Comprehensive deep learning-based framework for automatic organs-at-risk segmentation in head-and-neck and pelvis for MR-guided radiation therapy planning

Introduction: The excellent soft-tissue contrast of magnetic resonance imaging (MRI) is appealing for delineation of organs-at-risk (OARs) as it is required for radiation therapy planning (RTP). In the last decade there has been an increasing interest in using deep-learning (DL) techniques to shorten the labor-intensive manual work and increase reproducibility. This paper focuses on the automatic segmentation of 27 head-and-neck and 10 male pelvis OARs with deep-learning methods based on T2-weighted MR images.Method: The proposed method uses 2D U-Nets for localization and 3D U-Net for segmentation of the various structures. The models were trained using public and private datasets and evaluated on private datasets only.Results and discussion: Evaluation with ground-truth contours demonstrated that the proposed method can accurately segment the majority of OARs and indicated similar or superior performance to state-of-the-art models. Furthermore, the auto-contours were visually rated by clinicians using Likert score and on average, 81% of them was found clinically acceptable

ELMO1 Is Upregulated in AML CD34+ Stem/Progenitor Cells, Mediates Chemotaxis and Predicts Poor Prognosis in Normal Karyotype AML

Both normal as well leukemic hematopoietic stem cells critically depend on their microenvironment in the bone marrow for processes such as self-renewal, survival and differentiation, although the exact pathways that are involved remain poorly understood. We performed transcriptome analysis on primitive CD34+ acute myeloid leukemia (AML) cells (n = 46), their more differentiated CD34- leukemic progeny, and normal CD34+ bone marrow cells (n = 31) and focused on differentially expressed genes involved in adhesion and migration. Thus, Engulfment and Motility protein 1 (ELMO1) was identified amongst the top 50 most differentially expressed genes. ELMO1 is a crucial link in the signaling cascade that leads to activation of RAC GTPases and cytoskeleton rearrangements. We confirmed increased ELMO1 expression at the mRNA and protein level in a panel of AML samples and showed that high ELMO1 expression is an independent negative prognostic factor in normal karyotype (NK) AML in three large independent patient cohorts. Downmodulation of ELMO1 in human CB CD34+ cells did not significantly alter expansion, progenitor frequency or differentiation in stromal co-cultures, but did result in a decreased frequency of stem cells in LTC-IC assays. In BCR-ABL-transduced human CB CD34+ cells depletion of ELMO1 resulted in a mild decrease in proliferation, but replating capacity of progenitors was severely impaired. Downregulation of ELMO1 in a panel of primary CD34+ AML cells also resulted in reduced long-term growth in stromal co-cultures in two out of three cases. Pharmacological inhibition of the ELMO1 downstream target RAC resulted in a severely impaired proliferation and survival of leukemic cells. Finally, ELMO1 depletion caused a marked decrease in SDF1-induced chemotaxis of leukemic cells. Taken together, these data show that inhibiting the ELMO1-RAC axis might be an alternative way to target leukemic cells

Depletion of SAM50 Specifically Targets BCR-ABL-Expressing Leukemic Stem and Progenitor Cells by Interfering with Mitochondrial Functions

A high proliferation rate of malignant cells requires an increased energy production, both by anaerobic glucose metabolism and mitochondrial respiration. Moreover, increased levels of mitochondria-produced reactive oxygen species (ROS) promote survival of transformed cells and contribute to the disease progression both in solid tumors and leukemia. Consequently, interfering with mitochondrial metabolism has been used as a strategy to specifically target leukemic cells. SAM50 is a mitochondrial outer membrane protein involved in the formation of mitochondrial intermembrane space bridging (MIB) complex. Although the importance of SAM50 in maintaining MIB integrity and in the assembly of mitochondrial respiratory chain complexes has been described, its specific role in the normal and leukemic hematopoietic cells remains unknown. We observed that human leukemic cells display a specific dependency on SAM50 expression, as downregulation of SAM50 in BCR-ABL-expressing, but not normal CD34(+) human hematopoietic stem and progenitor cells (HSPCs) caused a significant decrease in growth, colony formation, and replating capacity. Mitochondrial functions of BCR-ABL-expressing HSPCs were compromised, as seen by a decreased mitochondrial membrane potential and respiration. This effect of SAM50 downregulation was recapitulated in normal HSPCs exposed to cytokine-rich culture conditions that stimulate proliferation. Both oncogene-transduced and cytokine-stimulated HSPCs showed increased mitochondrial membrane potential and increased ROS levels compared to their normal counterparts. Therefore, we postulate that human leukemic HSPCs are highly dependent on the proper functioning of mitochondria and that disruption of mitochondrial integrity may aid in targeting leukemic cells

Global inhibition of RAC activity severely impairs proliferation and survival of leukemic cells while ELMO1 depletion strongly affects migration.

<p>(A) CB CD34⁺ cells were transduced with BCR-ABL-expressing vector, sorted and plated on MS5 stroma. Cells were allowed to proliferate for 5 days after which RAC inhibitor was added to the following concentrations: 20 µM, 40 µM or 100 µM. Co-cultures were demi-depopulated on indicated days for analysis. Cumulative cell count is shown representative of 3 independent experiments. (B) CD34⁺ cells were sorted from BC CML patient sample and plated on MS5 stroma. Cells were allowed to proliferate for 5 days after which RAC inhibitor was added to the following concentrations: 20 µM, 40 µM or 100 µM. Co-cultures were demi-depopulated on indicated days for analysis. Cumulative cell count is shown representative of 3 independent experiments. (C) BCR-ABL transduced cells as described in (A) were treated with 50 µM NSC for 3 days after which suspension cells were harvested and stained with Annexin V to assess apoptosis. (D) TF-1 and THP-1 cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1) and migration was evaluated in a transwell assay. The percentage of migrating cells relative to control is shown as an average of 3 independent experiments.</p

Summary of clinical parameters of AML patients used in this study.

<p>Abbreviations: <i>FLT3</i>, fms-related tyrosine kinase 3; ITD, internal tandem duplication; NK, normal karyotype; <i>NPM1</i>, nucleophosmin 1; NPMc⁺, cytoplasmic dislocalization of NPM1; wt, wild type.</p><p>Summary of clinical parameters of AML patients used in this study.</p

BCR-ABL-transformed human CB CD34⁺ show proliferative disadvantage and markedly reduced replating capacity upon ELMO1 depletion.

<p>(A) CB CD34⁺ stem/progenitor cells were double-transduced with BCR-ABL and either control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1). FACS plots of transduction efficiency are shown. (B) 3×10⁵ double-transduced cells per group were plated in liquid culture and followed for 35 day. Cumulative cell count is shown representative of 3 independent experiments. (C) 5×10³ double-transduced cells were sorted per group and plated on MS5 stromal cells; cultures were demi-depopulated on indicated days for analysis and replated when stroma showed signs of detaching. Cumulative cell growth is shown for a representative experiment of 3 independent experiments and the average of those 3 experiments is shown in (D). (E) Suspension cells from MS5 co-cultures as described in panel B were analyzed for progenitor frequency by CFC assay. 10³ freshly transduced cells or 10⁴ cells from each co-culture were plated in a CFC assay in methylcellulose in duplicate, and colonies were evaluated 2 weeks after plating. CFC cells were harvested and 10⁵ cells were replated to assess secondary CFC formation. Total CFC numbers are shown from a representative of 3 independent experiments; error bars indicate standard deviation. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

ELMO1 expression is increased in AML CD34⁺ cells and predicts poor prognosis in normal karyotype AML patients.

<p>(A) VENN diagram displaying the overlap in AML CD34⁺ specific and AML CD34− specific transcriptomes compared to NBM CD34⁺ cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111568#pone.0111568-Bonardi1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111568#pone.0111568-deJonge1" target="_blank">[18]</a>. (B) 1677 AML CD34⁺-specific genes were subjected to Gene Ontology (GO) analysis for Cellular Component (CC) of which several CCs are shown. (C) 1677 AML CD34⁺-specific genes were subjected to GO analysis for terms associated with adhesion and migration. (D) ELMO1 mRNA was significantly higher expressed in AML CD34⁺ (n = 46) as compared to AML CD34⁻ cells (<i>p</i><0.0001), as well as to NBM CD34⁺ cells (n = 31) (<i>p</i><0.0001). (E) Increased expression of ELMO1 was further confirmed by independent Q-PCRs in a panel of 11 AML and 6 NBM samples. (F, G) The increase in ELMO1 mRNA was paralleled by increased protein levels in two representative cases. (H) High expression of ELMO1 predicts poor survival in a cohort of NK AML patients (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111568#pone.0111568-Valk1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111568#pone.0111568-deJonge2" target="_blank">[29]</a>) (p = 0.0034).</p

ELMO1 depletion in primary AML CD34⁺ cells impairs long-term expansion on stroma.

<p>AML CD34⁺ cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1). After washing away the virus all the cells were plated on MS5 stroma; cultures were demi-depopulated on indicated days for analysis. Cumulative cell growth is shown for each AML sample studied.</p