Impact of additional genetic abnormalities at diagnosis of chronic myeloid leukemia for first-line imatinib-treated patients receiving proactive treatment intervention.

The BCR::ABL1 gene fusion initiates chronic myeloid leukemia (CML); however, evidence has accumulated from studies of highly selected cohorts that variants in other cancer-related genes are associated with treatment failure. Nevertheless, the true incidence and impact of additional genetic abnormalities (AGA) at diagnosis of chronic phase (CP)-CML is unknown. We sought to determine whether AGA at diagnosis in a consecutive imatinib-treated cohort of 210 patients enrolled in the TIDEL-II trial influenced outcome despite a highly proactive treatment intervention strategy. Survival outcomes including overall survival, progression-free survival, failure-free survival, and BCR::ABL1 kinase domain mutation acquisition were evaluated. Molecular outcomes were measured at a central laboratory and included major molecular response (MMR, BCR::ABL1 ≤0.1%IS), MR4 (BCR::ABL1 ≤0.01%IS), and MR4.5 (BCR::ABL1 ≤0.0032%IS). AGA included variants in known cancer genes and novel rearrangements involving the formation of the Philadelphia chromosome. Clinical outcomes and molecular response were assessed based on the patient's genetic profile and other baseline factors. AGA were identified in 31% of patients. Potentially pathogenic variants in cancer-related genes were detected in 16% of patients at diagnosis (including gene fusions and deletions) and structural rearrangements involving the Philadelphia chromosome (Ph-associated rearrangements) were detected in 18%. Multivariable analysis demonstrated that the combined genetic abnormalities plus the EUTOS long-term survival clinical risk score were independent predictors of lower molecular response rates and higher treatment failure. Despite a highly proactive treatment intervention strategy, first-line imatinib-treated patients with AGA had poorer response rates. These data provide evidence for the incorporation of genomically-based risk assessment for CML.

Novel modes of MPL activation in triple-negative myeloproliferative neoplasms.

The identification of a somatic mutation associated with myeloid malignancy is of diagnostic importance in myeloproliferative neoplasms (MPNs). Individuals with no mutation detected in common screening tests for variants in JAK2, CALR, and MPL are described as 'triple-negative' and pose a diagnostic challenge if there is no other evidence of a clonal disorder. To identify potential drivers that might explain the clinical phenotype, we used an extended sequencing panel to characterise a cohort of 44 previously diagnosed triple-negative MPN patients for canonical mutations in JAK2, MPL and CALR at low variant allele frequency (found in 4/44 patients), less common variants in the JAK-STAT signalling pathway (12 patients), or other variants in recurrently mutated genes from myeloid malignancies (18 patients), including hotspot variants of potential clinical relevance in eight patients. In one patient with thrombocytosis we identified biallelic germline MPL variants. Neither MPL variant was activating in cell proliferation assays, and one of the variants was not expressed on the cell surface, yet co-expression of both variants led to thrombopoietin hypersensitivity. Our results highlight the clinical value of extended sequencing including germline variant analysis and illustrate the need for detailed functional assays to determine whether rare variants in JAK2 or MPL are pathogenic.

Hypofractionation with simultaneous integrated boost after breast-conserving surgery: Long term results of two phase-II trials.

PURPOSE: To analyze long-term results of two multicenter prospective single-arm trials (ARO-2010-01 and ARO-2013-04) investigating adjuvant hypofractionated radiotherapy (HF) with simultaneous integrated boost (SIB) after breast-conserving surgery (BCS). METHODS: Eligible patients had histopathologically confirmed unifocal breast cancer planned for whole breast irradiation plus boost radiotherapy to the tumor bed. In both studies, a total dose of 40 Gy was applied to the whole breast and of 48 Gy to the tumor bed in 16 fractions of 2.5 and 3.0 Gy. Radiotherapy could be given either as three-dimensional conformal radiotherapy (3D-CRT) or as intensity-modulated radiotherapy (IMRT). The primary study objectives were feasibility and security within an observation period of six months. The current investigation focuses on long-term efficacy and toxicities. RESULTS: Between 2011 and 2014, both trials enrolled 300 patients in total. Data from 274 of these patients could be used for the current analysis. The median follow-up time was 60 months and the 5-year disease-free survival 92.1%. Three patients suffered a local recurrence (after 36-72 months) while a regional recurrence occurred in one patient (after 17 months). The 5-year local control rate in the breast was 99.6%. 63.5% of all patients did not report any late radiation-related toxicity, 28.5% reported grade 1 and 7.3% grade 2 toxicities. The highest late toxicity was grade 3 in 2 women (0.7%, telangiectasia and lymphedema of the breast). CONCLUSION: Our analysis demonstrates favorable efficacy and low rates of long-term side effects of HF with SIB after BCS. Randomized controlled phase III trials are ongoing.

RNA-Based Targeted Gene Sequencing Improves the Diagnostic Yield of Mutant Detection in Chronic Myeloid Leukemia.

Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mutation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia. Variants included single-nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assessed. A method was developed that targeted 130 genes relevant for myeloid and lymphoid leukemia. Retrospective samples with 121 precharacterized variants were tested using total RNA and/or DNA. Concordance of detection of precharacterized variants using RNA or DNA was 96%, whereas the enhanced sensitivity identified additional variants. Comparison between 24 matched DNA and RNA samples demonstrated 95.3% of 170 variants detectable using DNA were detected using RNA, including all but one variant predicted to activate nonsense-mediated decay. RNA identified an additional 10 variants, including fusion transcripts. Furthermore, the true effect of splice variants on RNA splicing was only evident using RNA. In conclusion, capture sequencing using total RNA alone is suitable for detecting a range of variants relevant in chronic myeloid leukemia and may be more broadly applied to other hematological malignancies where diverse variant types define risk groups.

Dynamic Structural Changes and Thermodynamics in Phase Separation Processes of an Intrinsically Disordered-Ordered Protein Model.

Elastin-like proteins (ELPs) are biologically important proteins and models for intrinsically disordered proteins (IDPs) and dynamic structural transitions associated with coacervates and liquid-liquid phase transitions. However, the conformational status below and above coacervation temperature and its role in the phase separation process is still elusive. Employing matrix least-squares global Boltzmann fitting of the circular dichroism spectra of the ELPs (VPGVG)20 , (VPGVG)40 , and (VPGVG)60 , we found that coacervation occurs sharply when a certain number of repeat units has acquired ß-turn conformation (in our sequence setting a threshold of approx. 20 repeat units). The character of the differential scattering of the coacervate suspensions indicated that this fraction of ß-turn structure is still retained after polypeptide assembly. Such conformational thresholds may also have a role in other protein assembly processes with implications for the design of protein-based smart materials.

Targeted gene panels identify a high frequency of pathogenic germline variants in patients diagnosed with a hematological malignancy and at least one other independent cancer.

The majority of studies assessing the contribution of pathogenic germline variants (PGVs) to cancer predisposition have focused on patients with single cancers. We analyzed 45 known cancer predisposition genes (CPGs) in germline samples of 202 patients with hematological malignancies (HMs) plus one or more other independent cancer managed at major tertiary medical centers on two different continents. This included 120 patients with therapy-related myeloid neoplasms (t-MNs), where the HM occurred after cytotoxic treatment for a first malignancy, and 82 patients with multiple cancers in which the HM was not preceded by cytotoxic therapy (MC-HM). Using American College of Medical Genetics/Association for Molecular Pathology variant classification guidelines, 13% of patients had PGVs, most frequently identified in CHEK2 (17% of PGVs), BRCA1 (13%), DDX41 (13%), and TP53 (7%). The frequency of PGVs in MC-HM was higher than in t-MN, although not statistically significant (18 vs. 9%; p = 0.085). The frequency of PGVs in lymphoid and myeloid HM patients was similar (19 vs. 17.5%; p > 0.9). Critically, patients with PGVs in BRCA1, BRCA2 or TP53 did not satisfy current clinical phenotypic criteria for germline testing. Our data suggest that a personal history of multiple cancers, one being a HM, should trigger screening for PGVs.

Adjuvant hypofractionated radiotherapy with simultaneous integrated boost after breast-conserving surgery: results of a prospective trial.

PURPOSE: We report results of a multicenter prospective single-arm phase II trial (ARO-2013-04, NCT01948726) of moderately accelerated hypofractionated radiotherapy with a simultaneous integrated boost (SIB) in patients with breast cancer receiving adjuvant radiotherapy after breast-conserving surgery. METHODS: The eligibility criteria included unifocal breast cancer with an indication for adjuvant radiotherapy to the whole breast and boost radiotherapy to the tumor bed. The whole breast received a dose of 40 Gy and the tumor bed a total dose of 48 Gy in 16 fractions of 2.5 and 3 Gy, respectively. Radiotherapy could be given either as 3D conformal RT (3D-CRT) or as intensity-modulated radiotherapy (IMRT). The study was designed as a prospective single-arm trial to evaluate the acute toxicity of the treatment regimen. The study hypothesis was that the frequency of acute skin reaction grade ≥2 would be 20% or less. RESULTS: From November 2013 through July 2014, 149 patients were recruited from 12 participating centers. Six patients were excluded, leaving 143 patients for analysis. Eighty-four patients (58.7%) were treated with 3D-CRT and 59 (41.3%) with IMRT. Adherence to the treatment protocol was high. The rate of grade ≥2 skin toxicity was 14.7% (95% confidence interval 9.8-21.4%). The most frequent grade 3 toxicity (11%) was hot flashes. CONCLUSION: This study demonstrated low toxicity of and high treatment adherence to hypofractionated adjuvant radiotherapy with SIB in a multicenter prospective trial, although the primary hypothesis was not met.

RUNX1 mutations in blast-phase chronic myeloid leukemia associate with distinct phenotypes, transcriptional profiles, and drug responses.

Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations, RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1mut BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1mut CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1mut blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1mut patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1-/- and heterozygous RUNX1-/mut BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1mut BP-CML patients.

Impact of guideline changes on adoption of hypofractionation and breast cancer patient characteristics in the randomized controlled HYPOSIB trial.

PURPOSE: Hypofractionated radiotherapy is the standard of care for adjuvant whole breast radiotherapy (RT). However, adoption has been slow. The indication for regional nodal irradiation has been expanded to include patients with 0-3 involved lymph nodes. We investigated the impact of the publication of the updated German S3 guidelines in 2017 on adoption of hypofractionation and enrollment of patients with lymph node involvement within a randomized controlled phase III trial. METHODS: In the experimental arm of the HYPOSIB trial (NCT02474641), hypofractionated RT with simultaneous integrated boost (SIB) was used. In the standard arm, RT could be given as hypofractionated RT with sequential boost (HFseq), normofractionated RT with sequential boost (NFseq), or normofractionated RT with SIB (NFSIB). The cutoff date for the updated German S3 guidelines was December 17, 2017. Temporal trends were analyzed by generalized linear regression models. Multiple logistic regression models were used to investigate the influence of time (prior to/after guideline) and setting (university hospital/other institutions) on the fractionation patterns. RESULTS: Enrollment of patients with involved lymph nodes was low throughout the trial. Adoption of HFseq increased over time and when using the guideline publication date as cutoff. Results of the multiple logistic regressions showed an interaction between time and setting. Furthermore, the use of HFseq was significantly more common in university hospitals. CONCLUSION: The use of HFseq in the standard arm increased over the course of the HYPOSIB trial and after publication of the S3 guideline update. This was primarily driven by patients treated in university hospitals. Enrolment of patients with lymph node involvement was low throughout the trial.

Anisotropic 3D Multi-Stream CNN for Accurate Prostate Segmentation from Multi-Planar MRI.

BACKGROUND AND OBJECTIVE: Accurate and reliable segmentation of the prostate gland in MR images can support the clinical assessment of prostate cancer, as well as the planning and monitoring of focal and loco-regional therapeutic interventions. Despite the availability of multi-planar MR scans due to standardized protocols, the majority of segmentation approaches presented in the literature consider the axial scans only. In this work, we investigate whether a neural network processing anisotropic multi-planar images could work in the context of a semantic segmentation task, and if so, how this additional information would improve the segmentation quality. METHODS: We propose an anisotropic 3D multi-stream CNN architecture, which processes additional scan directions to produce a high-resolution isotropic prostate segmentation. We investigate two variants of our architecture, which work on two (dual-plane) and three (triple-plane) image orientations, respectively. The influence of additional information used by these models is evaluated by comparing them with a single-plane baseline processing only axial images. To realize a fair comparison, we employ a hyperparameter optimization strategy to select optimal configurations for the individual approaches. RESULTS: Training and evaluation on two datasets spanning multiple sites show statistical significant improvement over the plain axial segmentation (p<0.05 on the Dice similarity coefficient). The improvement can be observed especially at the base (0.898 single-plane vs. 0.906 triple-plane) and apex (0.888 single-plane vs. 0.901 dual-plane). CONCLUSION: This study indicates that models employing two or three scan directions are superior to plain axial segmentation. The knowledge of precise boundaries of the prostate is crucial for the conservation of risk structures. Thus, the proposed models have the potential to improve the outcome of prostate cancer diagnosis and therapies.

Aberrant Splicing of SDHC in Families With Unexplained Succinate Dehydrogenase-Deficient Paragangliomas.

CONTEXT: Germline mutations in the succinate dehydrogenase genes (SDHA/B/C/D, SDHAF2-collectively, "SDHx") have been implicated in paraganglioma (PGL), renal cell carcinoma (RCC), gastrointestinal stromal tumor (GIST), and pituitary adenoma (PA). Negative SDHB tumor staining is indicative of SDH-deficient tumors, usually reflecting an underlying germline SDHx mutation. However, approximately 20% of individuals with SDH-deficient tumors lack an identifiable germline SDHx mutation. METHODS: We performed whole-exome sequencing (WES) of germline and tumor DNA followed by Sanger sequencing validation, transcriptome analysis, metabolomic studies, and haplotype analysis in 2 Italian-Australian families with SDH-deficient PGLs and various neoplasms, including RCC, GIST, and PA. RESULTS: Germline WES revealed a novel SDHC intronic variant, which had been missed during previous routine testing, in 4 affected siblings of the index family. Transcriptome analysis demonstrated aberrant SDHC splicing, with the retained intronic segment introducing a premature stop codon. WES of available tumors in this family showed chromosome 1 deletion with loss of wild-type SDHC in a PGL and a somatic gain-of-function KIT mutation in a GIST. The SDHC intronic variant identified was subsequently detected in the second family, with haplotype analysis indicating a founder effect. CONCLUSIONS: This is the deepest intronic variant to be reported among the SDHx genes. Intronic variants beyond the limits of standard gene sequencing analysis should be considered in patients with SDH-deficient tumors but negative genetic test results.

Comprehensive Analysis of Tumour Sub-Volumes for Radiomic Risk Modelling in Locally Advanced HNSCC.

Imaging features for radiomic analyses are commonly calculated from the entire gross tumour volume (GTV entire). However, tumours are biologically complex and the consideration of different tumour regions in radiomic models may lead to an improved outcome prediction. Therefore, we investigated the prognostic value of radiomic analyses based on different tumour sub-volumes using computed tomography imaging of patients with locally advanced head and neck squamous cell carcinoma. The GTV entire was cropped by different margins to define the rim and the corresponding core sub-volumes of the tumour. Subsequently, the best performing tumour rim sub-volume was extended into surrounding tissue with different margins. Radiomic risk models were developed and validated using a retrospective cohort consisting of 291 patients in one of the six Partner Sites of the German Cancer Consortium Radiation Oncology Group treated between 2005 and 2013. The validation concordance index (C-index) averaged over all applied learning algorithms and feature selection methods using the GTVentire achieved a moderate prognostic performance for loco-regional tumour control (C-index: 0.61 ± 0.04 (mean ± std)). The models based on the 5 mm tumour rim and on the 3 mm extended rim sub-volume showed higher median performances (C-index: 0.65 ± 0.02 and 0.64 ± 0.05, respectively), while models based on the corresponding tumour core volumes performed less (C-index: 0.59 ± 0.01). The difference in C-index between the 5 mm tumour rim and the corresponding core volume showed a statistical trend (p = 0.10). After additional prospective validation, the consideration of tumour sub-volumes may be a promising way to improve prognostic radiomic risk models.

Hypofractionated Versus Standard Fractionated Radiotherapy in Patients With Early Breast Cancer or Ductal Carcinoma In Situ in a Randomized Phase III Trial: The DBCG HYPO Trial.

PURPOSE: Given the poor results using hypofractionated radiotherapy for early breast cancer, a dose of 50 Gy in 25 fractions (fr) has been the standard regimen used by the Danish Breast Cancer Group (DBCG) since 1982. Results from more recent trials have stimulated a renewed interest in hypofractionation, and the noninferiority DBCG HYPO trial (ClincalTrials.gov identifier: NCT00909818) was designed to determine whether a dose of 40 Gy in 15 fr does not increase the occurrence of breast induration at 3 years compared with a dose of 50 Gy in 25 fr. PATIENTS AND METHODS: One thousand eight hundred eighty-two patients > 40 years of age who underwent breast-conserving surgery for node-negative breast cancer or ductal carcinoma in situ (DCIS) were randomly assigned to radiotherapy at a dose of either 50 Gy in 25 fr or 40 Gy in 15 fr. The primary end point was 3-year grade 2-3 breast induration assuming noninferiority regarding locoregional recurrence. RESULTS: A total of 1,854 consenting patients (50 Gy, n = 937; 40 Gy, n = 917) were enrolled from 2009-2014 from eight centers. There were 1,608 patients with adenocarcinoma and 246 patients with DCIS. The 3-year rates of induration were 11.8% (95% CI, 9.7% to 14.1%) in the 50-Gy group and 9.0% (95% CI, 7.2% to 11.1%) in the 40-Gy group (risk difference, -2.7%; 95% CI, -5.6% to 0.2%; P = .07). Systemic therapies and radiotherapy boost did not increase the risk of induration. Telangiectasia, dyspigmentation, scar appearance, edema, and pain were detected at low rates, and cosmetic outcome and patient satisfaction with breast appearance were high with either no difference or better outcome in the 40-Gy cohort compared with the 50-Gy cohort. The 9-year risk of locoregional recurrence was 3.3% (95% CI, 2.0% to 5.0%) in the 50-Gy group and 3.0% (95% CI, 1.9% to 4.5%) in the 40-Gy group (risk difference, -0.3%; 95% CI, -2.3% to 1.7%). The 9-year overall survival was 93.4% (95% CI, 91.1% to 95.1%) in the 50-Gy group and 93.4% (95% CI, 91.0% to 95.2%) in the 40-Gy group. The occurrence of radiation-associated cardiac and lung disease was rare and not influenced by the fractionation regimen. CONCLUSION: Moderately hypofractionated breast irradiation of node-negative breast cancer or DCIS did not result in more breast induration compared with standard fractionated therapy. Other normal tissue effects were minimal, with similar or less frequent rates in the 40-Gy group. The 9-year locoregional recurrence risk was low.

2D and 3D convolutional neural networks for outcome modelling of locally advanced head and neck squamous cell carcinoma.

For treatment individualisation of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with primary radiochemotherapy, we explored the capabilities of different deep learning approaches for predicting loco-regional tumour control (LRC) from treatment-planning computed tomography images. Based on multicentre cohorts for exploration (206 patients) and independent validation (85 patients), multiple deep learning strategies including training of 3D- and 2D-convolutional neural networks (CNN) from scratch, transfer learning and extraction of deep autoencoder features were assessed and compared to a clinical model. Analyses were based on Cox proportional hazards regression and model performances were assessed by the concordance index (C-index) and the model's ability to stratify patients based on predicted hazards of LRC. Among all models, an ensemble of 3D-CNNs achieved the best performance (C-index 0.31) with a significant association to LRC on the independent validation cohort. It performed better than the clinical model including the tumour volume (C-index 0.39). Significant differences in LRC were observed between patient groups at low or high risk of tumour recurrence as predicted by the model ([Formula: see text]). This 3D-CNN ensemble will be further evaluated in a currently ongoing prospective validation study once follow-up is complete.

A novel single-cell based method for breast cancer prognosis.

Breast cancer prognosis is challenging due to the heterogeneity of the disease. Various computational methods using bulk RNA-seq data have been proposed for breast cancer prognosis. However, these methods suffer from limited performances or ambiguous biological relevance, as a result of the neglect of intra-tumor heterogeneity. Recently, single cell RNA-sequencing (scRNA-seq) has emerged for studying tumor heterogeneity at cellular levels. In this paper, we propose a novel method, scPrognosis, to improve breast cancer prognosis with scRNA-seq data. scPrognosis uses the scRNA-seq data of the biological process Epithelial-to-Mesenchymal Transition (EMT). It firstly infers the EMT pseudotime and a dynamic gene co-expression network, then uses an integrative model to select genes important in EMT based on their expression variation and differentiation in different stages of EMT, and their roles in the dynamic gene co-expression network. To validate and apply the selected signatures to breast cancer prognosis, we use them as the features to build a prediction model with bulk RNA-seq data. The experimental results show that scPrognosis outperforms other benchmark breast cancer prognosis methods that use bulk RNA-seq data. Moreover, the dynamic changes in the expression of the selected signature genes in EMT may provide clues to the link between EMT and clinical outcomes of breast cancer. scPrognosis will also be useful when applied to scRNA-seq datasets of different biological processes other than EMT.

Correction to: Long-term results achieved by guideline-based stage-dependent management of anal cancer in a non-HIV population.

The authors of the article mentioned above found out errors on the Table 4 concerning the number and survival rates of patients with incomplete radiotherapy.

RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML.

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.