancer tissue, the consistency of the original cancer tissue was assessed by histopathological examination. As a result, it was confirmed that the shape and internal structure of gastric cancer-PDO were derived from the original gastric cancer cells, and the tumor specificity of gastric cancer-PDO was confirmed through Periodic acid-Schiff (PAS) and polyclonal carcinoembryonic antigen antibody staining. These results demonstrate that gastric cancer-PDO models show the characteristics of primary tumors and have potential for drug screening and providing a personalized medicine platform.     

Investigative Study of Myofibroblasts and Cytokines in Peri-Implant Tissue of Silicone Breast Expander by RT-PCR in a Rat Model

The excessive collagen deposition around silicone breast implants followed by contracture and development of severe pain is a major clinical problem. This study was conducted to investigate the profibrotic and antifibrotic cytokines secreted by inflammatory cells and development of myofibroblasts at the tissue material interface around silicone breast expander and ultra-high-molecular-weight polyethylene (UHMWPE). Both materials were implanted in rats for 30, 90 and 180 days. Inflammatory cells and collagen deposition at the material–tissue interface were assessed with Haematoxylin-Eosin and Masson's Trichrome stain. Gene expression of TGFβ, IL-1β, IFNγ, IL10 and α-SMA was quantitated by real-time (RT)-PCR in the peri-implant tissue. Results indicate a difference in collagen deposition and myofibroblast development around both materials with involvement of TGFβ, IFNγ and IL10. The results emphasise the need for further investigation into the molecular mechanisms of protomyofibroblast and myofibroblast formation around silicone implants, which would provide information on these target cells for inhibitory therapy in the clinical situation.     

Expression of MMP2, MMP9 and MMP3 in Breast Cancer Brain Metastasis in a Rat Model

In order to study the expression of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain. Immunohistochemistry and Western Blotting analyses showed that MMP-2, -3 and -9 proteins expressions are consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. These results were confirmed by RT-PCR. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. Together our results suggest that MMP-2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain.     

利多卡因下调ABCG2抑制人乳腺癌细胞的活性和耐药性

目的 探究利多卡因体外下调ATP结合转运蛋白G家族成员2(ATP binding cassette transporter G family member 2,ABCG2)抑制人乳腺癌细胞的活性及顺铂耐药性的可能机制.方法 选取MDA-MB-231、MCF-7细胞,使用CCK-8、EdU、TUNEL法染色验证利多卡因对...     

Weekly Paclitaxel and Trastuzumab as a First-Line Therapy in Patients with HER2-Overexpressing Metastatic Breast Cancer: Magnitude of HER2/neu Amplification as a Predictive Factor for Efficacy

We evaluated the efficacy and safety of weekly paclitaxel plus trastuzumab as firs-tline chemotherapy in women with HER2-overexpressing metastatic breast cancer (MBC), and we investigated the prognostic factors including magnitude of HER2/neu amplification in this population. We analyzed 54 patients with HER2-overexpressing MBC that were treated with weekly paclitaxel plus trastuzumab as first-line chemotherapy from February 2004 to December 2006. At a median follow-up of 28 months, median time to progression (TTP) was 16.6 months (95% CI, 9.4 to 23.7 months) and median overall survival was 25.6 months (95% CI, 21.8 to 27.3 months). Therapy was generally well tolerated, although three patients (5.5%) experienced reversible, symptomatic heart failure. Of the 27 patients evaluable for the HER2 FISH, patients with a HER2/CEP17 ratio of ≤4.0 had significantly shorter TTP than those with a HER2/CEP17 ratio of >4.0 (10.8 vs. 23.2 months, P=0.034). A HER2/CEP17 ratio of >4.0 was identified as significant predictive factor of TTP by multivariate analysis (P=0.032). The combination of weekly paclitaxel plus trastuzumab as first-line chemotherapy is an effective regimen in patients with HER2-FISH-positive MBC. Furthermore, the magnitude of HER2 amplification is an independent predictive factor of TTP.     

Comparison of Her-2, EGFR and Cyclin D1 in Primary Breast Cancer and Paired Metastatic Lymph Nodes: An Immunohistochemical and Chromogenic In Situ Hybridization Study

The significant advance in the development of molecular-targeting drugs has made an evaluation of Her-2, EGFR, and cyclin D1 an important clinical issue in breast cancer patients. This study compared the Her-2, EGFR, and cyclin D1 status of primary tumors as well as their matching lymph node metastases using immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in 73 breast cancer patients. Her-2, EGFR, and cyclin D1 protein showed a concordance between the primary lesion and the metastatic regional lymph nodes in 82%, 90%, and 63%, respectively. CISH also revealed 92%, 93%, and 85% concordance in the gene amplification status of Her-2, EGFR, and cyclin D1, showing a reasonable agreement between primary tumors and metastatic regional lymph nodes. Although a statistically significant agreement was found in Her-2 expression, a relatively high discordance rate (18%) raises a little concern. Our findings suggest that the Her-2 status can be reliably assessed on primary tumor but a possible difference can be found in Her-2, EGFR, and cyclin D1 status between the primary and the metastatic sites and this possibility should be concerned in patients considering molecular targeted therapy or patients with progress of disease.     

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a

Detection and Identification of Yeasts from Formalin-Fixed,Paraffin-Embedded Tissue by Use of PCR-Electrospray Ionization Mass Spectrometry

Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.