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Posts Tagged ‘cancer metabolic pathways’


Tumor Ammonia Recycling: How Cancer Cells Use Glutamate Dehydrogenase to Recycle Tumor Microenvironment Waste Products for Biosynthesis

Reporter: Stephen J. Williams, PhD

A feature of the tumorigenic process is the rewiring of the metabolic processes that provides a tumor cell the ability to grow and thrive in conditions of limiting nutrients as well as the ability to utilize waste products in salvage pathways for production of new biomass (amino acids, nucleic acids etc.) required for cellular growth and division 1-8.  A Science article from Spinelli et al. 9 (and corresponding Perspective article in the same issue by Dr. Chi V. Dang entitled Feeding Frenzy for Cancer Cells 10) describes the mechanism by which estrogen-receptor positive (ER+) breast cancer cells convert glutamine to glutamate, release ammonia  into the tumor microenvironment, diffuses into tumor cells and eventually recycle this ammonia by reductive amination of a-ketoglutarate by glutamate dehydrogenase (GDH) to produce glutamic acid and subsequent other amino acids needed for biomass production.   Ammonia can accumulate in the tumor microenvironment in poorly vascularized tumor. Thus ammonia becomes an important nitrogen source for tumor cells.

Mammalian cells have a variety of mechanisms to metabolize ammonia including

  • Glutamate synthetase (GS) in the liver can incorporate ammonia into glutamate to form glutamine
  • glutamate dehydrogenase (GDH) converts glutamate to a-ketoglutarate and ammonia under allosteric regulation (discussed in a post on this site by Dr. Larry H. Berstein; subsection Drugging Glutaminolysis)
  • the reverse reaction of GDH, which was found to occur in ER+ breast cancer cells, a reductive amination of a-ketoglutarate to glutamate11, is similar to the reductive carboxylation of a-ketoglutarate to citrate by isocitrate dehydrogenase (IDH) for fatty acid synthesis (IDH is overexpressed in many tumor types including cancer stem cells 12-15), and involved in immune response and has been developed as a therapeutic target for various cancers. IDH mutations were shown to possess the neomorphic activity to generate the oncometabolite, 2-hydroxyglutarate (2HG) 16-18. With a single codon substitution, the kinetic properties of the mutant IDH isozyme are significantly altered, resulting in an obligatory sequential ordered reaction in the reverse direction 19.

 

In the Science paper, Spinelli et al. report that ER+ breast cancer cells have the ability to utilize ammonia sources from their surroundings in order to produce amino acids and biomass as these ER+ breast cancer cells have elevated levels of GS and GDH with respect to other breast cancer histotypes.

GDH was elevated in ER+ luminal cancer cells and the quiescent epithelial cells in organoid culture

However proliferative cells were dependent on transaminases, which transfers nitrogen from glutamate to pyruvate or oxaloacetate to form a-ketoglutarate and alanine or aspartate. a-ketoglutarate is further metabolized in the citric acid cycle.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.    Reductive amination and transamination reactions of glutamic acid.  Source http://www.biologydiscussion.com/organism/metabolism-organism/incorporation-of-ammonia-into-organic-compounds/50870

Spinelli et al. showed GDH is necessary for ammonia reductive incorporation into a-ketoglutarate and also required for ER+ breast cancer cell growth in immunocompromised mice.

In addition, as commented by Dr. Dang in his associated Perspectives article, (quotes indent)

The metabolic tumor microenvironment produced by resident cells, such as fibroblasts and macrophages, can create an immunosuppressive environment 20.  Hence, it will be of great interest to further understand whether products such as ammonia could affect tumor immunity or induce autophagy  (end quote indent)

 

 

 

Figure 2.  Tumor ammonia recycling.  Source:  From Chi V. Dang Feeding Frenzy for cancer cells.  Rights from RightsLink (copyright.com)

Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass

Jessica B. Spinelli1,2, Haejin Yoon1, Alison E. Ringel1, Sarah Jeanfavre2, Clary B. Clish2, Marcia C. Haigis1 *

1.      1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. 2.      2Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

* *Corresponding author. Email: marcia_haigis@hms.harvard.edu

Science  17 Nov 2017:Vol. 358, Issue 6365, pp. 941-946 DOI: 10.1126/science.aam9305

Abstract

Ammonia is a ubiquitous by-product of cellular metabolism; however, the biological consequences of ammonia production are not fully understood, especially in cancer. We found that ammonia is not merely a toxic waste product but is recycled into central amino acid metabolism to maximize nitrogen utilization. In our experiments, human breast cancer cells primarily assimilated ammonia through reductive amination catalyzed by glutamate dehydrogenase (GDH); secondary reactions enabled other amino acids, such as proline and aspartate, to directly acquire this nitrogen. Metabolic recycling of ammonia accelerated proliferation of breast cancer. In mice, ammonia accumulated in the tumor microenvironment and was used directly to generate amino acids through GDH activity. These data show that ammonia is not only a secreted waste product but also a fundamental nitrogen source that can support tumor biomass.

 

 

References

1          Strickaert, A. et al. Cancer heterogeneity is not compatible with one unique cancer cell metabolic map. Oncogene 36, 2637-2642, doi:10.1038/onc.2016.411 (2017).

2          Hui, S. et al. Glucose feeds the TCA cycle via circulating lactate. Nature 551, 115-118, doi:10.1038/nature24057 (2017).

3          Mashimo, T. et al. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell 159, 1603-1614, doi:10.1016/j.cell.2014.11.025 (2014).

4          Sousa, C. M. et al. Erratum: Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature 540, 150, doi:10.1038/nature19851 (2016).

5          Sousa, C. M. et al. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature 536, 479-483, doi:10.1038/nature19084 (2016).

6          Commisso, C. et al. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 497, 633-637, doi:10.1038/nature12138 (2013).

7          Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57-70 (2000).

8          Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646-674, doi:10.1016/j.cell.2011.02.013 (2011).

9          Spinelli, J. B. et al. Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass. Science 358, 941-946, doi:10.1126/science.aam9305 (2017).

10        Dang, C. V. Feeding frenzy for cancer cells. Science 358, 862-863, doi:10.1126/science.aaq1070 (2017).

11        Smith, T. J. & Stanley, C. A. Untangling the glutamate dehydrogenase allosteric nightmare. Trends in biochemical sciences 33, 557-564, doi:10.1016/j.tibs.2008.07.007 (2008).

12        Metallo, C. M. et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380-384, doi:10.1038/nature10602 (2011).

13        Garrett, M. et al. Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities. Cancer & metabolism 6, 4, doi:10.1186/s40170-018-0177-4 (2018).

14        Calvert, A. E. et al. Cancer-Associated IDH1 Promotes Growth and Resistance to Targeted Therapies in the Absence of Mutation. Cell reports 19, 1858-1873, doi:10.1016/j.celrep.2017.05.014 (2017).

15        Sciacovelli, M. & Frezza, C. Metabolic reprogramming and epithelial-to-mesenchymal transition in cancer. The FEBS journal 284, 3132-3144, doi:10.1111/febs.14090 (2017).

16        Dang, L. et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739-744, doi:10.1038/nature08617 (2009).

17        Gross, S. et al. Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations. The Journal of experimental medicine 207, 339-344, doi:10.1084/jem.20092506 (2010).

18        Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer cell 17, 225-234, doi:10.1016/j.ccr.2010.01.020 (2010).

19        Rendina, A. R. et al. Mutant IDH1 enhances the production of 2-hydroxyglutarate due to its kinetic mechanism. Biochemistry 52, 4563-4577, doi:10.1021/bi400514k (2013).

20        Zhang, X. et al. IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligand expression. Neuro-oncology 18, 1402-1412, doi:10.1093/neuonc/now061 (2016).

 

Other articles on this Open Access Journal on Cancer Metabolism Include:

 

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

 

Accumulation of 2-hydroxyglutarate is not a biomarker for malignant progression of IDH-mutated low grade gliomas

 

 

Protein-binding, Protein-Protein interactions & Therapeutic Implications [7.3]

Is the Warburg effect an effect of deregulated space occupancy of methylome?

Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

New Insights on the Warburg Effect [2.2]

The Inaugural Judith Ann Lippard Memorial Lecture in Cancer Research: PI 3 Kinase & Cancer Metabolism

Renal (Kidney) Cancer: Connections in Metabolism at Krebs cycle and Histone Modulation

Warburg Effect and Mitochondrial Regulation- 2.1.3

Refined Warburg Hypothesis -2.1.2

 

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Metabolic Response Heterogeneity

Larry H Bernstein, MD, FCAP, Curator

LFBI

 

The Prognostic Significance of Metabolic Response Heterogeneity in Metastatic Colorectal Cancer

PLoS One. 2015; 10(9): e0138341.

Published online 2015 Sep 30. doi:  10.1371/journal.pone.0138341

PMCID: PMC4589397

Alain Hendlisz,1,* Amelie Deleporte,1 Thierry Delaunoit,2 Raphaël Maréchal,3 Marc Peeters,4 Stéphane Holbrechts,6Marc Van den Eynde,7 Ghislain Houbiers,9 Bertrand Filleul,2 Jean-Luc Van Laethem,3 Sarah Ceyssens,5 Anna-Maria Barbuto,6 Renaud Lhommel,8 Gauthier Demolin,9 Camilo Garcia,10 Hazem El Mansy,1,2,3,4,5,6,7,8,9,10 Lieveke Ameye,11 Michel Moreau,11 Thomas Guiot,10 Marianne Paesmans,11 Martine Piccart,1 and Patrick Flamen10

Daniele Santini, Editor

Author information ► Article notes ► Copyright and License information ►

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Abstract

Background Tumoral heterogeneity is a major determinant of resistance in solid tumors. FDG-PET/CT can identify early during chemotherapy non-responsive lesions within the whole body tumor load. This prospective multicentric proof-of-concept study explores intra-individual metabolic response (mR) heterogeneity as a treatment efficacy biomarker in chemorefractory metastatic colorectal cancer (mCRC).

Methods Standardized FDG-PET/CT was performed at baseline and after the first cycle of combined sorafenib (600mg/day for 21 days, then 800mg/day) and capecitabine (1700 mg/m²/day administered D1-14 every 21 days). MR assessment was categorized according to the proportion of metabolically non-responding (non-mR) lesions (stable FDG uptake with SUV-max decrease <15%) among all measurable lesions.

Results Ninety-two patients were included. The median overall survival(OS) and progression-free survival (PFS) were 8.2months (95%CI:6.8–10.5) and 4.2months (95%CI:3.4–4.8) respectively. In the 79 assessable patients, early PET-CT showed no metabolically refractory lesion in 47%, a heterogeneous mR with at least one non-mR lesion in 32%, and a consistent non-mR or early disease progression in 21%. On exploratory analysis, patients without any non-mR lesion showed a significantly longer PFS (HR 0.34; 95% CI: 0.21–0.56, P-value 0.02) compared to the other patients. The proportion of non-mR lesions within the tumor load did not impact PFS/OS.

Conclusion The presence of at least one metabolically refractory lesion is associated with a poorer outcome in advanced mCRC patients treated with combined sorafenib-capecitabine. Early detection of treatment-induced mR heterogeneity may represent an important predictive efficacy biomarker in mCRC.

Trial Registration ClinicalTrials.gov NCT01290926

 

Background

Tumoral heterogeneity is a major determinant of resistance in solid tumors. FDG-PET/CT can identify early during chemotherapy non-responsive lesions within the whole body tumor load. This prospective multicentric proof-of-concept study explores intra-individual metabolic response (mR) heterogeneity as a treatment efficacy biomarker in chemorefractory metastatic colorectal cancer (mCRC).

Methods

Standardized FDG-PET/CT was performed at baseline and after the first cycle of combined sorafenib (600mg/day for 21 days, then 800mg/day) and capecitabine (1700 mg/m²/day administered D1-14 every 21 days). MR assessment was categorized according to the proportion of metabolically non-responding (non-mR) lesions (stable FDG uptake with SUVmax decrease <15%) among all measurable lesions.

Results

Ninety-two patients were included. The median overall survival (OS) and progression-free survival (PFS) were 8.2 months (95% CI: 6.8–10.5) and 4.2 months (95% CI: 3.4–4.8) respectively. In the 79 assessable patients, early PET-CT showed no metabolically refractory lesion in 47%, a heterogeneous mR with at least one non-mR lesion in 32%, and a consistent non-mR or early disease progression in 21%. On exploratory analysis, patients without any non-mR lesion showed a significantly longer PFS (HR 0.34; 95% CI: 0.21–0.56, P-value <0.001) and OS (HR 0.58; 95% CI: 0.36–0.92, P-value 0.02) compared to the other patients. The proportion of non-mR lesions within the tumor load did not impact PFS/OS.

Conclusion

The presence of at least one metabolically refractory lesion is associated with a poorer outcome in advanced mCRC patients treated with combined sorafenib-capecitabine. Early detection of treatment-induced mR heterogeneity may represent an important predictive efficacy biomarker in mCRC.

Trial Registration

ClinicalTrials.gov NCT01290926

Introduction

The development of new therapeutics for solid tumors is currently strained by increasing regulatory demands to better define subpopulations bearing resistant diseases in order to spare patients from useless toxicities and the society from unaffordable costs in case of ineffective treatments.

Tumor heterogeneity through the existence of resistant subclones (genetic drift) or local environmental factors is nowadays accepted as a major determinant of treatment resistance. However, sensitive biomarkers of tumoral heterogeneity are lacking.[13] Current response assessment methods using morphology (RECIST using MRI/CT) or metabolism (PERCIST using FGD-PET/CT) do not allow the description of tumor heterogeneity because dichotomization of response (versus non-response) requires summing of measurements or the selection of the one single most representative lesion.[4] Moreover most of the new biological therapies render response evaluation even more challenging by the infrequency of tumor shrinkage.[58]

Imaging tumour metabolism using 18F-Fluorodeoxyglucose positron emission tomography coupled with computed tomography (FDG-PET/CT) allows rapid identification of treatment-refractory lesions with a high negative predictive value (NPV).[914] FDG-PET is currently central in the international recommendations for response assessment for Hodgkin’s disease and aggressive non-Hodgkin’s lymphoma, in which medical conditions it is used commonly as a basis for therapeutic decisions. [1417] In contrast, solid tumors are frequently more refractory to treatment and reveal smaller and slower changes in FDG uptake under therapy leading to the existence of different criteria for metabolic response assessment at the lesion as well as at the patient level.[18,19] This ongoing discussion explain why metabolic imaging has still not acquired a biomarker status in solid tumors.

Metabolic imaging provides a whole-body quantitative assessment of treatment-induced changes in tumoral glycolysis early after treatment initiation, before any morphological changes are observed. It has therefore the potential to detect tumoral heterogeneity by revealing how distinct tumor sites behave in response to treatment.

Several trials suggest meaningful clinical activity of combined sorafenib-capecitabine in metastatic breast and colorectal cancer. However the significant toxicity of the combination renders its use practically incompatible with a palliative setting, further underscoring the need to identify a sensitive biomarker for patient selection.[20,21] Preliminary reports in lung and renal cancer suggest that FDG-PET-based metabolic response assessment could be used as a predictive biomarker of sorafenib.[22,23]

The trial is a proof-of-concept study designed to explore intra-individual mR heterogeneity as a prognostic biomarker for this combination of a biological and a cytotoxic agent in mCRC.

 

 

Material and Methods

Belgian competent authorities and ethical committees of the 6 participating centres approved the study (EudraCT 2010-023695-91, clinicaltrials.gov NCT01290926), designed as a prospective multicentric single-arm phase II, with one-stage accrual.

Patients with histologically proven unresectable metastatic CRC failing all standard treatments but not necessarily bevacizumab were eligible. Exclusion criteria were contraindications for capecitabine and sorafenib, ECOG performance status (PS) > 1, age < 18 years, and cerebral metastasis. Normal organ and bone marrow function, a life expectancy >12 weeks, and a signed informed consent were required.

Both drugs were given orally on an outpatient basis: sorafenib 200mg in the morning and 400 mg in the evening every day for the first cycle, then 400 mg twice a day every day; capecitabine 850 mg/m2 twice a day on days 1 to 14, every 21 days. One cycle was defined as a 21-day period. Adverse events were reported according to the National Cancer Institute Criteria, version 3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf). Study medications were to be stopped at disease progression or when unacceptable toxicity occurred. RECIST 1.1-radiological response was assessed locally every two cycles (6weeks). Patients were followed until objective disease progression and every 3 months thereafter for survival assessment.

FDG-PET/CT Imaging

For the FDG-PET/CT, patients were referred to one of the 5 participating PET/CT centres, previously approved for participation based on FDG-PET phantom imaging study for quality’s central assessment [24]. An independent academic molecular imaging core laboratory (OriLab) centralized all FDG-PET/CT images through anonymized CD-Rom transfers, checked image’s quality, DICOM headers, compliance to the Standard Procedures Imaging Manual and imaging case report forms.

Baseline FDG-PET/CT was performed within 7 days preceding chemotherapy initiation and repeated under the same technical and patient conditions on day (D)21 (range D19-D23), with D1 as the first day of chemotherapy administration. Prior to FDG injection, fasting ≥ 6 hours and glycemia levels <120 mg/dL for non-diabetic patients, and <180 mg/dL for diabetic patients were required. Short-acting insulin use on the day of FDG-PET/CT was not allowed.

The PET/CT was initiated 60 to 90 minutes after intravenous injection of 3.7 to 7.4 MBq/kg FDG, optimized for body weight. Similar FDG activity (+/-15%) and time window (+/- 15 min) were used for the second PET/CT.

Whole body scanning with a low dose CT (without intravenous or oral contrast, from proximal femur to skull) was performed, immediately followed by the PET acquisition. Imaging acquisition and reconstruction remained stable over the whole study period. The second FDG-PET/CT was strictly blinded to the investigators, and was not added to the patient’s (electronic) medical records.

The standard uptake value (SUV) of FDG used was the lean body mass-based maximal SUV value within the lesion (SUVmax, g/ml).

All FDG-PET/CT images were analysed in batches using the same software (PETVcar version 4.6, General Electric, USA) and display techniques. Two senior nuclear medicine physicians (PF, CG) performed independent mR analyses using a predefined 3-step methodology.[13] First, on the baseline PET/CT, target lesions were identified according to the following criteria: transaxial diameter (measured on the CT of the PET/CT) > 15 mm, intense FDG uptake (> 2 x normal liver parenchym uptake) and with an unequivocally neoplastic basis. Each target lesion was then classified as non-responding (decrease of SUVmax on second PET-CT<15%) or responding. Second, the patients were classified according to the lesional distribution of mR; class I: absence of any metabolically non-responding lesion, class II: a minor part of whole body tumour load shows a non-response, class III: major part of whole body target tumour load does not respond, and, class IV: all target lesions are non-responding, or presence of a progressive lesion (progression defined as >25% increase of SUVMax, or appearance of a new lesion). (Fig 1) Finally, different methods of patient response dichotomization (metabolic responders versus non-responders) were explored.

Fig 1

Classes of metabolic responses. Class1: no metabolic unresponsive lesion; Class2: minority of unresponsive lesions among whole body target tumour load; Class3: majority of whole body target tumour load does not respond; Class4: all target lesions are non-responding,or, presence of progressive lesions [progression defined as > 25% increase of FDG up take on second PET or appearance of a new lesion]  http://dx.doi.org:/10.1371/journal.pone.0138341.g001

 

Classes of metabolic responses.

Statistical considerations

A first co-primary objective defined the minimal clinical activity necessary to explore the negative predictive value of metabolic response imaging on OS as a survival rate at 6 months > 30% according to the existing literature on chemorefractory CRC. To reject the null hypothesis that the 6 month-OS rate would be <30% using a binomial distribution, a 1-sided test with α = 0.025 and a power of 90% in case of a true 6 months-OS ≥ 50% was used, requiring a sample size of 66 eligible patients followed for at least 6 months. An intent-to-treat (ITT) approach was used.

The second co-primary objective was the prognostic value of mR classification. Based on a previous study,[13] and anticipating a 95% eligibility rate, a 50% early PET/CT non-responders rate, and a hazard ratio (HR) around 0.385 for comparison between the survival distributions, 54 events were needed for a 90% power and a two-sided logrank test at the 2.5% level.

Because the mR rate monitored during the study was higher than expected, the number of events to be observed was increased to 62. This decision was taken without changing the HR to be detected and without estimating this HR during study conduct.

Secondary objectives were to describe PFS, objective response rate and toxicity and to determine the predictive value of early MR on PFS.

For the first co-primary objective, the 6 month-OS, median (m)OS and mPFS were calculated from the patient’s inclusion. For the second co-primary objective, the predictive value assessment of mR on OS and PFS was done from the time of the second FDG-PET/CT on patients having undergone the second FDG-PET/CT in order to control for guarantee-time bias.[25] PFS was calculated up to the time of disease progression or death, whichever occurred first. Kaplan-Meier estimates were used to characterise PFS and OS, and the log-rank test to investigate comparisons between survival curves. Cox’s proportional hazards model was used to calculate HR and their 95% CI

The multivariate analysis was performed using Cox’s proportional hazard model. Variables with a univariate P-value < 0.20 were considered as possible predictors in the multivariate model. We performed stepwise forward selection of variables, i.e. forward selection but at each step variables already in the model could be dropped if their associated p-value became >0.05. To verify the final model, also backward selection of variables was performed on all variables with univariate p-value<0.20, resulting in the same set of variables.[26]

All statistical analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 6 software.

Patients found with an early metabolic progressive disease (class IV) were not excluded from the statistical analyses as the objectives of the paper were to show the predictive value of early metabolic response on OS and PFS, which implies the necessity of an intent-to-treat analysis. The event “progression” in the definition of PFS is moreover a radiological progression. Patients belonging to class IV do not meet this definition of radiological progression, which remains an event to be predicted.

Results

Between February and October 2011, 97 consecutive patients were enrolled in 6 clinical centres. The CONSORT diagram details the reasons for considering 5 patients as ineligible, excluding them from all analysis (Fig 2). The eligible patients (N = 92), median age 63 (range 28–83), male/female ratio of 54/46, PS 0 (55%) or 1(45%) received a median of 5 (range 0–44+) cycles of sorafenib-capecitabine after an history of a median of 3 (range 1–6) prior therapeutic lines including bevacizumab in 55% of patients. Codons 12–13 KRAS mutations were present in 52%.

Fig 2

Consort Diagram.  http://dx.doi.org:/10.1371/journal.pone.0138341.g002

Toxicity (Table 1)

Table 1

Most important (>10%) side effects in the 88 patients who received treatment according to Common Toxicity Criteria CTC3.0.  http://dx.doi.org:/10.1371/journal.pone.0138341.t001

Patients presented a median of 7 (Q1 = 4, Q3 = 9) different adverse reactions during therapy. All but one patient experienced at least one toxicity of any grade, of whom 61.4% with at least one grade III-IV. Grade III-IV side effects were mainly fatigue (21.6%), hand-foot skin reactions (HFSR) (15.9%), and diarrhoea (12.5%). No toxic death was observed. Toxicity led to dose modifications in 63.6% and therapy discontinuation in 5.7% of cases.

Survival data and radiological response

The mOS and mPFS were 8.2 months (95% CI: 6.8–10.5) and 4.2 months (95% CI: 3.4–4.8) respectively. The OS rate at 6 months was 71% (65/92) (95% CI: 61%-79%), significantly higher than the 30% minimal efficiency level predefined in the statistical plan (p-value <0.001), meeting the clinical co-primary endpoint.

According to RECIST, partial response was observed in 7/92 patients (7.6%, 95%CI 2.2–13.0). In the 79 assessable patients, disease control at first evaluation (partial responses and stable diseases according to RECIST) was noted in 32/37 (80%) of the patients with consistent mR versus 24/42 (57%) in other patients (p-value 0.006) (Table 2).

Table 2

RECIST1.1 versus Metabolic Response classes in patients for whom both mR and RECIST assessment of response are available.  http://dx.doi.org:/10.1371/journal.pone.0138341.t002

Metabolic response analysis

MR data were available for 79 patients: 37 (46.8%) were classified as class I; 14 (17.7%) as class II; 11 (13.9%) as class III; and 17 (21.5%) as class IV. Within Class IV, 8 patients (10%) showed early metabolic disease progression.

Patients without any metabolically non-responding lesions (Class I) performed better than patients with heterogeneous responses (Class II and III) or with a consistent non-response or progressive disease (Class IV). The difference between the four classes is statistically significant for mPFS (p-value <0.001) but not for mOS (p-value = 0.13). (Fig 3A and 3B)

Fig 3

PFS* (A) and OS* (B) distribution according to the 4 classes of metabolic response.  Class1: no metabolic unresponsive lesion; Class2: minority of unresponsive lesions among whole body target tumour load; Class3: majority of whole body target tumour load does not respond; Class 4: all target lesions are nonresponding, or, presence of progressive lesions [progression defined as >25% increase of FDG uptake on second PET, or appearance of a new lesion].*from date of the second FDG PET-CT.

Two classifications were considered for reporting response in a dichotomized way according to mR heterogeneity among lesions: classes (I and II) versus classes (III and IV),[13] and classes (I) versus classes (II+III+IV). The first compares outcome according to the dominance of non-mR lesions within the tumor load, the second according to the consistence of mR (Table 3Fig 4). “Using the “dominance” classification to define early metabolic non response, the second co-primary objective, which was to identify a prognostic value on survival for early metabolic assessment, was not met while it was successful to discriminate patients according to their outcome using the exploratory “consistence” classification.“Five of the 42 patients (12%) with at least one non-responding lesion remained free of disease progression at 6 months, versus 15 of the 37 class I patients (41%) (p-value 0.005).

http://dx.doi.org;/10.1371/journal.pone.0138341.g003

 

Table 3 Correlation of mPFS and mOS with Dominance and Consistency of metabolic response.  http://dx.doi.org:/10.1371/journal.pone.0138341.t003

 

Fig 4   PFS and OS distribution according to the dichotomized mR classifications.  http://dx.doi.org:/10.1371/journal.pone.0138341.g004

Multivariate analysis after stepwise variable selection of age, PS, number of previous chemotherapy lines, bevacizumab pretreatment, sex, Body Mass Index (BMI), HFSR occurrence and mR retained the absence of metabolically resistant lesion (class I) as the only variable significantly correlated with both mOS and mPFS (Table 4).

Table 4  Univariate and multivariate analysis for OS and PFS.   http://dx.doi.org:/10.1371/journal.pone.0138341.t004

Discussion

Tumoral heterogeneity, described as the coexistence of genomically different subclones within a patient tumor load or to local environmental aspects, is recognized as a major determinant of resistance to treatment in solid tumors.[13] However, interlesional tumor heterogeneity in metastatic setting is not covered by current response assessment methods because of the analysis’ methodology performing averaging of responses among lesions. This prospective multicentric proof-of-concept study explored interlesional mR heterogeneity as a biomarker of treatment resistance in advanced solid tumors.

As previously reported in several solid tumors, FDG-PET/CT response assessment after one therapy cycle allows a rapid identification of non-responding lesions/patients, fulfilling the necessary conditions to become potentially a good predictive biomarker, which is crucial to avoid useless toxicity.[4,912,22,27] Moreover, significant progresses and implementation of standardized methodology for FDG-PET/CT imaging, including homogenization of imaging procedures and patient’s preparation, quality control and independent central analysis, now allows its use in multicentric trials.[24,27,28]

Studying tumoral heterogeneity requires assessing the response of the whole baseline metastatic tumor load without restriction in number nor site. However, existing morphological (WHO, RECIST) and metabolic (EORTC, PERCIST) response assessment methods do not take into account this response heterogeneity because they only consider a limited number of operator-selected target lesions and/or perform summing or averaging of response variables.[4,19,29,30] Moreover, being classically performed late during treatment, these assessment criteria measure response, while from a clinical point of view, it is the presence of non-response that triggers the need for treatment adaptation. For this, based on prior research, in order to optimize the negative predictive value (NPV) of mR assessment, a 15% cut-off value of SUVmax decrease instead of the standard 25–30% response cut-off value was chosen.[18,31] Such low cut-off value maximally avoids unjustified denial to a potentially active treatment regimen.

With regard to the characterization of response heterogeneity among lesions, this study adopted a multistep descriptive procedure. First, a lesion-by-lesion response analysis of all measurable lesions on baseline FDG-PET/CT without restriction of their number was performed applying the 15% cut-off for non-response. Then, a patient-based 4-class classification was applied, describing the presence and proportion of metabolically non-responding lesions among the whole-body tumor load.[13]

Using such methodology, 22% of the patients showed overall treatment resistance of whom 10% showed early metabolic disease progression at 3 weeks. This observation indicates the importance of performing the baseline FDG-PET/CT as close as possible before the start of the tested drug administration, because rapid disease progression during this timeframe could lead to false negative mR assessment.

On the other hand, after one treatment cycle, 32% of the patients showed heterogeneous metabolic responses combining resistant with potentially responding lesions (Class II and III). Of these, 18% showed non-mR in the minor, while 14% showed a non-mR in the major part of the tumor load. The proportion of heterogeneous response observed in this study is considerable, confirming earlier observation in an independent mCRC patient group treated with chemotherapy, where heterogeneity of mR was described in 67% of patients.[13] Other comparisons are impossible because information about heterogeneity is lacking in most available literature, which apply dichotomization to response assessment.[3234]

Indeed, for clinical decision-making, the response assessment is generally reported dichotomously, because clinicians have to decide whether to continue or adapt the initiated treatment. Such information-reducing response reporting may only be adequate in case of homogeneous mR, but blurs useful information in case of response heterogeneity.

Outcome analysis in this study indicated that mPFS and mOS are comparable in patients bearing one or more metabolically resistant lesion. Only patients without any resistant lesion (class I) seemed to have a better outcome (mPFS and mOS) compared to all others. Therefore it seems that the presence but not the number/proportion of non-responding lesions is the most important prognostic determinant. Moreover, its value is reinforced by a multivariate analysis showing absence of any metabolically treatment resistant lesion as an independent prognostic factor for both PFS and OS.

A valid assessment of a predictive biomarker requires a significant level of activity of the regimen under study. This was achieved, as 71% of the included patients were still alive at 6 months, which was significantly higher than the minimal activity predefined in the study design. ITT analysis of the 92 eligible patients showed a mPFS of 4.2 months and a mOS of 8.2 months respectively, suggesting an overall beneficial effect for this drug combination compared to recent historical data with 2 months mPFS and 4–6 months mOS in the same clinical setting.[6,31,3537]

Moreover, this study confirms the need for an effective predictive response biomarker for a sorafenib-containing regimen, because of the high toxicity profile together with the poor sensitivity of morphology-based imaging (CT/MRI) for detecting responses (only 8% of partial response according to RECIST) during treatment.[7,8,38]

A major application of standardized metabolic imaging is expected in early drug development (phase I-II) for two reasons: (i) as FDG-PET response analysis seems to be correlated with prognosis, it provides a rapid appraisal of the new drug activity even in small patient populations, and (ii) image-guided biopsies of resistant lesions could identify the molecular basis of treatment resistance by demonstrating genomic or epigenomic heterogeneity.

In this study for instance, half (47%) of the patients didn’t demonstrate any resistant lesion, indicating a remarkable activity level for such a heavily pre-treated patients population, unsuspected by classical morphological imaging.

Furthermore, in the metastatic setting, FDG-PET/CT may provide a tool for the identification of patients with one or very few metastatic sites resisting to treatment for whom the continuation of unchanged therapy carries a grim prognosis. This raises the potential of adding locoregional ablative treatments guided by the imaging of metabolic response, in order to achieve homogeneity of disease control and restore prognosis. If the current observation is confirmed by an ongoing multicentric trial, (clinicaltrials.gov NCT01929616), randomized prospective trials using early FDG-PET/CT response assessment as an interventional tool for targeting locoregional therapy (eg. surgery, radioembolization, radiofrequency ablation) will be justified.

Finally, in the absence of randomized data based on PET response, it remains to be proven whether the presence of metabolically non-responding lesions is a biomarker identifying more heterogeneous diseases with intrinsically a worse prognosis, or a genuine therapeutic predictive tool for a given treatment.

 

Conclusions

Metabolic response assessment allows the early identification of treatment-resistant tumor sites. The presence of metabolically refractory lesions seems to negatively impact overall treatment outcome whatever their number, adding to the mounting evidence that tumour heterogeneity is a key element in cancer management.

Early assessment of mR heterogeneity is a potentially powerful predictive biomarker enabling the personalization of anticancer treatments by increasing their cost-effectiveness and sparing useless toxicities.

Go to:

Supporting Information

S1 Protocol

Study protocol.

(PDF)

Click here for additional data file.(1.1M, pdf)

S1 TREND Checklist

TREND Checklist.

(PDF)

Click here for additional data file.(1.3M, pdf)

References

  1. McDermott U, Downing JR, Stratton MR (2011) Genomics and the continuum of cancer care. N Engl J Med 364: 340–350. doi: 10.1056/NEJMra0907178[PubMed]
  2. Greaves M, Maley CC (2012) Clonal evolution in cancer. Nature 481: 306–313. doi:10.1038/nature10762[PMC free article] [PubMed]
  3. Aparicio S, Caldas C (2013) The implications of clonal genome evolution for cancer medicine. N Engl J Med 368: 842–851. doi: 10.1056/NEJMra1204892[PubMed]
  4. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. (2009) NSew response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45: 228–247.[PubMed]
  5. Grothey A, Hedrick EE, Mass RD, Sarkar S, Suzuki S, Ramanathan RK, et al. (2008) Response-independent survival benefit in metastatic colorectal cancer: a comparative analysis of N9741 and AVF2107.J Clin Oncol 26: 183–189. doi: 10.1200/JCO.2007.13.8099[PubMed]
  6. Grothey A, Van Cutsem E, Sobrero A, Siena S, Falcone A, Ychou M, et al. (2012) Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet 381: 303–312. doi: 10.1016/S0140-6736(12)61900-X[PubMed]
  7. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359: 378–390. doi: 10.1056/NEJMoa0708857[PubMed]
  8. Awada A, Gil T, Whenham N, Van Hamme J, Besse-Hammer T, Brendel E, et al. (2011) Safety and pharmacokinetics of sorafenib combined with capecitabine in patients with advanced solid tumors: results of a phase 1 trial. J Clin Pharmacol 51: 1674–1684. doi: 10.1177/0091270010386226[PubMed]
  9. Ott K, Weber WA, Lordick F, Becker K, Busch R, Herrmann K, et al. (2006) Metabolic imaging predicts response, survival, and recurrence in adenocarcinomas of the esophagogastric junction. J Clin Oncol 24: 4692–4698. [PubMed]
  10. Hoekstra CJ, Stroobants SG, Smit EF, Vansteenkiste J, van Tinteren H, Postmus PE, et al. (2005)Prognostic relevance of response evaluation using [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography in patients with locally advanced non-small-cell lung cancer. J Clin Oncol 23: 8362–8370. [PubMed]
  11. Rousseau C, Devillers A, Sagan C, Ferrer L, Bridji B, Campion L, et al. (2006) Monitoring of early response to neoadjuvant chemotherapy in stage II and III breast cancer by [18F]fluorodeoxyglucose positron emission tomography. J Clin Oncol 24: 5366–5372. [PubMed]
  12. de Geus-Oei LF, van Laarhoven HW, Visser EP, Hermsen R, van Hoorn BA, Kamm YJ, et al. (2008)Chemotherapy response evaluation with FDG-PET in patients with colorectal cancer. Ann Oncol 19: 348–352. [PubMed]
  13. Hendlisz A, Golfinopoulos V, Garcia C, Covas A, Emonts P, Ameye L, et al. (2012) Serial FDG-PET/CT for early outcome prediction in patients with metastatic colorectal cancer undergoing chemotherapy.Ann Oncol 23: 1687–1693. doi: 10.1093/annonc/mdr554[PubMed]
  14. Barrington SF, Mikhaeel NG, Kostakoglu L, Meignan M, Hutchings M, Mueller SP, et al. (2014) Role of Imaging in the Staging and Response Assessment of Lymphoma: Consensus of the International Conference on Malignant Lymphomas Imaging Working Group. J Clin Oncol. [PubMed]
  15. Cheson BD, Pfistner B, Juweid ME, Gascoyne RD, Specht L, Horning SJ, et al. (2007) Revised response criteria for malignant lymphoma. J Clin Oncol 25: 579–586. [PubMed]

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2.0 Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Writer and Curator: Larry H Bernstein, MD, FCAP

This is the second article in a series concerning genomic expression, The first of which was concerned with the expanded technologies in use for study of genomic expression.  This portion will also cover more of genetic errors as well as methodologies, but not all examples are in the realm of cancer.

I shall start with a New York Times editorial on July 24, 2015 by Angelina Jolie Pitt on her experience with BRCA1 gene and her family history.  It is very instructive on how she worked through her experience.

http://www.nytimes.com/2015/03/24/opinion/angelina-jolie-pitt-diary-of-a-surgery.html?

Two years ago she was found to have a positive test for BRCA1, carrying an 87 percent risk for breast cancer and a 50 percent risk for ovarian cancer.  At that time she had a preventive mastectomy.  The decision was not easy, but it also brought into consideration that her mother and grandmother both died of breast cancer.  She did not have an oophorectomy at that time because on considering the advice of medical experts, she would have been left with no estrogen support. She wanted to delay her early vegetative senescence.  She has reached the age of 39 years and on the advice of medical expert opinion, she proceeded with salpingo-oophorectomy, at age 39 years, a decade before  her  mother had developed cancer.  But her delay was to allow her to recover and adjust emotionally to her ongoing situation, with a remaining risk for ovarian cancer.

She tested negative for CA-1251-5 at this time prior to surgery. But the CA-125 test could well be negative with early onset ovarian cancer. It may be considered a better test for following treatment than for early diagnosis. Her choice was to sacrifice early menopause to the ability to live through her childrens’ childhood development.  This was a well thought out decision.  In addition, there were abnormal inflammatory markers that were not specific for cancer rsik, but were worth taking into account.  The procedure itself was simpler than the mastectomy.

23op-ed-thumbStandard

http://static01.nyt.com/images/2015/03/23/opinion/23op-ed/23op-ed-master315.jpg

2.1  CA-125 and Ovarian Cancer

2.1.1  lmmunoradiometric Assay of CA 125 in Effusions: Comparison with Carcinoembryonic Antigen

Marguerite M. Pinto, MD,‘ Larry H. Bernstein, MD,* Dennis A. Brogan, MPH, MT

and Elaine Criscuolo, CT(ASCP) CMIACS

The levels of CA 125 antigen were measured in 167 effusions from 150 patients using radioimmunoassay, and the results compared with the levels of carcinoembryonic antigen (CEA) in the fluids. The results indicate that an elevated fluid CA 125 level (>14,000 U/ml-68,000 U/ml) and a negative fluid CEA level (4 ng/ml) is suggestive of serous and endometrioid carcinoma of ovary, and adenocarcinoma of the endometrium and fallopian tube. Alternatively, an elevated fluid CEA level (14 ng/ml-600 ng/ml) and a negative CA 125 level (20-5000 U/ml) is seen in metastatic carcinomas of breast, lung, gastrointestinal tract, and mucinous ystadenocarcinoma. Lymphomas, melanomas, and benign effusions are negative for both antigens. The combined use of CEA and CA 125 antigen in fluids is useful in the differential diagnosis of adenocarcinoma of unknown primary. Cancer 59:218-222, 1987.

2.1.2 CA-125 in fine-needle aspirates of solid tumors: comparison with cytologic diagnosis and carcinoembryonic antigen (CEA) assay.

Marguerite M. Pinto, S Kotta

Diagnostic Cytopathology 03/1996; 14(2):121-5.
http://dx.doi.org:/10.1002/(SICI)1097-0339(199603)14:2<121::AID-DC4>3.0.CO;2-M

One hundred and twenty-two fine needle aspirates (FNA) from female patients were studied to determine whether CA-125 assay contributed to cytologic diagnosis and CEA assay. Cytologic examination was done on Papanicolaou-stained smears and cell blocks, CEA by EIA (Abbott Laboratory, > 5 ng/ml cutoff) and CA-125 by RIA (Abbott Laboratory, North Chicago, IL, > 66 mu/ml cutoff). Final diagnosis were correlated with histologic diagnosis when available, clinical, radiologic studies, and follow-up. Results: 29 benign, 93 malignant. Sensitivities and specificities: cytology, 91%, 100%; CEA: 59%, 86%; CA-125, 50%, 55%. CEA plus cytology sensitivity, 97%. CA-125 content was highest in endometrial/ovarian carcinoma (39,899 mu/ml) and < 5,000 mu/ml in other tumors and benign FNA in contrast to CEA which showed highest levels in carcinomas of colon, pancreas, and lung (> 280 ng/ml). While elevated CEA enhances the sensitivity of cytologic diagnosis of carcinomas of the colon, pancreas, and lung, low CEA and high CA-125 content supports an ovarian/endometrial primary.

2.1.3  Diagnostic efficiency of carcinoembryonic antigen and CA125 in the cytological evaluation of effusions.

Pinto MM, Bernstein LH, Rudolph RA, Brogan DA, Rosman M.
Arch Pathol Lab Med. 1992 Jun; 116(6):626-31.

In our previous study, the combination of the concentrations of carcinoembryonic antigen (CEA) and CA125 and the findings from cytological examination in 189 benign and malignant pleural and peritoneal effusions was useful in the diagnosis/classification of malignant effusions. Sensitivity of CEA (level, greater than 5 ng/mL) was 68%; specificity was 99% for the diagnosis of malignant effusions secondary to carcinoma of the lung, breast, gastrointestinal tract, and mucinous carcinoma of the ovary. Sensitivity of CA125 (level, greater than 5000 U/mL) was 85%; specificity was 96% for the diagnosis of malignant effusions in carcinoma of the ovary, fallopian tube, and endometrium. We now expanded the study to include 840 pleural and peritoneal effusions (benign, n = 520; malignant, n = 320) and analyzed the data by the statistical method of Rudolph and colleagues. Based on new cutoff values, ie, CEA level at 6.3 ng/mL and CA125 level at 3652 U/mL, the sensitivities for detection of malignant effusions secondary to carcinomas of the lung, breast, and gastrointestinal tract and mucinous carcinoma of the ovary varied between 75% and 100%; specificity was 98%. Sensitivity of CA125 for detection of malignant effusions from müllerian epithelial carcinoma was 71%; specificity was 99%. The elevated CEA fluid level alone helped to diagnose malignant effusions of the gastrointestinal tract in 54%, breast in 19%, and lung in 16%. The high CA125 fluid level was predictive of müllerian epithelial carcinoma. Adjunctive use of CEA and CA125 levels in fluid enhances the sensitivity of cytological diagnosis and may be predictive of the primary site in patients who present with carcinoma of an unknown primary source.

2.2 Carcinoembryonic antigen in diagnostics

2.2.1 Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Pinto MM1, Ha DJ.
Acta Cytol. 1992 May-Jun; 36(3):277-82

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.

2.2.2  Relationship between serum CA125 half life and survival in ovarian cancer

Table
Gupta and Lis Journal of Ovarian Research 2009 2:13
http://dx.doi.org:/10.1186/1757-2215-2-13

First Author, Year, Study Place Data Collection Study
Design
Sample
Size
RR/HR, (95% CI),
P-Value
Riedinger JM, 2006, France 1988 to
1996
R 553 2.04 (1.58-2.63), < 0.0001
Gadducci A, 2004, Italy 1996 to2002 R 71 3.11 (1.22-7.98), 0.0181
Munstedt K, 1997, Germany 1987 to1994 R 85 0.6184
Gadducci A, 1995, Italy 1986 to1992 R 225 2.13 (1.23-3.68), 0.0073
Rosman M, 1994, Connecticut 1985 to
1989
R 51 3.6 (1.8-7.4), < 0.001
Yedema C A, 1993, Netherlands 1984 to
1990
R 60 9.17 (1.49-56.3), 0.01
Hawkins RE, 1989, London NA P 29 3.7 (0.7-20.1), 0.001;27.8 (4.0-193), 0.001

1CA125 half-life was independent prognostic indicator for survival
2FIGO stage, tumor grade, residual disease, CA125
http://www.ovarianresearch.com/content/2/1/13/table/T6

3.3.0      DNA double strand breaks

2.3.1.  Collaboration and competition – DNA double-strand break repair pathways

Kass EM, Jasin M
FEBS Letters 2010; 584:3703-3708
http://dx.doi.org:/jfebslet.2010.07.057

DNA double-strand breaks occur in replication and exogenous sources pose risk to genome stability. There are two pathways to repair.  They are non-homologous end joining and homologous recombination. Both pathways cooperate and compete at double-strand break sites.

2.3.2 DNA Double-Strand Break Repair Inhibitors as Cancer Therapeutics

Srivastava M, Rashavan SC
Chem & Biol 2015 Jan; pp17-29
http://dx.doi.org:/10.1016/jchembiol.2014.11.013

Homologous recombination and non-homologous end joining are the two major repair pathways expressed in eukaryotes.  For double-strand breaks, and the DSB repair gene is vulnerable to chemotherapy and radiation therapy, accounting for treatment resistance. Therefore, targeting DSB repair is attractive. Blocking the residual repair using inhibitors can potentiate treatment.

2.3.3  Animation published in DNA Repair: Helleday T, Lo J, van Gent DC, Engelward BP. DNA double-strand break repair: From mechanistic understanding to cancer treatment. DNA Repair. (14 Mar 2007)
2.3.3.1 http://web.mit.edu/engelward-lab/animations/DSBR.html

2.3.3.2 https://www.youtube.com/watch?v=eg8rpYFsqCA

2.3.4 Homology-dependent double strand break repair. Oxford Academic (Oxford University Press)

https://www.youtube.com/watch?v=86JCMM5kb2A

2.4.0 Managing DNA data sets

2.4.1 Bionimbus –  a cloud for managing, analyzing and sharing large genomics datasets

The Bionimbus Protected Data Cloud (PDC) is a collaboration between the Open Science Data Cloud (OSDC) and the IGSB (IGSB,) the Center for Research Informatics (CRI), the Institute for Translational Medicine (ITM), and the University of Chicago Comprehensive Cancer Center (UCCCC). The PDC allows users authorized by NIH to compute over human genomic data from dbGaP in a secure compliant fashion. Currently, selected datasets from the The Cancer Genome Atlas (TCGA) are available in the PDC.

https://bionimbus-pdc.opensciencedatacloud.org/

2.4.1.2 Accounting for uncertainty in DNA sequencing data

O’Rawe JA, Ferson S, Lyon GJ
Trends in Genetics 2015 Feb; 31(2):61-66
http://dx.doi.org:/10.101/jtig.2014.12.002

This article reviews uncertainty in quantification in DNA sequency applications and sources of error propagation, and it proposes methods to account for errors and uncertainties.

2.5.0 Linking Traits to Mechanisms and UPR response/proteostasis

2.5.1 Stress-Independent Activation of XBP1s and/or ATF6 Reveals –Three Linking traits based on their shared molecular mechanisms

Shoulders MD, Ryno LM, Genereux JC,…Wiseman BL
Cell Reports 2013 Apr; 3, pp 1279-1292
http://dx.doi.org:/10.1016/j.celrep.2013.03.024

The unfolded protein response (UPR) maintains ER proteostasis through the transcription factors XP1s and ATF6. This study measured orthogonal small molecule-mediated activation of transcription factors nXP1s and/or ATF6 using transcriptomics and quantitative proteomics. The finding is that three ER proteostasis environmants differentially influence

  1. Folding
  2. Traffiking, and
  3. Degradation of destabilized ER client proteins

Without affecting endogenous proteome. The proteostasis network is remodeled with the potential for selective restoration of the aberrant ER proteostasis.

2.5.2 Biological and chemical approaches to diseases of proteostasis deficiency.

Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE
Annu Rev Biochem. 2009; 78:959-91.
http://dx.doi.org:/10.1146/annurev.biochem.052308.114844

Many diseases appear to be caused by the misregulation of protein maintenance. Such diseases of protein homeostasis, or “proteostasis,” include loss-of-function diseases (cystic fibrosis) and gain-of-toxic-function diseases (Alzheimer’s, Parkinson’s, and Huntington’s disease). Proteostasis is maintained by the proteostasis network, which comprises pathways that control protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. The decreased ability of the proteostasis network to cope with inherited misfolding-prone proteins, aging, and/or metabolic/environmental stress appears to trigger or exacerbate proteostasis diseases. Herein, we review recent evidence supporting the principle that proteostasis is influenced both by an adjustable proteostasis network capacity and protein folding energetics, which together determine the balance between folding efficiency, misfolding, protein degradation, and aggregation. We review how small molecules can enhance proteostasis by binding to and stabilizing specific proteins (pharmacologic chaperones) or by increasing the proteostasis network capacity (proteostasis regulators). We propose that such therapeutic strategies, including combination therapies, represent a new approach for treating a range of diverse human maladies.

2.5.3 Extracellular Chaperones and Proteostasis

Amy R. Wyatt, Justin J. Yerbury, Heath Ecroyd, and Mark R. Wilson
Annual Review of Biochemistry 2013 Jun; 82: 295-322
http://dx.doi.org:/10.1146/annurev-biochem-072711-163904

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world’s most debilitating, costly, and intractable diseases.

http://www.proteostasis.com/our-technology/proteostasis-network.html

proteostasis model

http://www.proteostasis.com/images/stories/technology/illustration1.gif

2.6.0 Transcription

2.6.1 Looping Back to Leap Forward. Transcription Enters a New Era

Levine M, Cattoglio C, Tijan R
Cell 2014 Mar; 157: 13-22.
http://dx.doi.org:/10.1016/j.cell.2014.02.009

Organism complexity is not in gene number, but lies in gene regulation. The human genbome contains hundreds of thousands of enhancers, and genes are embedded in a milieu of enhancers . Proliferation of cis-regulatory DNAs is accompanied by complexity and functional diversity of transcription machinery recognizing distal enhancers and promotors, and high-order spatial organization. This article reviews the dynamic communication of remote enhancers with target promoters.

2.6.2 Activating gene expression in mammalian cells with promoter-targeted duplex RNAs.

Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR.
Nat Chem Biol. 2007 Mar; 3(3):166-73
http://dx.doi.org:/10.1038/nchembio860

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.
2.6.3 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

M Gossen and H Bujard
Proc Natl Acad Sci U S A. 1992 Jun 15; 89(12): 5547–5551.

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of “on/off” situations for such genes in a reversible way.

Diagrams of two regulatable gene expression systems.

Diagrams of two regulatable gene expression systems.

http://www.intechopen.com/source/html/16788/media/image5.jpeg

schematic-representation-of-transgenic-mouse-breeding-scheme-h2b-gfp-mice-should-not-express-gfp-in-the-absence-of-a-tetracycline-regulatable-transactivator

schematic-representation-of-transgenic-mouse-breeding-scheme-h2b-gfp-mice-should-not-express-gfp-in-the-absence-of-a-tetracycline-regulatable-transactivator

http://openi.nlm.nih.gov/imgs/512/321/2408727/2408727_pone.0002357.g001.png

2.7.0 Epigenetics and Cancer

2.7.1 Epigenetics and cancer metabolism

Johnson C, Warmoes MO, Shen X, Locasale JW
Cancer Letters 2015;  356:309-314.
http://dx.doi.org:/10.1016/j.canlet.2013.09.043

Cancer is characterized by adaptive metabolic changes for proliferation and survival of the neoplastic cell, which is accompanied by dysfunctional metabolic enzyme changes in a specific nutrient supplied environment. The oncogenic change uses epigenetic level enzymes that catalyze posttranslational modifications of the DNA/histone expression with metabolites including cofactors and substrates for reactions. This interaction of epigenetics and metabolism provides new insights for anti-cancer therapy.

2.7.2 Cancer Epigenetics. From Mechanism to Therapy

Dawson MA, Konzarides T
Cell 2012 Jul; 150:12-27
http://dx.doi.org:/10.1016/j.cell.2012.06.013

Carcinogenesis requires all of the following:

  • DNA methylation
  • Histone modification
  • Nucleosome remodeling
  • RNA mediated targeting

This article reviews basic principles of epigenetic pathways that are dysregulated in carcinogenesis.

2.7.4 A subway review of cancer pathways

Hahn WC, Weinberg RA
Nature Reviews: Cancer
http://www.nature.com/nrc/poster/subpathways/index.html

Cancer arises from the stepwise accumulation of genetic changes that confer upon an incipient neoplastic cell the properties of unlimited, self-sufficient growth and resistance to normal homeostatic regulatory mechanisms. Advances in human genetics and molecular and cellular biology have identified a collection of cell phenotypes � the main destinations in the subway map below � that are required for malignant transformation1. Specific molecular pathways (subway lines) are responsible for programming these behaviours. Although the connections between cancer-cell wiring and function remain incompletely explored and specified � hence the many lines under construction � the broad outlines of the molecular circuitry of the cancer cell can now be sketched. Further advances in understanding these pathways and their interconnections will accelerate the development of molecularly targeted therapies that promise to change the practice of oncology.

cancer subway map

cancer subway map

http://www.nature.com/nrc/poster/subpathways/images/map.gif

Subway map designed by Claudia Bentley.

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