RNA Disruption Research Articles

Neoadjuvant trastuzumab deruxtecan (T-DXd) with response-directed definitive therapy in early stage HER2-positive breast cancer: a phase II study protocol (SHAMROCK study)

BackgroundThe current standard of care in the neoadjuvant setting for high-risk HER2-positive (HER2 +) breast cancer is to combine systemic chemotherapy with dual HER2 blockade, trastuzumab and pertuzumab. Targeted therapies have significantly improved outcomes for patients with HER2-positive breast cancer. To improve treatment-associated toxicity, chemotherapy-sparing approaches are currently being investigated. Trastuzumab deruxtecan (T-DXd) is an HER2-directed antibody–drug-conjugate (ADC) with promising results in the metastatic setting for HER2-positive breast cancer. The SHAMROCK study investigates neoadjuvant T-DXd in early stage HER2-positive breast cancer, using pathological complete response (pCR) rate as the primary endpoint.MethodsThis is a phase II open-label, single arm, adaptive multi-centre trial of T-DXd in the neoadjuvant setting in stage 2–3 HER2-positive breast cancer. Eligible patients will receive 5.4 mg/kg of T-DXd intravenously every 3 weeks for up to 6 cycles. A repeat biopsy will performed after 2 cycles for the RNA disruption index (RDI) score assessment. According to their likelihood of pCR, as determined by the RDI score, patients will either undergo 4 or 6 cycles of T-DXd prior to imaging. Patients with imaging complete response (iCR) after either 4 or 6 cycles will proceed to surgery. Patients who do not achieve iCR will either undergo further systemic therapy or proceed to surgery.DiscussionThe SHAMROCK study is a chemotherapy-sparing approach to curative intent treatment, investigating neoadjuvant T-DXd. We hypothesise that neoadjuvant T-DXd will have a high pCR rate and be associated low toxicity in early stage HER2-positive breast cancer.Trial registrationEudraCT Number: 2022–002485-32; ClinicalTrials.gov identifier: NCT05710666; Cancer Trials Ireland study number: CTRIAL-IE 22–01.

Low RNA disruption during neoadjuvant chemotherapy predicts pathologic complete response absence in patients with breast cancer.

In previously reported retrospective studies, high tumor RNA disruption during neoadjuvant chemotherapy predicted for post-treatment pathologic complete response (pCR) and improved disease-free survival at definitive surgery for primary early breast cancer. The BREVITY (Breast Cancer Response Evaluation for Individualized Therapy) prospective clinical trial (NCT03524430) seeks to validate these prior findings. Here we report training set (Phase I) findings, including determination of RNA disruption index (RDI) cut points for outcome prediction in the subsequent validation set (Phase II; 454 patients). In 80 patients of the training set, maximum tumor RDI values for biopsies obtained during neoadjuvant chemotherapy were significantly higher in pCR responders than in patients without pCR post-treatment (P = .008). Moreover, maximum tumor RDI values ≤3.7 during treatment predicted for a lack of pCR at surgery (negative predictive value = 93.3%). These findings support the prospect that on-treatment tumor RNA disruption assessments may effectively predict post-surgery outcome, possibly permitting treatment optimization.

RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed “RNA disruption”. Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on aconsensus promoter.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor n}{}sigma to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein–DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with n}{}sigma and the RNAP n}{}beta subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

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Abstract P1-08-09: High mid-treatment RNA disruption in patients with HER2-negative breast cancer predicts survival benefit after neoadjuvant chemotherapy

Abstract BACKGROUND: Ribosomal RNA fragmentation (RNA disruption) measured mid-therapy in breast cancer tumors has been shown to correlate with pCR and improved disease-free survival. Data from the NeoAva clinical trial was used to evaluate RNA disruption index values (RDI) for measurement of therapy efficacy. METHODS: The randomized phase II clinical trial NeoAva investigated the effects of neoadjuvant chemotherapy (FEC) in HER2-negative breast cancer patients with or without bevacizumab. Serial biopsies were taken prior to treatment and after 12 and 25 weeks of treatment. The majority of patients in this study were diagnosed with ER positive tumors (88% of the total 12-week samples). RNA isolation from frozen tumor tissue was carried out using the AllPrep DNA/RNA Mini kit with QIACube (Qiagen), and the quality of the RNA assessed (Agilent 2100 Bioanalyzer). Treatment response was measured using both pathological complete response (pCR) and residual cancer burden (RCB) at the time of surgery, and median follow-up time was 6.8 years. Using the RNA electropherogram data (Agilent Bioanalyzer) generated in this study, RDI values were assessed in pre-therapy samples (n=109), 12-week samples (n=98) and 25-week samples (n=106). RESULTS: RDI values were measured at three timepoints, however only the RDI values assessed from 12 week samples correlated with clinical outcomes. RDI values were higher in patients that achieved a RCB class of either 0 or 1 (n=28) (median RDI=2.6) compared with those patients that had an RCB class of 3 (n=17) (median RDI=1.3) (Mann-Whitney p=0.006). Out of the samples that were analyzed by the RNA disruption assay (RDA), 18 patients achieved a pCR and had a median RDI value of 2.6 compared with a median RDI of 1.8 for those who did not achieve a pCR (n=80); this finding did not reach statistical significance. RDI values measured in samples taken prior to therapy or at 25-weeks did not correlate with either RCB class or subsequent pCR. With 93% and 94% of patients having mid-therapy RDI values greater than 1.1 for RCB class of 0/1 or pCR, respectively, this was chosen as a cut point to generate Kaplan-Meier curves of recurrence free survival. These curves illustrated statistically significant improved survival (HR =2.6; 95% CI 0.8-8.1) for patients with a RDI > 1.1 (p=0.05). Similarly, Kaplan-Meier curves of breast cancer specific survival also demonstrated improved survival (HR = 3.1; 95% CI 0.8-11.7) for patients with a RDI > 1.1 (p=0.03). Kaplan-Meier curves based on pCR/no pCR were generated from the set of patients that had 12-week RDI values assessed and did not demonstrate improved survival in this small, predominantly ER+ group. No survival benefit was found based on RDI values measured prior to therapy or at 25 weeks. Patients that received bevacizumab (n=45) had significantly higher RDI values at 12 weeks (median RDI = 2.5) than patients that did not receive the drug (median RDI = 1.4) (n=53) (Mann-Whitney test p=0.0005). This overall increase in RDI values with bevacizumab resulted in improved survival for patients at a higher RDI cutoff values of 1.6 (p=0.03)(HR = 4.4; 95% CI 0.8-25.1). CONCLUSION: Taken together, our findings suggest that increased RNA disruption measured in tumor biopsies during neoadjuvant chemotherapy correlates with a survival benefit for patients and may be a more useful indicator of therapy efficacy than pCR, particularly for patients with ER positive tumors. Citation Format: Laura B Pritzker, Mads Haugland Haugen, Hedda von der Lippe Gythfeldt, Twinkle Masilamani, Gabriel Theriault, Renee St-Onge, Lavina D'costa, Ole Christian Lingjaerde, Amadeo Parissenti, Olav Engebraaten. High mid-treatment RNA disruption in patients with HER2-negative breast cancer predicts survival benefit after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-09.

Abstract OT2-03-01: RNA disruption assay (RDA) - Breast cancer response evaluation for individualized therapy (brevity/brevity-02)

Abstract Background & Rationale Identifying non-responders to chemotherapy with reliable predictive tests is crucial to response-guided neoadjuvant therapy. BREVITY aims to support RDA as a response assessment tool in breast cancer.RDA is based on RNA disruption, a qualitative and quantitative alteration of ribosomal RNA that correlates with chemotherapy response. RDA stratifies patients into 3 zones: non-response zone 1, partial-response zone 2 and response zone 3. Survival data in MA.22 trial showed significantly superior 5-year DFS KM-curves for zone 3 patients compared to zones 1 & 2. MA.22 and other studies indicated a potentially valuable clinical utility to be pursued. Trial Design. Study type: Prospective 2-phase interventional study. Samples: 2 core needle biopsy specimens taken at 2 time points during chemotherapy. 1 Follow-up: 60 months from date of study enrolment. Incl. Criteria. •Women 18+ years old and able to provide informed consent. •Newly diagnosed clinical stage I, II or III breast cancer with complete surgical excision after neoadjuvant therapy as treatment goal. •Confirmation of invasive breast cancer of any subtype or grade, tumor size > 1cm. •Scheduled for neoadjuvant chemotherapy +/- antibodies and +/- other drugs according to SoC. •Willing to have research core needle biopsies at 2 timepoints during neoadjuvant treatment. Excl. Criteria. •Prior local or systemic therapy for current breast cancer. •Participation in another interventional clinical trial with experimental drugs during neoadjuvant therapy. •Stage IV breast cancer. •Bilateral, multifocal or multicentric breast cancer. •Prior malignant disease except curatively treated basalioma of the skin or pTis of the cervix uteri. •Pregnancy or breast feeding. Trial Objectives & Endpoints Statistical Methods. Phase 1: training set will be used to estimate and visualize operating characteristics as functions of RDI. These include NPV, PPV, sensitivity, specificity, and related concepts. Suitable cut-offs will be determined that optimize NPV of zone 1 and PPV of zone 3. Phase 1 powered to estimate 25th RDI percentile as candidate for cut-off c1 with 9.5% precision E. Phase 2: analysis by interval-estimation of performance characteristics on an independent validation set, given cut-offs selected in phase 1. Primary target of validation is NPV of zone 1 that corresponds to RDA’s main clinical utility. PPV of zone 3 to be established for selected subgroups. Sample size for validation chosen to yield 95% CI estimate for NPV with 5.25% precision. Present and Target Accrual. Present AccrualTarget Accrual(must be fully evaluable). Phase 1 (Training set):71 (target 80). Phase 2 (Validation set):454 Other Contacts. Sponsor: Rna Diagnostics Inc Biopsy Collection Time Points for RDABiopsy(2 specimens)1st core biopsy2nd core biopsyTiming35 +/- 4 daysIf no drug change:55 +/- 5 days. If drug change:2-3 weeks after start of new drugs:•3-weekly: at 16 +/- 2 days•Bi-weekly: at day of 2nd dose•Weekly: at day of 4th dose Primary ObjectivesDescriptionEndpointsi.1.Phase 1: Determine 2 RDI cut-offs to have a diagnostic test optimized in terms of predictive values2.Phase 2: Establish performance characteristics for first cut-off in terms of NPVNPVPPVSecondary Objectivesi.Assess NPV in subgroups by subtypeNPVii.Assess PPV in HER2+PPViii.Compare pCR prevalence and DFS in zones 1-3 for all patients and by subgrouppCRDFSv.Test for association btw. cancer subgroups and test resultTest result (zones 1-3)vi.Compare RDA and other biomarker results (Ki67, tumor grade) Citation Format: Maureen Trudeau, Joke Tio, Foluso Ademuyiwa, Thierry Petit, Bryan Hennessy, Marina Cazzaniga, Daniele Generali. RNA disruption assay (RDA) - Breast cancer response evaluation for individualized therapy (brevity/brevity-02) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-03-01.

The RNA disruption assay is superior to conventional drug sensitivity assays in detecting cytotoxic drugs

Conventional drug sensitivity assays used to screen prospective anti-cancer agents for cytotoxicity monitor biological processes associated with active growth and proliferation, used as proxies of cell viability. However, these assays are unable to distinguish between growth-arrested (but otherwise viable) cells and non-viable/dead cells. As a result, compounds selected based on the results of these assays may only be cytostatic, halting or slowing tumour progression temporarily, without tumour eradication. Because agents capable of killing tumour cells (cytotoxic drugs) are likely the most promising in the clinic, there is a need for drug sensitivity assays that reliably identify cytotoxic compounds that induce cell death. We recently developed a drug sensitivity assay, called the RNA disruption assay (RDA), which measures a phenomenon associated with tumour cell death. In this study, we sought to compare our assay’s performance to that of current commonly used drug sensitivity assays (i.e, the clonogenic, the cell counting kit-8 and the Trypan blue exclusion assays). We found that RNA disruption occurred almost exclusively when total cell numbers decreased (cytotoxic concentrations), with little to no signal detected until cells had lost viability. In contrast, conventional assays detected a decrease in their respective drug sensitivity parameters despite cells retaining their viability, as assessed using a recovery assay. We also found that the RDA can differentiate between drug-sensitive and -resistant cells, and that it can identify agents capable of circumventing drug resistance. Taken together, our study suggests that the RDA is a superior drug discovery tool, providing a unique assessment of cell death.

Quantification of immune cell-mediated destruction of tumor cells in vitro using the RNA disruption assay.

e15017 Background: Immune checkpoint inhibitors (ICIs) are increasingly being used in the treatment of human cancers, often in combination with cytotoxic chemotherapy drugs. However, current ICIs are costly and are only effective against a fraction of patient tumours. Thus, a tool that could reliably quantify tumour response to ICIs and predict outcome post-treatment would be highly valuable. Patients with non-responding tumours could be spared the costs and toxic side-effects of the ineffective drugs and moved promptly to alternate treatments. We have observed that a variety of structurally and mechanistically distinct chemotherapy agents induce the degradation of ribosomal RNA (rRNA) into a large number of high molecular weight fragments (between the 28S and 18S rRNAs)---a phenomenon we have termed “RNA disruption”. High tumour RNA disruption during treatment, as quantified using the RNA disruption assay (RDA), has been shown to predict pathologic complete response and improved disease-free survival in breast cancer patients. Methods: To assess whether RDA could be used to monitor immune cell killing of tumour cells, we collected peripheral blood mononuclear cells (PBMCs) from consenting healthy human donors and enriched the cell population for natural killer (NK) cells to 90% using a negative selection approach. The cells, with or without IL-2 pre-treatment, were incubated with K562 cells, after which K562 cell killing was quantified over time using a standard immunocytotoxicity assay and K562 cell RNA disruption was measured using RDA. Results: Freshly isolated human NK cells when incubated with K562 chronic myeloid leukemia cells induced cell destruction and RNA disruption in K562 cells in a dose-dependent manner. Pre-incubation with IL-2 augmented both NK cell-mediated RNA disruption and NK-mediated cytotoxicity in K562 cells. Interestingly, the pattern of K562 RNA disruption fragments generated by NK cells was similar to that generated by chemotherapy drugs. Conclusions: RDA was successfully used to quantify immune cell-mediated destruction of tumour cells, raising the prospect of its possible use to monitor tumour destruction by immune cells in vivo and to predict response to ICIs (alone or in concert with cytotoxic chemotherapy drugs).

Specific Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein Deduced from Hairpin Unfolding.

RNA and DNA hairpin formation and disruption play key regulatory roles in a variety of cellular processes. The 59-nucleotide transactivation response (TAR) RNA hairpin facilitates the production of full-length transcripts of the HIV-1 genome. Yet the stability of this long, irregular hairpin becomes a liability during reverse transcription as 24 base pairs must be disrupted for strand transfer. Retroviral nucleocapsid (NC) proteins serve as nucleic acid chaperones that have been shown to both destabilize the TAR hairpin and facilitate strand annealing with its complementary DNA sequence. Yet it has remained difficult to elucidate the way NC targets and dramatically destabilizes this hairpin while only weakly affecting the annealed product. In this work, we used optical tweezers to measure the stability of TAR and found that adding NC destabilized the hairpin and simultaneously caused a distinct change in both the height and location of the energy barrier. This data was matched to an energy landscape predicted from a simple theory of definite base pair destabilization. Comparisons revealed the specific binding sites found by NC along the irregular TAR hairpin. Furthermore, specific binding explained both the unusual shift in the transition state and the much weaker effect on the annealed product. These experiments illustrate a general method of energy landscape transformation that exposes important physical insights.

RNA disruption indicates CHOP therapy efficacy in canine lymphoma

BackgroundAssessment of the efficacy of a multi-agent chemotherapy protocol in which cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are administered in canine lymphoma is generally performed by physical measurement of lymph node diameter. However, no consistent correlation has been made with prognostic indicators and the length or absence of clinical remission based on lymph node size. RNA disruption measured mid-therapy has been correlated with increased disease-free survival in recent studies of human cancer and was assessed in this study of canine lymphoma patients. Fine needle aspirate samples were taken before treatment and at weeks 3, 6, and 11 of CHOP therapy. RNA was isolated from these samples and assessed using an Agilent Bioanalyzer. RNA disruption assay (RDA) analysis was performed on the data from the resulting electropherograms.ResultsAn increased RNA disruption index (RDI) score was significantly associated with improved progression-free survival.ConclusionsPredicting the risk of early relapse during chemotherapy could benefit veterinary patients by reducing ineffective treatment and could allow veterinary oncologists to switch earlier to a more effective drug regimen.

MA 15.14 Long Non-Coding RNA Disruption in Lung Adenocarcinoma Reveals Novel Mechanisms of Metastasis

Identifying the drivers of metastasis will yield new molecular targets for prognostics and therapeutics. Long non-coding RNAs (lncRNAs) are known to regulate gene transcription through their influence on the expression of nearby (cis) and distant (trans) genes. Emerging evidence suggests that lncRNAs are involved in key cellular processes, presenting an opportunity for large-scale identification of lncRNA genes critical to lung cancer progression. Here we investigate the contribution of this class of non-coding RNA to lung adenocarcinoma (LUAD) metastasis.

Abstract P1-09-19: Tumor RNA disruption index as a tool to predict response to neoadjuvant chemotherapy in breast cancer: Optimizing timing of biopsy

Abstract BACKGROUND. Early detection of tumor response to neoadjuvant therapy (NAT) could be used to tailor therapy and lower toxicity from ineffective treatments. The CCTG MA.22 trial has shown that RNA disruption is associated with breast cancer response to NAT when measured by image guided core biopsy mid-treatment. The objectives of this study were: 1) to determine the optimal time to measure the tumor RNA Disruption Index (RDI) after initiation of NAT when assessed by fine needle aspiration (FNA) in an office setting and 2) to determine if RDI could predict response to a second chemotherapy agent in patients that had a suboptimal response. METHODOLOGY. We performed a prospective pilot study including patients with palpable biopsy-proven breast cancer eligible for NAT. Chemotherapy and surgery were at the discretion of the treating physician. Two FNAs after cycles 1, 2, 3 and after initiation of a new chemotherapy agent were collected in RNA Protect Cell Reagent and sent to Rna Diagnostics Inc. to assess RDI. Prospectively recorded clinical tumor measurements and surgical pathology reports were obtained. Tumor pathological response (pR) after NAT was measured by pathological complete response (pCR: no invasive disease in breast) and residual cancer burden index (RCBI). RESULTS. 30 patients were accrued to the study. One patient withdrew consent, one patient was found to have metastatic disease and did not undergo surgery, and one patient had bilateral breast cancer (n= 29 evaluable tumors). ER+HER2-: 38% (11/29), ER-Her2-: 28% (8/29) and HER2+: 34% (10/29). 89% (25/28) of patients received taxane and anthracycline containing regimens. All HER2+ received trastuzumab. Our pCR and RCBI 0-1 rates were 24% (7/29) & 38% (11/29) respectively. At cycles 1, 2 and 3, RDI could be evaluated in 72% (21/29), 73% (16/22) and 44% (7/16) of palpable tumors. After the switch to a new agent, RDI could only be evaluated in 30% (3/10) of patients with palpable tumors. Using a tumor RDA cutoff at 5 (non-responder RDI < 5 (NR) and responder RDI ≥ 5 (R)), responder status between cycle 1 and 2 was concordant in 73% (11/15). After 1 cycle, NR vs. R was associated with numerically lower pCR (13% (2/15) vs. 33% (2/6), p=0.54) and RCBI 0-1 at surgery (20% (3/15) vs. 33% (2/6), p=0.60). These findings were similar after cycle 2. Non-analyzable samples (NAS) because of absence of RNA were associated with high pR (pCR: 38% (3/8) and RBCI 0-1: 75% (6/8)). The 3 NR at cycle 1 that achieved a significant pR had either non-palpable tumors or NAS after switching to a new chemotherapy agent. CONCLUSION. RDI can be measured by FNA in an office setting and could be helpful to identify early non-responders to NAT. The optimal time to perform RDI is after 1 or 2 cycles of treatment, which should be considered in ongoing and future trials. This study was underpowered to detect a statistically significant correlation between RDI and pR. NAS is associated with high pR and could represent responders to treatment. This early data suggests that RDI is unlikely to be helpful in assessing response to a second chemotherapy agent after receiving 4 cycles of standard chemotherapy. The use of RDI to tailor NAT needs to be evaluated in larger prospective trials. Citation Format: Samkari A, Chung W, Parissenti A, Pritzker L, Trabulsi N, Basik M, Boileau J-F. Tumor RNA disruption index as a tool to predict response to neoadjuvant chemotherapy in breast cancer: Optimizing timing of biopsy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-19.

RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.

BackgroundCellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are.MethodsOvarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor.ResultsAll chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3’-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption.ConclusionsSupporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2197-1) contains supplementary material, which is available to authorized users.

Tumor RNA disruption predicts survival benefit from breast cancer chemotherapy.

In a prior substudy of the CAN-NCIC-MA.22 clinical trial (ClinicalTrials.gov identifier NCT00066443), we observed that neoadjuvant chemotherapy reduced tumor RNA integrity in breast cancer patients, a phenomenon we term "RNA disruption." The purpose of the current study was to assess in the full patient cohort the relationship between mid-treatment tumor RNA disruption and both pCR post-treatment and, subsequently, disease-free survival (DFS) up to 108 months post-treatment. To meet these objectives, we developed the RNA disruption assay (RDA) to quantify RNA disruption and stratify it into 3 response zones of clinical importance. Zone 1 is a level of RNA disruption inadequate for pathologic complete response (pCR); Zone 2 is an intermediate level, while Zone 3 has high RNA disruption. The same RNA disruption cut points developed for pCR response were then utilized for DFS. Tumor RDA identified >fourfold more chemotherapy non-responders than did clinical response by calipers. pCR responders were clustered in RDA Zone 3, irrespective of tumor subtype. DFS was about 2-fold greater for patients with tumors in Zone 3 compared to Zone 1 patients. Kaplan-Meier survival curves corroborated these findings that high tumor RNA disruption was associated with increased DFS. DFS values for patients in zone 3 that did not achieve a pCR were similar to that of pCR recipients across tumor subtypes, including patients with hormone receptor positive tumors that seldom achieve a pCR. RDA appears superior to pCR as a chemotherapy response biomarker, supporting the prospect of its use in response-guided chemotherapy.

RNA Disruption and Drug Response in Breast Cancer Primary Systemic Therapy.

As there is now evidence that switching clinical nonresponders early in primary systemic therapy to alternate treatment regimens can enhance survival in some breast cancer patients, the need for a robust intermediate endpoint that can guide treatment response across all tumor subtypes is urgent. Recently, chemotherapy drugs have been shown to induce a decrease in RNA quality in tumor cells from breast cancer biopsies in some patients at midtherapy, and that this has been associated with subsequent achievement of pathological complete response (pCR). The decrease in RNA quality has been shown to be associated with RNA disruption; aberrant RNA bands visualized by RNA electrophoresis have been associated with subsequent tumor cell death. The objectives of these studies are to show that a new assay based on induction of RNA disruption in tumor cells by chemotherapy can stratify at midtherapy, pCR responders from non-pCR responders irrespective of clinical response and to present early evidence that clinically useful RNA disruption can be detected as early as 14 days after initiation of treatment. RNA disruption in tumor cells was quantified by analysis of the RNA electrophoresis banding pattern and expressed as an RNA disruption index (RDI). To develop the RNA disruption assay (RDA), RDI was correlated with clinical outcome (pCR) from the NCIC-CTG MA.22 breast cancer clinical trial (ClinicalTrials.gov NCT00066443). RDA Zones were established by stratifying patients using RDI values into Zone 1, Zone 2, and Zone 3. Zone 3 included seven out of eight pCR responders, whereas Zone 1 contained no pCR responders. An intermediate zone (Zone 2) was established which contained one pCR. Subsequently, to determine early drug response, RNA disruption was examined by RDI after 14 days exposure to trastuzumab, zoledronic acid, or letrozole + cyclophosphamide ± sorafenib therapy. In MA.22, RDA stratified 23 of 85 patients in Zone 1 as pCR nonresponders, 24 patients in Zone 2, an intermediate zone, and 38 patients in Zone 3, pCR responders and non-pCR patients who share RDI comparable to those achieving pCR. In the early response studies, after 14 days exposure to chemotherapy, some RNA disruption as measured by RDI elevation could be detected in 3/12 trastuzumab, 7/15 zoledronic acid, 5/29 letrozole + cyclophosphamide, and 5/23 letrozole + cyclophosphamide + sorafenib patients. RDA is a novel intermediate endpoint that has promise for clinical utility for breast cancers early in response-guided primary systemic therapy.

Non-coding RNAs in lung cancer.

The discovery that protein-coding genes represent less than 2% of all human genome, and the evidence that more than 90% of it is actively transcribed, changed the classical point of view of the central dogma of molecular biology, which was always based on the assumption that RNA functions mainly as an intermediate bridge between DNA sequences and protein synthesis machinery. Accumulating data indicates that non-coding RNAs are involved in different physiological processes, providing for the maintenance of cellular homeostasis. They are important regulators of gene expression, cellular differentiation, proliferation, migration, apoptosis, and stem cell maintenance. Alterations and disruptions of their expression or activity have increasingly been associated with pathological changes of cancer cells, this evidence and the prospect of using these molecules as diagnostic markers and therapeutic targets, make currently non-coding RNAs among the most relevant molecules in cancer research. In this paper we will provide an overview of non-coding RNA function and disruption in lung cancer biology, also focusing on their potential as diagnostic, prognostic and predictive biomarkers.

RETRACTED: α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells.

Pure anti-tumor effect of zoledronic acid in naïve bone-only metastatic and locally advanced breast cancer: proof from the “biological window therapy”

The study investigated the anti-tumour effect of zoledronic acid (ZA) administered alone in a biological window therapy in naïve bone-only metastatic and locally advanced breast cancer (LABC) patients. 33 patients with LABC (Group 1) and 20 patients with a first diagnosis of bone metastasis only (Group 2) received 4 mg single dose of ZA, 14 days (biological window) before starting any treatment. In Group 1, Ki67, CD34, p53/bcl-2 and caspase 3 expression along with the adenosine triphosphate (ATP) levels and RNA disruption index were evaluated as markers of tumor growth in tumour specimens obtained before and after ZA administration (basal, day 14). In Group 2, the total enumeration of circulating tumour cells (CTCs), and of M30+ve CTCs along with the soluble marker of cell death (M30/M65) were carried-out as markers of tumor dissemination at baseline, at 48 h and day 14th. In Group 1, there was a significant reduction in Ki67, CD34, bcl-2 expression after 14 days ZA based-treatment (p = 0.0032; p = 0.0001, p < 0.00001 respectively). ZA showed a significant increase of RNA disruption (p < 0.0076). In Group 2, we observed a significant reduction of CTCs number after 48 h (p = 0.0012), followed by a significant rebound at 14 days (p = 0.012). The apoptotic CTCs/M30+ve and M65 levels significantly increased under treatment (p = 0.018 and p = 0.039 respectively) after drug administration when compared to the baseline. These results are the first prospective in vivo data showing the direct pure anti-tumour effect (either on the tumour cell or on CTCs) of ZA.

Abstract P1-08-24: High tumor RNA disruption is associated with increased survival in patients with locally advanced breast cancer

Abstract Background: We recently reported that epirubicin/docetaxel treatment of locally advanced breast cancer patients in the MA.22 clinical trial reduced tumor RNA integrity and that low mid-treatment tumor RNA integrity was associated with a pathologic complete response (pCR) post-treatment [Breast Cancer Res. Treat. 119:347-356]. A novel method for detection of chemotherapy-dependent loss of RNA integrity, the RNA disruption assay (RDA), has since been developed, which stratifies RNA disruption into 3 zones. Zone 1 represents drug effect inadequate to obtain a pCR [RNA disruption index (RDI) ≤ 10, NPV&amp;gt;0.99]. Zone 2, an intermediate zone (RDI &amp;gt;10 and ≤35) had 1 pCR in our study, while Zone 3 (RDI &amp;gt;35) had the most pCRs (n = 7). Methods: We have now evaluated the relationship between tumor RNA disruption and both treatment response and patient survival in all evaluable MA.22 patients (n = 85). RNA was isolated from 2-3 tumor biopsies/patient prior to and after 3 (Schedule A) or 4 (Schedule B) cycles of chemotherapy at 3- or 2-weekly intervals, respectively. RNA quality was assessed on an Agilent 2100 Bioanalyzer and RNA disruption quantified using RDA. Results: Chemotherapy exposure reduced maximum tumor RNA content from 197 ± 24 ng/μl (mean ± standard error) pre-treatment to 94.0 ± 12.8 ng/μl mid-treatment, with maximum tumor RDI values increasing from 26.0 ± 5.8 to 88.1 ± 19.1 (p&amp;lt;0.01 for both observations by Sign test). At the time of assessment, there were similar numbers of deceased patients across tumor RDA zones 1, 2, and 3 mid-treatment (7, 8, and 9 patients, respectively). In contrast, there were 2.9-fold more living patients with tumors in RDA zone 3 (29) than in zone 1 (10). Disease-free survival (DFS) was significantly greater (by 23.6 months) for zone 3 patients (56.9 ± 5.6 months) compared to zone 1 patients (33.3 ± 6.8 months) (p = 0.0096 by Mann Whitney Wilcoxon Test for this and all subsequent statistical tests). Living patients with tumor RDA values in zone 3 exhibited 24.5 months greater DFS than living patients with tumors in zone 1 (68.2 ± 5.5 months versus 43.7 ± 9.5 months, respectively; p = 0.015). Given their high frequency within the patient population, we were also able to conclude that greater DFS (an additional 28.6 months) could be seen for patients with estrogen receptor positive (ER+) tumors in zone 3 (59.2 ± 7.2 months) compared to patients with similar tumors in zone 1 (30.6 ± 6.3 months; p = 0.0088). Living patients with ER+ tumors in zone 3 had even greater DFS (+42.5 months) compared to living patients with ER+ tumors in zone 1 (71.8 ± 5.8 months versus 29.3 ± 8.7 months, respectively; p = 0.0012). There were also 2.5-fold more living patients with ER+ tumor RDA values in zone 3 (15) than in zone 1 (6). None of the above significant differences were observed when pre-treatment tumor RDI values or pre-treatment patient characteristics were assessed. Irrespective of breast tumor receptor subtype, chemotherapy-dependent RNA disruption mid-treatment was typically higher in patients that achieved a pCR post-treatment than for patients that did not. Conclusions: Our findings suggest that tumor RNA disruption measurements during treatment will be highly useful to predict response and survival for locally advanced breast cancer patients. Our data demonstrate that non-responding patients (zone 1 by RDA) have considerably reduced DFS with very little chance of achieving a pCR. Such patients can be spared the toxicities associated with continuing an ineffective regimen and can be considered quickly for alternate treatments, including surgery, radiation therapy, or other anti-cancer drugs. The impact of treatment schedule and dose on RDA's ability to predict patient response and survival is currently being assessed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-24.