Risk of developing chronic myeloid neoplasms in well-differentiated thyroid cancer patients treated with radioactive iodine.

Exposure to ionizing radiation increases the risk of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), but such risks are not known in well-differentiated thyroid cancer (WDTC) patients treated with radioactive iodine (RAI). A total of 148 215 WDTC patients were identified from Surveillance, Epidemiology and End Results registries between 1973 and 2014, of whom 54% underwent definitive thyroidectomy and 46% received adjuvant RAI. With a median follow-up of 6.6 years, 77 and 66 WDTC patients developed MDS and MPN, respectively. Excess absolute risks for MDS and MPN from RAI treatment when compared to background rates in the US population were 6.6 and 8.1 cases per 100 000 person-years, respectively. Compared to background population rates, relative risks of developing MDS (3.85 (95% confidence interval, 1.7-7.6); P=0.0005) and MPN (3.13 (1.1-6.8); P=0.012) were significantly elevated in the second and third year following adjuvant RAI therapy, but not after thyroidectomy alone. The increased risk was significantly associated with WDTC size ⩾2 cm or regional disease. Development of MDS was associated with shorter median overall survival in WDTC survivors (10.3 vs 22.5 years; P<0.001). These data suggest that RAI treatment for WDTC is associated with increased risk of MDS with short latency and poor survival.

The relationship between eligibility criteria and adverse events in randomized controlled trials of hematologic malignancies.

To minimize adverse events (AEs) unrelated to drugs and maximize the likelihood of drug approvals, eligibility criteria for randomized controlled trials (RCTs) may be overly restrictive. The purpose of this study was to determine if RCTs in hematologic malignancies exclude patients irrespective of known toxicities or observed AEs. MEDLINE was searched from 1/2010 to 1/2015 for RCTs published in high-impact journals. Of 97 trials, 33% were conducted in leukemia, 28% in lymphoma, 34% in multiple myeloma and 5% in myelodysplastic syndromes or myelofibrosis. Expected toxicities at thresholds of ⩾10%, ⩾5% and <5% were not correlated with cardiac, hepatic or renal eligibility criteria (logistic regression). To explore this lack of correlation we tested the concordance of expected toxicities and eligibility criteria using a modified version of McNemar's test: at each threshold, hepatic, renal and cardiac expected toxicities were significantly discordant with eligibility criteria. Hepatic and renal eligibility criteria were also not correlated with observed AEs, P=0.69 and P=0.77, respectively, but a significant correlation was detected between cardiac eligibility criteria and observed AEs, P=0.02. Thus, the analyzed RCTs excluding patients with organ dysfunction do not reflect expected toxicities, based on prescription drug labels/prior experience, or reported AEs on the trials.

Genomic determinants of chronic myelomonocytic leukemia.

The biology, clinical phenotype and progression rate of chronic myelomonocytic leukemia (CMML) are highly variable due to diverse initiating and secondary clonal genetic events. To determine the effects of molecular features including clonal hierarchy in CMML, we studied whole-exome and targeted next-generation sequencing data from 150 patients with robust clinical and molecular annotation assessed cross-sectionally and at serial time points of disease evolution. To identify molecular lesions unique to CMML, we compared it to the related myeloid neoplasms (N=586), including juvenile myelomonocytic leukemia, myelodysplastic syndromes (MDS) and primary monocytic acute myeloid leukemia and discerned distinct molecular profiles despite similar pathomorphological features. Within CMML, mutations in certain pathways correlated with clinical classification, for example, proliferative vs dysplastic features. While most CMML patients (59%) had ancestral (dominant/co-dominant) mutations involving TET2, SRSF2 or ASXL1 genes, secondary subclonal hierarchy correlated with clinical phenotypes or outcomes. For example, progression was associated with acquisition of new expanding clones carrying biallelic TET2 mutations or RAS family, or spliceosomal gene mutations. In contrast, dysplastic features correlated with mutations usually encountered in MDS (for example, SF3B1 and U2AF1). Classification of CMML based on hierarchies of ancestral and subclonal mutational events may correlate strongly with clinical features and prognosis.

Incorporation of molecular data into the Revised International Prognostic Scoring System in treated patients with myelodysplastic syndromes.

The Revised International Prognostic Scoring System (IPSS-R) was developed for untreated myelodysplastic syndrome (MDS) patients based on clinical data. We created and validated a new model that incorporates mutational data to improve the predictive capacity of the IPSS-R in treated MDS patients. Clinical and mutational data from treated MDS patients diagnosed between January 2000 and January 2012 were used to develop the new prognostic system. A total of 508 patients were divided into training (n=333) and validation (n=175) cohorts. Independent significant prognostic factors for survival included age, IPSS-R, EZH2, SF3B1 and TP53. Weighted coefficients for each factor were used to build the new linear predictive model, which produced four prognostic groups: low, intermediate-1, intermediate-2 and high with a median overall survival of 37.4, 23.2, 19.9 and 12.2 months, respectively, P<0.001. Significant improvement in the C-index of the new model (0.73) was observed compared with the IPSS-R (0.69). The new model predicted outcome both in a separate validation cohort and in another cohort of patients with paired samples at different time points during their disease course. The addition of mutational data to the IPSS-R makes it dynamic and enhances its predictive ability in treated MDS patients regardless of their initial or subsequent therapies.

The complexity of interpreting genomic data in patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by the accumulation of complex genetic alterations responsible for the initiation and progression of the disease. Translating genomic information into clinical practice remained challenging with conflicting results regarding the impact of certain mutations on disease phenotype and overall survival (OS) especially when clinical variables are controlled for when interpreting the result. We sequenced the coding region for 62 genes in 468 patients with secondary AML (sAML) and primary AML (pAML). Overall, mutations in FLT3, DNMT3A, NPM1 and IDH2 were more specific for pAML whereas UTAF1, STAG2, BCORL1, BCOR, EZH2, JAK2, CBL, PRPF8, SF3B1, ASXL1 and DHX29 were more specific for sAML. However, in multivariate analysis that included clinical variables, only FLT3 and DNMT3A remained specific for pAML and EZH2, BCOR, SF3B1 and ASXL1 for sAML. When the impact of mutations on OS was evaluated in the entire cohort, mutations in DNMT3A, PRPF8, ASXL1, CBL EZH2 and TP53 had a negative impact on OS; no mutation impacted OS favorably; however, in a cox multivariate analysis that included clinical data, mutations in DNMT3A, ASXL1, CBL, EZH2 and TP53 became significant. Thus, controlling for clinical variables is important when interpreting genomic data in AML.

Defining AML and MDS second cancer risk dynamics after diagnoses of first cancers treated or not with radiation.

Risks of acute myeloid leukemia (AML) and/or myelodysplastic syndromes (MDS) are known to increase after cancer treatments. Their rise-and-fall dynamics and their associations with radiation have, however, not been fully characterized. To improve risk definition we developed SEERaBomb R software for Surveillance, Epidemiology and End Results second cancer analyses. Resulting high-resolution relative risk (RR) time courses were compared, where possible, to results of A-bomb survivor analyses. We found: (1) persons with prostate cancer receiving radiation therapy have increased RR of AML and MDS that peak in 1.5-2.5 years; (2) persons with non-Hodgkin lymphoma (NHL), lung and breast first cancers have the highest RR for AML and MDS over the next 1-12 years. These increased RR are radiation specific for lung and breast cancer but not for NHL; (3) AML latencies were brief compared to those of A-bomb survivors; and (4) there was a marked excess risk of acute promyelocytic leukemia in persons receiving radiation therapy. Knowing the type of first cancer, if it was treated with radiation, the interval from first cancer diagnosis to developing AML or MDS, and the type of AML, can improve estimates of whether AML or MDS cases developing in this setting are due to background versus other processes.

GFI1 as a novel prognostic and therapeutic factor for AML/MDS.

Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting histone deacetylases to target genes. We present data that low expression of GFI1 is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1 expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. Leukemic cells from animals that express low levels of GFI1 show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1 expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KD mice and from AML patients with low GFI1 levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1 expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.

Clinical and biological implications of ancestral and non-ancestral IDH1 and IDH2 mutations in myeloid neoplasms.

Mutations in isocitrate dehydrogenase 1/2 (IDH1/2(MT)) are drivers of a variety of myeloid neoplasms. As they yield the same oncometabolite, D-2-hydroxyglutarate, they are often treated as equivalent, and pooled. We studied the validity of this approach and found IDH1/2 mutations in 179 of 2119 myeloid neoplasms (8%). Cross-sectionally, the frequencies of these mutations increased from lower- to higher risk disease, thus suggesting a role in clinical progression. Variant allelic frequencies indicated that IDH1(MT) and IDH2(MT) are ancestral in up to 14/74 (19%) vs 34/99 (34%; P=0.027) of cases, respectively, illustrating the pathogenic role of these lesions in myeloid neoplasms. IDH1/2(MT) was associated with poor overall survival, particularly in lower risk myelodysplastic syndromes. Ancestral IDH1(MT) cases were associated with a worse prognosis than subclonal IDH1(MT) cases, whereas the position of IDH2(MT) within clonal hierarchy did not impact survival. This may relate to distinct mutational spectra with more DNMT3A and NPM1 mutations associated with IDH1(MT) cases, and more ASXL1, SRSF2, RUNX1, STAG2 mutations associated with IDH2(MT) cases. Our data demonstrate important clinical and biological differences between IDH1(MT) and IDH2(MT) myeloid neoplasms. These mutations should be considered separately as their differences could have implications for diagnosis, prognosis and treatment with IDH1/2(MT) inhibitors of IDH1/2(MT) patients.

Misrejoining of double-strand breaks after X irradiation: relating moderate to very high doses by a Markov model.

Misrejoining of double-strand breaks (DSBs) detected with pulsed-field gel electrophoresis (PFGE) after X irradiation of human cells at very high doses (80-160 Gy) is related to dose-response relationships for chromosome aberrations at moderate doses (1-5 Gy) by the Sax-Markov binary eurejoining/misrejoining (SMBE) model. The SMBE model applies Sax's breakage-and-reunion hypothesis to a subset of DSBs active in binary misrejoining and in binary eurejoining (accidental restitution). The model is numerically consistent with both data on chromosome aberrations and the data obtained by PFGE if proximity effects (restrictions on the range of interactions of DSB free ends) are present. Proximity effects are modeled by partitioning the cell's nucleus into approximately 400 interaction sites, with two active DSB free ends capable of rejoining only if they were produced within the same site. Neglecting one-track action, the SMBE model predicts a quadratic-linear dose-response relationship for DSB misrejoining after exposure to low-LET radiation; i.e., there is a quadratic response at moderate doses which becomes linear as the dose becomes large, rather than vice versa. The linear region results because at very high doses almost all of the active DSB free ends misrejoin rather than eurejoin.

Recent data obtained by pulsed-field gel electrophoresis suggest two types of double-strand breaks.

The temporal evolution of unrejoined and misrejoined DNA double-strand breaks (DSBs) produced by high doses (80-160 Gy) of X rays has been estimated using pulsed-field gel electrophoresis (PFGE) (Löbrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). We attempted to fit these data to three models. An RBM ("Revell binary misrejoining") model, based on the usual repair-misrepair and lethal-potentially lethal models, appears to be inconsistent with the data. The main discrepancies are the following: (1) The RBM model predicts that 90% of the misrejoined DSBs form by the time 75% of the DSBs have disappeared, while the data indicate that only 50% are formed by this time; and (2) the model predicts an increasing fraction of DSBs misrejoined at 160 Gy compared to 80 Gy, while the data support approximately equal fractions misrejoined. These discrepancies are alleviated in the Sax subset (SS) and Revell subset (RS) models. In the SS and RS models, two types (or subsets) of DSBs exist: those that are active in misrejoining and those that are not. In the SS model, active DSBs misrejoin by the breakage-and-reunion mechanism described by Sax; in the RS model, active DSBs either repair, or misrejoin according to the complete exchange misrejoining mechanism described by Revell. Both models are consistent with the data set considered.

Modeling the low-LET dose-response of BCR-ABL formation: predicting stem cell numbers from A-bomb data.

Formation of the BCR-ABL chromosomal translocation t(9;22)(q34;q11) is essential to the genesis of chronic myeloid leukemia (CML). An interest in the dose-response of radiation induced CML therefore leads naturally to an interest in the dose-response of BCR-ABL formation. To predict the BCR-ABL dose-response to low-linear energy transfer (LET) ionizing radiation, three models valid over three different dose ranges are examined: the first for doses greater than 80 Gy, the second for doses less than 5 Gy and the third for doses greater than 2 Gy. The first of the models, due to Holley and Chatterjee, ignores the accidental binary eurejoining of DNA double-strand break (DSB) free ends ('eurejoining' refers to the accidental restitution of DSB free ends with their own proper mates). As a result, the model is valid only in the limit of high doses. The second model is derived directly from cytogenetic data. This model has the attractive feature that it implicitly accounts for single-track effects at low doses. The third model, based on the Sax-Markov binary eurejoining/misrejoining (SMBE) algorithm, does not account for single-track effects and is therefore limited to moderate doses greater than approximately 2 Gy. Comparing the second model to lifetime excess CML risks expected after 1 Gy, estimates of the number of hematopoietic stem cells capable of causing CML were obtained for male and female atomic bomb survivors in Hiroshima and Nagasaki. The stem cell number estimates lie in the range of 5 x 10(7)-3 x 10(8) cells.

Size distributions of misrejoining DNA fragments in irradiated cells.

When ionizing radiation strikes a cell it induces DNA double strand breaks (DSBs). Subsequently, some of the DSBs misrejoin and thus cause alterations in the size distribution of the DNA fragments. We derive a system of non-linear integro-differential equations describing the misrejoining interactions of five classes of DNA fragments, including rings and various types of linear fragments. The fragment classes are represented by density functions; the shape of a density function determines the probability that a fragment has a particular size and the amplitude (integral) equals the expected number of such fragments per cell. The equations are solved: analytically for exponentially distributed initial fragment sizes (corresponding to high doses) and numerically for arbitrary initial conditions. Computed final fragment size distributions are applied to situations representative of flow karyotypes and pulsed-field gel assays. For human flow karyotypes, the model can be used to obtain misrejoining estimates at doses too high for conventional methods of data analysis. For pulsed-field gel assays in which human chromosomes are digested with restriction endonucleases to form 'cut-somes' (restriction fragments), the model provides a means of misrejoining estimation when the cut-some sizes are non-random. The model suggests that if the cut-some size distribution for unirradiated cells is completely random, misrejoining of radiation-induced DSBs will not be detectable in the final size distribution.

A mathematical model of human thymidine kinase 2 activity.

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative cooperativity) for dT and ∼1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.

p53 independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia.

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin-modifying enzyme DNA methyl-transferase 1 without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine upregulated the key late differentiation factors CCAAT enhancer-binding protein ɛ and p27/cyclin dependent kinase inhibitor 1B (CDKN1B), induced cellular differentiation and terminated AML cell cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xenotransplanted AML cells was abrogated but normal hematopoietic stem cell engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S phase specific decitabine therapy. In xenotransplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared with conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy.

How to use the computing environment R to analyze ATP-induced ribonucleotide reductase R1 hexamerization data.

R is an object oriented free and open source statistical computing environment. The R package Combinatorially Complex Equilibrium Model Selection is being developed to meet the analysis challenges of ribonucleotide reductase (RNR). An example of its use is given here. This example involves ATP-induced R1 hexamerization dynamic light scattering data that suggests that R1 hexamers have two types of a-sites, one that binds ATP and another that does not (here, R1 is the large subunit of RNR).

A combinatorial multicomponent plug mixer for systems chemistry.

We report the construction and testing of a combinatorial multicomponent plug mixer (CMPM) chip that generates a large number of mix ratios. The CMPM chip has been designed to study ribonucleotide reductase (RNR) protein-protein/protein-ligand interaction networks. The 4-component chip is capable of 5400 different combinations in a 30 plug cycle. CMPM chips were tested producing fluorescent dye and dihydrofolate reductase NADPH/MX mixtures with plug lengths of 2 mm.