The DNA damage response pathway regulates the expression of the immune checkpoint CD47.

CD47 is a cell surface ligand expressed on all nucleated cells. It is a unique immune checkpoint protein acting as "don't eat me" signal to prevent phagocytosis and is constitutively overexpressed in many tumors. However, the underlying mechanism(s) for CD47 overexpression is not clear. Here, we show that irradiation (IR) as well as various other genotoxic agents induce elevated expression of CD47. This upregulation correlates with the extent of residual double-strand breaks (DSBs) as determined by γH2AX staining. Interestingly, cells lacking mre-11, a component of the MRE11-RAD50-NBS1 (MRN) complex that plays a central role in DSB repair, or cells treated with the mre-11 inhibitor, mirin, fail to elevate the expression of CD47 upon DNA damage. On the other hand, both p53 and NF-κB pathways or cell-cycle arrest do not play a role in CD47 upregualtion upon DNA damage. We further show that CD47 expression is upregulated in livers harvested from mice treated with the DNA-damage inducing agent Diethylnitrosamine (DEN) and in cisplatin-treated mesothelioma tumors. Hence, our results indicate that CD47 is upregulated following DNA damage in a mre-11-dependent manner. Chronic DNA damage response in cancer cells might contribute to constitutive elevated expression of CD47 and promote immune evasion.

DSP107 combines inhibition of CD47/SIRPα axis with activation of 4-1BB to trigger anticancer immunity.

BACKGROUND: Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPα and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPα 'don't eat me' signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. METHODS: Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. RESULTS: Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPα-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. CONCLUSIONS: DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients.

Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

Cardioprotection by placenta-derived stromal cells in a murine myocardial infarction model.

BACKGROUND: Autologous cells for cell therapy of ischemic cardiomyopathy often display age- and disease-related functional impairment, whereas an allogenic immunotolerant cell product would allow off-the-shelf application of uncompromised donor cells. We investigated the cardiac regeneration potential of a novel, clinical-grade placenta-derived human stromal cell product (PLX-PAD). METHODS: PLX-PAD cells derived from human donor placentas and expanded in a three-dimensional bioreactor system were tested for surface marker expression, proangiogenic, anti-inflammatory, and immunomodulatory properties in vitro. In BALB/C mice, the left anterior descending artery was ligated and PLX-PAD cells (n = 10) or vehicle (n = 10) were injected in the infarct border zone. Four weeks later, heart function was analyzed by two-dimensional and M-mode echocardiography. Scar size, microvessel density, extracellular matrix composition, myocyte apoptosis, and PLX-PAD cell retention were studied by histology. RESULTS: In vitro, PLX-PAD cells displayed both proangiogenesis and anti-inflammatory properties, represented by the secretion of both vascular endothelial growth factor and angiopoietin-1 that was upregulated by hypoxia, as well as by the capacity to suppress T-cell proliferation and augment IL-10 secretion when co-cultured with peripheral blood mononuclear cells. Compared with control mice, PLX-PAD-treated hearts had better contractile function, smaller infarct size, greater regional left ventricular wall thickness, and less apoptosis after 4 wk. PLX-PAD stimulated both angiogenesis and arteriogenesis in the infarct border zone, and periostin expression was upregulated in PLX-PAD-treated hearts. CONCLUSIONS: Clinical-grade PLX-PAD cells exert beneficial effects on ischemic myocardium that are associated with improved contractile function, and may be suitable for further evaluation aiming at clinical pilot trials of cardiac cell therapy.

Novel microRNA-based assay demonstrates 92% agreement with diagnosis based on clinicopathologic and management data in a cohort of patients with carcinoma of unknown primary.

BACKGROUND: Cancer of unknown or uncertain primary is a major diagnostic and clinical challenge, since identifying the tissue-of-origin of metastases is crucial for selecting optimal treatment. MicroRNAs are a family of non-coding, regulatory RNA molecules that are tissue-specific, with a great potential to be excellent biomarkers. METHODS: In this study we tested the performance of a microRNA-based assay in formalin-fixed paraffin-embedded samples from 84 CUP patients. RESULTS: The microRNA based assay agreed with the clinical diagnosis at presentation in 70% of patients; it agreed with the clinical diagnosis obtained after patient management, taking into account response and outcome data, in 89% of patients; it agreed with the final clinical diagnosis reached with supplemental immunohistochemical stains in 92% of patients, indicating a 22% improvement in agreement from diagnosis at presentation to the final clinical diagnosis. In 18 patients the assay disagreed with the presentation diagnosis and was in agreement with the final clinical diagnosis, which may have resulted in the administration of more effective chemotherapy. In three out of four discordant cases in which supplemental IHC was performed, the IHC results validated the assay's molecular diagnosis. CONCLUSIONS: This novel microRNA-based assay shows high accuracy in identifying the final clinical diagnosis in a real life CUP patient cohort and could be a useful tool to facilitate administration of optimal therapy.

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

BACKGROUND: Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. METHODS: We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. RESULTS: Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. CONCLUSION: The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

MicroRNAs as a potential prognostic factor in gastric cancer.

AIM: To compare the microRNA (miR) profiles in the primary tumor of patients with recurrent and non-recurrent gastric cancer. METHODS: The study group included 45 patients who underwent curative gastrectomies from 1995 to 2005 without adjuvant or neoadjuvant therapy and for whom adequate tumor content was available. Total RNA was extracted from formalin-fixed paraffin-embedded tumor samples, preserving the small RNA fraction. Initial profiling using miR microarrays was performed to identify potential biomarkers of recurrence after resection. The expression of the differential miRs was later verified by quantitative real-time polymerase chain reaction (qRT-PCR). Findings were compared between patients who had a recurrence within 36 mo of surgery (bad-prognosis group, n = 14, 31%) and those who did not (good-prognosis group, n = 31, 69%). RESULTS: Three miRs, miR-451, miR-199a-3p and miR-195 were found to be differentially expressed in tumors from patients with good prognosis vs patients with bad prognosis (P < 0.0002, 0.0027 and 0.0046 respectively). High expression of each miR was associated with poorer prognosis for both recurrence and survival. Using miR-451, the positive predictive value for non-recurrence was 100% (13/13). The expression of the differential miRs was verified by qRT-PCR, showing high correlation to the microarray data and similar separation into prognosis groups. CONCLUSION: This study identified three miRs, miR-451, miR-199a-3p and miR-195 to be predictive of recurrence of gastric cancer. Of these, miR-451 had the strongest prognostic impact.

Prospective gene signature study using microRNA to identify the tissue of origin in patients with carcinoma of unknown primary.

PURPOSE: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response. RESULTS: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases. CONCLUSIONS: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis.

Accurate classification of metastatic brain tumors using a novel microRNA-based test.

BACKGROUND: Identification of the tissue of origin of a brain metastatic tumor is vital to its management. Carcinoma of unknown primary (CUP) is common in oncology, representing 3%-5% of all invasive malignancies. We aimed to validate a recently developed microRNA-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) test for identifying the tumor tissue of origin, first in a consecutive cohort of metastatic tumors of known origin and then in a cohort of CUP cases resected from the central nervous system (CNS). PATIENTS AND METHODS: One hundred two resected CNS metastatic tumors with known origin, previously classified based on the patient's clinical history and pathological data, as well as a second cohort of resected CNS tumors from 57 patients originally diagnosed as CUP were studied. A qRT-PCR diagnostic assay that measures the expression level of 48 microRNAs was used to classify the tissue of origin of these metastatic tumors. RESULTS: In this blinded study, the test predictions correctly identified the reference diagnosis of the samples of known origin, excluding samples from prostate origin, in 84% of cases. In the second CUP patient cohort, the test prediction was in agreement with the diagnosis that was later confirmed clinically or with pathological evaluation in 80% of cases. CONCLUSION: In a cohort of brain and spinal metastases, a previously developed test based on the expression of 48 microRNAs allowed accurate identification of the tumor tissue of origin in the majority of cases. The high accuracy of this test in identifying the tissue of origin of metastases of unknown primary is demonstrated for the first time and may have broad clinical application.

hsa-miR-191 is a candidate oncogene target for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.

A diagnostic assay based on microRNA expression accurately identifies malignant pleural mesothelioma.

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Pro-tumorigenic effects of miR-31 loss in mesothelioma.

The human genome encodes several hundred microRNA (miRNA) genes that produce small (21-23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3'-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome.

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin.

Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissue-specific expression patterns of microRNA and suggested that microRNA profiling would be useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT-PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT-PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.

hsa-miR-29c* is linked to the prognosis of malignant pleural mesothelioma.

The inability to forecast outcomes for malignant mesothelioma prevents clinicians from providing aggressive multimodality therapy to the most appropriate individuals who may benefit from such an approach. We investigated whether specific microRNAs (miR) could segregate a largely surgically treated group of mesotheliomas into good or bad prognosis categories. A training set of 44 and a test set of 98 mesothelioma tumors were analyzed by a custom miR platform, along with 9 mesothelioma cell lines and 3 normal mesothelial lines. Functional implications as well as downstream targets of potential prognostic miRs were investigated. In both the training and test sets, hsa-miR-29c* was an independent prognostic factor for time to progression as well as survival after surgical cytoreduction. The miR was expressed at higher levels in epithelial mesothelioma, and the level of this miR could segregate patients with this histology into groups with differing prognosis. Increased expression of hsa-miR-29c* predicted a more favorable prognosis, and overexpression of the miR in mesothelioma cell lines resulted in significantly decreased proliferation, migration, invasion, and colony formation. Moreover, major epigenetic regulation of mesothelioma is mediated by hsa-miR-29c* and was shown through downregulation of DNA methyltransferases as well as upregulation of demethylating genes. A single miR has the potential to be a prognostic biomarker in mesothelioma, and validation of these findings as well as investigation of its downstream targets may give insight for potential therapies in the future.

Accurate classification of non-small cell lung carcinoma using a novel microRNA-based approach.

PURPOSE: Advances in targeted lung cancer therapy now demand accurate classification of non-small cell lung cancer (NSCLC). MicroRNAs (miRNA) are recently discovered short, noncoding genes that play essential roles in tissue differentiation during normal development and tumorigenesis. For example, hsa-miR-205 is a miRNA that is highly expressed in lung squamous cell carcinomas (SqCC) but not in lung adenocarcinomas. The differential expression of miRNAs could be exploited to distinguish these tumor types. EXPERIMENTAL DESIGN: One hundred and two resected NSCLCs were classified as SqCC or adenocarcinoma based on their histologic features and immunohistochemical profiles. Corresponding preoperative biopsies/aspirates that had been originally diagnosed as poorly differentiated NSCLCs were available for 21 cases. A quantitative reverse transcription-PCR diagnostic assay that measures the expression level of hsa-miR-205 was used to classify the carcinomas as SqCC or adenocarcinoma based solely on expression levels. The two sets of diagnoses were compared. RESULTS: Using standard pathologic methods of classification (i.e., microscopy and immunohistochemistry), 52 resected lung carcinomas were classified as SqCCs and 50 as adenocarcinomas. There was 100% concordance between the diagnoses established by conventional and miRNA-based methods. MiRNA profiling also correctly classified 20 of the 21 preoperative biopsy specimens. CONCLUSIONS: MiRNA profiling is a highly reliable strategy for classifying NSCLCs. Indeed, classification is consistently accurate even in small biopsies/aspirates of poorly differentiated tumors. Confirmation of its reliability across the full range of tumor grades and specimen types represents an important step toward broad application.

Effects of rosiglitazone on intramyocellular lipid accumulation in Psammomys obesus.

OBJECTIVE: To examine the effects of rosiglitazone in intramyocellular lipid (IMCL) content in diabetic Psammomys obesus using novel electron microscopy technologies. BACKGROUND: P. obesus is an unique polygenic model of obesity and type 2 diabetes. Male diabetic P. obesus were treated daily with 5 mg/Kg Rosiglitazone by oral gavage for 14 days. Data were compared with a group of age-matched diabetic P. obesus treated with saline vehicle. METHODS: Assessment of insulin resistance and adiposity were determine before and after the treatment period by oral glucose tolerance test (oGTT) and dual energy X-ray absorptiometry (DEXA) analysis. We used a new scanning electron microscopy technology, (WETSEM) to investigate the effects of rosiglitazone administration on IMCL content, size and distribution in red gastrocnemius muscle. RESULTS: Rosiglitazone treatment improved glucose tolerance in P. obesus with no difference in the overall body fat content although a significant reduction in subscapular fat mass was observed. Rosiglitazone changed the distribution of lipid droplet size in skeletal muscle. Treated animals tended to have smaller lipid droplets compared with saline-treated controls. CONCLUSIONS: Since smaller IMCL droplets are associated with improvements in insulin sensitivity, we propose that this may be an important mechanism by which rosiglitazone affects glucose tolerance.

Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma.

PURPOSE: Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS: High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS: We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION: Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.

Serum microRNAs are promising novel biomarkers.

BACKGROUND: Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers. METHODS AND RESULTS: After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage. CONCLUSIONS AND SIGNIFICANCE: Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

MicroRNAs accurately identify cancer tissue origin.

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

Identification of a novel stress-responsive gene Hi95 involved in regulation of cell viability.

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (gamma- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H(2)O(2). Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.