FusionNW, a potential clinical impact assessment of kinases in pan-cancer fusion gene network.

Kinase fusion genes are the most active fusion gene group in human cancer fusion genes. To help choose the clinically significant kinase so that the cancer patients that have fusion genes can be better diagnosed, we need a metric to infer the assessment of kinases in pan-cancer fusion genes rather than relying on the sample frequency expressed fusion genes. Most of all, multiple studies assessed human kinases as the drug targets using multiple types of genomic and clinical information, but none used the kinase fusion genes in their study. The assessment studies of kinase without kinase fusion gene events can miss the effect of one of the mechanisms that enhance the kinase function in cancer. To fill this gap, in this study, we suggest a novel way of assessing genes using a network propagation approach to infer how likely individual kinases influence the kinase fusion gene network composed of ~5K kinase fusion gene pairs. To select a better seed of propagation, we chose the top genes via dimensionality reduction like a principal component or latent layer information of six features of individual genes in pan-cancer fusion genes. Our approach may provide a novel way to assess of human kinases in cancer.

Cost-Efficient Detection of NTRK1/2/3 Gene Fusions: Single-Center Analysis of 8075 Tumor Samples.

The majority of NTRK1, NTRK2, and NTRK3 rearrangements result in increased expression of the kinase portion of the involved gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5'/3'-end expression imbalances is potentially capable of detecting the entire spectrum of NTRK gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis. The 5'/3'-end expression imbalances in NTRK genes were analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific PCR for 44 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). cDNA of sufficient quality was obtained in 7424/8075 (91.9%) tumors. NTRK rearrangements were detected in 7/6436 (0.1%) lung carcinomas, 11/137 (8.0%) pediatric tumors, and 13/851 (1.5%) adult non-lung malignancies. The highest incidence of NTRK translocations was observed in pediatric sarcomas (7/39, 17.9%). Increased frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%), salivary gland carcinomas (5/93, 5.4%), and sarcomas (7/143, 4.9%). None of the 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5'/3'-end expression imbalances. Variant-specific PCR was performed for 744 tumors with a normal 5'/3'-end expression ratio: there were no rearrangements in 172 EGFR/ALK/ROS1/RET/MET-negative lung cancers and 125 pediatric tumors, while NTRK3 fusions were detected in 2/447 (0.5%) non-lung adult malignancies. In conclusion, this study describes a diagnostic pipeline that can be used as a cost-efficient alternative to conventional methods of NTRK1-3 analysis.

Standardized evidence-based approach for assessment of oncogenic and clinical significance of NTRK fusions.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

Therapeutic Advances in the Management of Patients with Advanced RET Fusion-Positive Non-Small Cell Lung Cancer.

OPINION STATEMENT: Screening for activating driver gene alterations at the time of diagnosis is the standard of care for advanced non-small cell lung cancer (NSCLC). Activating RET fusions are identified in approximately 1-2% of NSCLCs and have emerged as a targetable driver alteration. Selpercatinib and pralsetinib are RET-selective tyrosine kinase inhibitors (TKIs) with encouraging efficacy, intracranial activity, and tolerability that we recommend as first-line therapy. As with use of TKIs in other oncogene-addicted NSCLCs, development of acquired resistance is pervasive and should be specifically delineated through use of repeat tissue biopsy with genetic profiling at the time of disease progression. If an actionable resistance mechanism emerges for which there is a candidate targeted therapy, combination inhibition should be considered. Alternatively, or in the absence of such findings, platinum doublet chemotherapy or particularly platinum-pemetrexed therapy with or without bevacizumab demonstrates a moderate effect.We would not recommend the routine use of nonselective multi-targeted TKIs such as cabozantinib and vandetanib, which have modest activity but limited tolerability due to predictable off-target effects. Single-agent immunotherapy has minimal activity in RET fusion-positive NSCLC. The role of combination chemotherapy and immunotherapy requires further study but may be considered, particularly in the presence of an activating KRAS alteration. While further development of novel RET-selective TKIs may address common RET-specific resistance mutations, they will not have activity against off-target, RET-independent resistance mechanisms. This again highlights the importance of serial biopsy and next-generation sequencing for the rational choice of sequential therapy in RET fusion-positive NSCLC.

MSI-High RAS-BRAF wild-type colorectal adenocarcinomas with MLH1 loss have a high frequency of targetable oncogenic gene fusions whose diagnoses are feasible using methods easy-to-implement in pathology laboratories.

Targetable kinase fusions are extremely rare (<1%) in colorectal cancers (CRCs), making their diagnosis challenging and often underinvestigated. They have been shown particularly frequently among MSI-High, BRAF/KRAS/NRAS wild-type CRCs with MLH1 loss (MLH1loss MSI-High wild-type). We searched for NTRK1, NTRK2, NTRK3, ALK, ROS1, BRAF, RET, and NRG1 kinase fusions in CRCs using methods easy-to-implement in pathology laboratories: immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and fully automated real-time PCR targeted analyses. RNA-sequencing analyses were used for confirmation. Among 84 selected MLH1 deficient (IHC) CRCs cases, MLH1loss MSI-High wild-type CRCs consisted first in 19 cases after Idylla™ analyses and finally in 18 cases (21%) after RNA-sequencing (detection of one additional KRASG12D mutation). FISH (and when relevant, IHC) analyses concluded in 5 NTRK1, 3 NTRK3, 1 ALK, 2 BRAF, and 2 RET FISH positive tumors. ALK and NTRK1 rearranged tumors were IHC positive, but pan-TRK IHC was negative in the 3 NTRK3 FISH positive tumors. RNA-sequencing analyses confirmed 12 of 13 fusions with only one false positive RET FISH result. Finally, 12/18 (67%) of MLH1loss MSI-High wild-type CRCs contained targetable kinase fusions. Our study demonstrates the feasibility, but also the cost-effectiveness, of a multistep but rapid diagnostic strategy based on nonsequencing methods to identify rare and targetable kinase fusions in patients with advanced CRCs, as well as the high prevalence of these kinase fusions in MLH1loss MSI-High wild-type CRCs. Nevertheless, confirmatory RNA-sequencing analyses are necessary in case of low FISH positive nuclei percentage to rule out FISH false-positive results.

Precision medicine and its implementation in patients with NTRK fusion genes: perspective from developing countries.

Precision oncology is the field that places emphasis on the diagnosis and treatment of tumors that harbor specific genomic alterations susceptible to inhibition or modulation. Although most alterations are only present in a minority of patients, a substantial effect on survival can be observed in this subgroup. Mass genome sequencing has led to the identification of a specific driver in the translocations of the tropomyosin receptor kinase family (NTRK) in a subset of rare tumors both in children and in adults, and to the development and investigation of Larotrectinib. This medication was granted approval by the US Food and Drug Administration for NTRK-positive tumors, regardless of histology or age group, as such, larotrectinib was the first in its kind to be approved under the premise that molecular pattern is more important than histology in terms of therapeutic approach. It yielded significant results in disease control with good tolerability across a wide range of diseases including rare pediatric tumors, salivary gland tumors, gliomas, soft-tissue sarcomas, and thyroid carcinomas. In addition, and by taking different approaches in clinical trial design and conducting allocation based on biomarkers, the effects of target therapies can be isolated and quantified. Moreover, and considering developing nations and resource-limited settings, precision oncology could offer a tool to reduce cancer-related disability and hospital costs. In addition, developing nations also present patients with rare tumors that lack a chance of treatment, outside of clinical trials. This, in turn, offers the possibility for international collaboration, and contributes to employment, education, and health service provisions. The reviews of this paper are available via the supplemental material section.

Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods.

BACKGROUND: Accurate fusion transcript detection is essential for comprehensive characterization of cancer transcriptomes. Over the last decade, multiple bioinformatic tools have been developed to predict fusions from RNA-seq, based on either read mapping or de novo fusion transcript assembly. RESULTS: We benchmark 23 different methods including applications we develop, STAR-Fusion and TrinityFusion, leveraging both simulated and real RNA-seq. Overall, STAR-Fusion, Arriba, and STAR-SEQR are the most accurate and fastest for fusion detection on cancer transcriptomes. CONCLUSION: The lower accuracy of de novo assembly-based methods notwithstanding, they are useful for reconstructing fusion isoforms and tumor viruses, both of which are important in cancer research.

A Nanopore Sequencing-Based Assay for Rapid Detection of Gene Fusions.

Structural chromosomal rearrangements leading to gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions and for recognizing specific subtypes of neoplasms that can have different management and prognosis. Rapid identification of gene fusions is particularly critical for patients with acute leukemia who cannot wait more than a few days before initiating treatment and for whom treatment can be dramatically different depending on the leukemia subtype. We have developed an assay for rapid detection of oncogenic gene fusions (within 24 hours) that takes advantage of the long reads and real-time data generation of the Oxford Nanopore MinION sequencing system. By using a modification of the anchored multiplex PCR method for library construction, we confidently identified BCR-ABL1 fusion transcripts, with >100 reads within 15 minutes of sequencing. By using formalin-fixed, paraffin-embedded specimens routinely tested in our clinical molecular laboratory, fusions were successfully identified within 5 hours from acquisition of Illumina-ready libraries and 30 minutes of sequencing initiation, including cases diluted to a tumor fraction of 5%. In conclusion, we have developed a nanopore-based sequencing assay that can decrease turnaround time for detection of fusion oncogenes and may be a valid approach for laboratories with low specimen volume and for cases in need of rapid results.

A simple, rapid, low-cost technique for naked-eye detection of urine-isolated TMPRSS2:ERG gene fusion RNA.

The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarker alternatives to the controversial serum PSA. Current methods for detecting TMPRSS2:ERG are limited in terms of long processing time, high cost and the need for specialized equipment. Thus, there is an unmet need for less complex, faster, and cheaper methods to enable gene fusion detection in the clinic. We describe herein a simple, rapid and inexpensive assay which combines robust isothermal amplification technique with a novel visualization method for evaluating urinary TMPRSS2:ERG status at less than USD 5 and with minimal equipment. The assay is sensitive, and rapidly detects as low as 10(5) copies of TMPRSS2:ERG transcripts while maintaining high levels of specificity.

Comparative assessment of methods for the fusion transcripts detection from RNA-Seq data.

RNA-Seq made possible the global identification of fusion transcripts, i.e. "chimeric RNAs". Even though various software packages have been developed to serve this purpose, they behave differently in different datasets provided by different developers. It is important for both users, and developers to have an unbiased assessment of the performance of existing fusion detection tools. Toward this goal, we compared the performance of 12 well-known fusion detection software packages. We evaluated the sensitivity, false discovery rate, computing time, and memory usage of these tools in four different datasets (positive, negative, mixed, and test). We conclude that some tools are better than others in terms of sensitivity, positive prediction value, time consumption and memory usage. We also observed small overlaps of the fusions detected by different tools in the real dataset (test dataset). This could be due to false discoveries by various tools, but could also be due to the reason that none of the tools are inclusive. We have found that the performance of the tools depends on the quality, read length, and number of reads of the RNA-Seq data. We recommend that users choose the proper tools for their purpose based on the properties of their RNA-Seq data.

Assessment of long-term outcomes associated with urinary prostate cancer antigen 3 and TMPRSS2:ERG gene fusion at repeat biopsy.

BACKGROUND: In men with clinically localized prostate cancer who have undergone at least 1 previous negative biopsy and have elevated serum prostate-specific antigen (PSA) levels, long-term health outcomes associated with the assessment of urinary prostate cancer antigen 3 (PCA3) and the transmembrane protease, serine 2 (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) gene fusion (T2:ERG) have not been investigated previously in relation to the decision to recommend a repeat biopsy. METHODS: The authors performed a decision analysis using a decision tree for men with elevated PSA levels. The probability of cancer was estimated using the Prostate Cancer Prevention Trial Risk Calculator (version 2.0). The use of PSA alone was compared with the use of PCA3 and T2:ERG scores, with each evaluated independently, in combination with PSA to trigger a repeat biopsy. When PCA3 and T2:ERG score evaluations were used, predefined thresholds were established to determine whether the patient should undergo a repeat biopsy. Biopsy outcomes were defined as either positive (with a Gleason score of <7, 7, or >7) or negative. Probabilities and estimates of 10-year overall survival and 15-year cancer-specific survival were derived from previous studies and a literature review. Outcomes were defined as age-dependent and Gleason score-dependent 10-year overall and 15-year cancer-specific survival rates and the percentage of biopsies avoided. RESULTS: Incorporating the PCA3 score (biopsy threshold, 25; generated based on the urine PCA3 level normalized to the amount of PSA messenger RNA) or the T2:ERG score (biopsy threshold, 10; based on the urine T2:ERG level normalized to the amount of PSA messenger RNA) into the decision to recommend repeat biopsy would have avoided 55.4% or 64.7% of repeat biopsies for the base-case patient, respectively, and changes in the 10-year survival rate were only 0.93% or 1.41%, respectively. Multi-way sensitivity analyses suggested that these results were robust with respect to the model parameters. CONCLUSIONS: The use of PCA3 or T2:ERG testing for repeat biopsy decisions can substantially reduce the number of biopsies without significantly affecting 10-year survival.

Careless talk costs lives: fibroblast growth factor receptor signalling and the consequences of pathway malfunction.

Since its discovery 40 years ago, fibroblast growth factor (FGF) receptor (FGFR) signalling has been found to regulate fundamental cellular behaviours in a wide range of cell types. FGFRs regulate development, homeostasis, and repair and are implicated in many disorders and diseases; and indeed, there is extensive potential for severe consequences, be they developmental, homeostatic, or oncogenic, should FGF-FGFR signalling go awry, so careful control of the pathway is critically important. In this review, we discuss the recent developments in the FGF field, highlighting how FGFR signalling works in normal cells, how it can go wrong, how frequently it is compromised, and how it is being targeted therapeutically.

[Standard protocol of ALK fusion gene assessment by fluorescent in situ hybridization in non-small cell lung cancer].

OBJECTIVE: To investigate the standard protocol for anaplastic lymphoma kinase (ALK) fusion gene assessment by fluorescent in situ hybridization (FISH) in non-small cell lung cancer (NSCLC). METHODS: Tissue specimens of NSCLC cases were retrospectively collected from Jan. 2011 to July 2012. ALK fusion gene was examined by FISH using break-apart ALK gene probes (Vysis company). The identification of ALK fusion gene was determined by fluorescent signals under a fluorescence microscope. RESULTS: One hundred and forty-six eligible NSCLC tumor specimens were tested for ALK fusion gene by FISH. The specimens included 110 cases (75.4%) of surgically-removed tissues, 11 cases (7.5%) of biopsy, 19 cases (13.0%) of lymph node and 6 cases (4.1%) of other metastatic tissues. The positivity of ALK fusion gene was 8.9% (13/146). CONCLUSIONS: The assessment of ALK fusing gene by FISH using standard protocol for formalin-fixed, paraffin-embedded (FFPE) tissue is feasible. The protocol can used to test in surgically-removed tissues, biopsies, metastatic lymph nodes and other metastastic specimens.

PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification.

PURPOSE: To improve the risk stratification of patients with rhabdomyosarcoma (RMS) through the use of clinical and molecular biologic data. PATIENTS AND METHODS: Two independent data sets of gene-expression profiling for 124 and 101 patients with RMS were used to derive prognostic gene signatures by using a meta-analysis. These and a previously published metagene signature were evaluated by using cross validation analyses. A combined clinical and molecular risk-stratification scheme that incorporated the PAX3/FOXO1 fusion gene status was derived from 287 patients with RMS and evaluated. RESULTS: We showed that our prognostic gene-expression signature and the one previously published performed well with reproducible and significant effects. However, their effect was reduced when cross validated or tested in independent data and did not add new prognostic information over the fusion gene status, which is simpler to assay. Among nonmetastatic patients, patients who were PAX3/FOXO1 positive had a significantly poorer outcome compared with both alveolar-negative and PAX7/FOXO1-positive patients. Furthermore, a new clinicomolecular risk score that incorporated fusion gene status (negative and PAX3/FOXO1 and PAX7/FOXO1 positive), Intergroup Rhabdomyosarcoma Study TNM stage, and age showed a significant increase in performance over the current risk-stratification scheme. CONCLUSION: Gene signatures can improve current stratification of patients with RMS but will require complex assays to be developed and extensive validation before clinical application. A significant majority of their prognostic value was encapsulated by the fusion gene status. A continuous risk score derived from the combination of clinical parameters with the presence or absence of PAX3/FOXO1 represents a robust approach to improving current risk-adapted therapy for RMS.

Development of a chimeric recombinant disintegrin as a cost-effective anti-cancer agent with promising translational potential.

Vicrostatin (VCN) is a chimeric recombinant disintegrin generated in Origami B (DE3) Escherichia coli as a genetic fusion between the C-terminal tail of a viperid disintegrin echistatin and crotalid disintegrin contortrostatin (CN). The therapeutic modulation of multiple integrin pathways via soluble disintegrins was previously shown by us and others to elicit potent anti-angiogenic and anti-metastatic effects in several animal cancer models. Despite these favorable attributes, these polypeptides are notoriously difficult to produce recombinantly in significant quantity due to their structure which requires the correct pairing of multiple disulfide bonds for biological activity. In this report, we show that VCN can be reliably produced in large amounts (yields in excess of 200 mg of active purified disintegrin per liter of bacterial culture) in Origami B (DE3), an E. coli expression strain engineered to support the folding of disulfide-rich heterologous proteins directly in its oxidative cytoplasmic compartment. VCN retains the integrin binding specificity of both parental molecules it was derived from, but with a different binding affinity profile. While competing for the same integrin receptors that are preferentially upregulated in the tumor microenvironment, VCN exerts a potent inhibitory effect on endothelial cell (EC) migration and tube formation in a dose-dependent manner, by forcing these cells to undergo significant actin cytoskeleton reorganization when exposed to this agent in vitro. Moreover, VCN has a direct effect on breast cancer cells inhibiting their in vitro motility. In an effort to address our main goal of developing a clinically relevant delivery method for recombinant disintegrins, VCN was efficiently packaged in liposomes (LVCN) and evaluated in vivo in an animal breast cancer model. Our data demonstrate that LVCN is well tolerated, its intravenous administration inducing a significant delay in tumor growth and an increase in animal survival, results that can be partially explained by potent tumor apoptotic effects.

[Detection of fusion gene--integral part of the assessment of children with acute leukemia]. / Detectia genelor de fuziune--parte integranta a evaluarii copiilor cu leucemie acuta.

UNLABELLED: Acute leukemia is the most common malignancy in children, being mostly produced by such chromosomal abnormalities as translocations or inversions causing gene fusion. Different clinical studies showed that translocations identified in ALL (acute lymphoblastic leukemia) and AML (acute myeloblastic leukemia) may be used to classify patients into risk groups. AIM: To detect three fusion genes that have been proven very important in patient classification: t(9:22)p190, t(4:11) and t(12:21). MATERIAL AND METHOD: We conducted a prospective study on 30 patients with acute leukemia diagnosed in the interval September 2009 - September 2010 at the Iasi Hemato-Oncology Unit of Saint Mary Hospital for Children. RESULTS: We found t(9:22)p190 in two patients, t(4:11) in two patients and t(12:21) in one patient. From the total of 30 patients, 7 were considered at high-risk, 3 were diagnosed with AML, and 20 were considered at standard-risk. CONCLUSIONS: Day 33 can still be considered the reference time in the evaluation of treatment response; patients with BCR-ABL seem to have a worse prognosis than those who do not have this translocation. The detection of fusion genes is very important in patient classification.

Poly (A)+ transcriptome assessment of ERBB2-induced alterations in breast cell lines.

We report the first quantitative and qualitative analysis of the poly (A)⁺ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

Risk assessment in skin gene therapy: viral-cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors.

Cutaneous gene therapy can be envisioned through the use of keratinocyte stem cell clones in which retroviral genotoxic risks can be pre-assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been proven in experimental and clinical settings, the formation of chimeric viral-cellular transcripts originated by the inefficient termination (read-through) of retroviral transcripts remains to be studied in depth. We now demonstrate the widespread presence of viral-cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors. We have detected high molecular weight RNAs in northern blot analysis of retroviral vector expression in individual cell clones. Characterization of some of these transcripts revealed that they originate from genes located at the proviral integration sites. One class of transcripts corresponds to fusions of the viral vectors with intronic sequences, terminating at cryptic polyadenylation sites located in introns. A second class comprises fusion transcripts with coding sequences of genes at the integration sites. These are generated through splicing from a cryptic, not previously described donor site in the lentiviral vectors to exons of cellular genes, and have the potential to encode unintended open reading frames, although they are downregulated by cellular mechanisms. Our data contribute to a better understanding of the impact of SIN lentiviral vector integration on cellular gene transcription, and will be helpful in improving the design of this type of vectors.

Pre-clinical toxicity assessment of tumor-targeted interleukin-12 low-intensity electrogenetherapy.

This study's goal was to assess the safety of tumor-targeted interleukin-12 (ttIL-12) when administered by electrogenetherapy in C3H/HeJ mice by identifying an initial safe dose for human dose escalation schemes, toxicity target organs, markers of toxicity, and toxicity reversibility. Tumor-free mice receiving two doses of 0.45% NaCl, 1 µg ttIL-12 DNA in 0.45% NaCl or 5 µg ttIL-12 DNA in 0.45% NaCl, 10 days apart combined with low-intensity electroporation were compared with non-treatment controls over time. All mice had blood cell counts, serum chemistry profiles, plasma interleukin-12 and IFNγ determinations, necropsy and multi-organ histopathology. Mild treatment-associated changes included electroporation-associated muscle changes that resolved by 30 days; decreased total white blood cell counts and infectious disease in the 5 µg ttIL-12 group, but not in the 1 µg group, and liver changes in ttIL-12 groups that correlated with alanine transaminase levels and resolved by 30 days. Dystrophic cardiac calcification seen in older, 5 µg ttIL-12-treated mice was the only serious toxicity. Based on these results and the lack of any effect on wound healing when combined with surgery, low-intensity electrogenetherapy with ttIL-12 appears to be safe and well tolerated.