Relating pre-treatment non-Gaussian intravoxel incoherent motion diffusion-weighted imaging to human papillomavirus status and response in oropharyngeal carcinoma.

Background and purpose: Diffusion-weighted imaging (DWI) is a promising technique for response assessment in head-and-neck cancer. Recently, we optimized Non-Gaussian Intravoxel Incoherent Motion Imaging (NG-IVIM), an extension of the conventional apparent diffusion coefficient (ADC) model, for the head and neck. In the current study, we describe the first application in a group of patients with human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma. The aim of this study was to relate ADC and NG-IVIM DWI parameters to HPV status and clinical treatment response. Materials and methods: Thirty-six patients (18 HPV-positive, 18 HPV-negative) were prospectively included. Presence of progressive disease was scored within one year. The mean pre-treatment ADC and NG-IVIM parameters in the gross tumor volume were compared between HPV-positive and HPV-negative patients. In HPV-negative patients, ADC and NG-IVIM parameters were compared between patients with and without progressive disease. Results: ADC, the NG-IVIM diffusion coefficient D, and perfusion fraction f were significantly higher, while pseudo-diffusion coefficient D* and kurtosis K were significantly lower in the HPV-negative compared to HPV-positive patients. In the HPV-negative group, a significantly lower D was found for patients with progressive disease compared to complete responders. No relation with ADC was observed. Conclusion: The results of our single-center study suggest that ADC is related to HPV status, but not an independent response predictor. The NG-IVIM parameter D, however, was independently associated to response in the HPV-negative group. Noteworthy in the opposite direction as previously thought based on ADC.

Accounting for fractionation and heterogeneous dose distributions in the modelling of osteoradionecrosis in oropharyngeal carcinoma treatment.

BACKGROUND AND PURPOSE: Osteoradionecrosis (ORN) of the mandible is a severe complication following radiotherapy (RT). With a renewed interest in hypofractionation for head and neck radiotherapy, more information concerning ORN development after high fraction doses is important. The aim of this explorative study was to develop a model for ORN risk prediction applicable across different fractionation schemes using Equivalent Uniform Doses (EUD). MATERIAL AND METHODS: We performed a retrospective cohort study in 334 oropharyngeal squamous cell carcinoma (OPSCC) patients treated with either a hypofractionated Stereotactic Body Radiation Therapy (HF-SBRT) boost or conventional Intensity Modulated Radiation Therapy (IMRT). ORN was scored with the CTCAE v5.0. HF-SBRT and IMRT dose distributions were converted into equivalent dose in 2 Gy fractions (α/ß = 0.85 Gy) and analyzed using EUD. The parameter a that led to an EUD that best discriminated patients with and without grade ≥ 2 ORN was selected. Patient and treatment-related risk factors of ORN were analyzed with uni- and multivariable regression analysis. RESULTS: A total of 32 patients (9.6%) developed ORN grade ≥ 2. An EUD(a = 8) best discriminated between ORN and non-ORN (AUC = 0.71). In multivariable regression, pre-RT extractions (SHR = 2.34; p = 0.012), mandibular volume (SHR = 1.04; p = 0.003), and the EUD(a = 8) (SHR = 1.14; p < 0.001) were significantly associated with ORN. CONCLUSION: Risk models for ORN based on conventional DVH parameters cannot be directly applied to HF-SBRT fractionation schemes and dose distributions. However, after correcting for fractionation and non-uniform dose distributions using EUD, a single model can distinguish between ORN and non-ORN after conventionally fractionated radiotherapy and hypofractionated boost treatments.

Ex Vivo Functional Assay for Evaluating Treatment Response in Tumor Tissue of Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) displays a large heterogeneity in treatment response, and consequently in patient prognosis. Despite extensive efforts, no clinically validated model is available to predict tumor response. Here we describe a functional test for predicting tumor response to radiation and chemotherapy on the level of the individual patient. METHODS: Resection material of 17 primary HNSCC patients was cultured ex vivo, irradiated or cisplatin-treated, after which the effect on tumor cell vitality was analyzed several days after treatment. RESULTS: Ionizing radiation (IR) affected tumor cell growth and viability with a clear dose-response relationship, and marked heterogeneity between tumors was observed. After a single dose of 5Gy, proliferation in IR-sensitive tumors dropped below 30% of the untreated level, while IR-resistant tumors maintained at least 60% of proliferation. IR-sensitive tumors showed on average a twofold increase in apoptosis, as well as an increased number and size of DNA damage foci after treatment. No differences in the homologous recombination (HR) proficiency between IR-sensitive and -resistant tumors were detected. Cisplatin caused a decrease in proliferation, as well as induction of apoptosis, again with marked variation between the samples. CONCLUSIONS: Our functional ex vivo assay discriminated between IR-sensitive and IR-resistant HNSCC tumors, and may also be suitable for predicting response to cisplatin. Its predictive value is currently under investigation in a prospective clinical study.

Magnetic Resonance Imaging-Based Delineation of Organs at Risk in the Head and Neck Region.

Purpose: The aim of this article is to establish a comprehensive contouring guideline for treatment planning using only magnetic resonance images through an up-to-date set of organs at risk (OARs), recommended organ boundaries, and relevant suggestions for the magnetic resonance imaging (MRI)-based delineation of OARs in the head and neck (H&N) region. Methods and Materials: After a detailed review of the literature, MRI data were collected from the H&N region of healthy volunteers. OARs were delineated in the axial, coronal, and sagittal planes on T2-weighted sequences. Every contour defined was revised by 4 radiation oncologists and subsequently by 2 independent senior experts (H&N radiation oncologist and radiologist). After revision, the final structures were presented to the consortium partners. Results: A definitive consensus was reached after multi-institutional review. On that basis, we provided a detailed anatomic and functional description and specific MRI characteristics of the OARs. Conclusions: In the era of precision radiation therapy, the need for well-built, straightforward contouring guidelines is on the rise. Precise, uniform, delineation-based, automated OAR segmentation on MRI may lead to increased accuracy in terms of organ boundaries and analysis of dose-dependent sequelae for an adequate definition of normal tissue complication probability.

Aggressive unifocal bone Langerhans cell histiocytosis with soft tissue extension both responsive to radiotherapy: a case report.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare haematological neoplasm characterized by the accumulation of CD1a+, CD207/Langerin+ histiocytes within inflammatory lesions. LCH can involve any organ, but osteolytic bone lesions are most often encountered. Unifocal bone lesions may regress spontaneously after a thick needle biopsy has been taken. CASE PRESENTATION: In this case report, we describe the initial presentation of a single BRAFV600E mutated osteolytic LCH lesion in the left proximal humerus of a 46-year-old previously healthy woman. Despite multiple surgical interventions, she unexpectedly experienced progressive disease manifestation with significant soft tissue extension to the surrounding musculature, subcutis and epidermis. Because the disease manifestation remained loco-regional, radiotherapy (RT) (total dose of 20 Gy in 10 fractions) was initiated. CONCLUSION: The patient achieved a complete remission without any side effects. This case highlights that RT is a rational and relative mild local treatment option for patients with aggressive LCH affecting the bone and surrounding soft tissue.

The COMPLETE trial: HolistiC early respOnse assessMent for oroPharyngeaL cancEr paTiEnts; Protocol for an observational study.

INTRODUCTION: The locoregional failure (LRF) rate in human papilloma virus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) remains disappointingly high and toxicity is substantial. Response prediction prior to or early during treatment would provide opportunities for personalised treatment. Currently, there are no accurate predictive models available for correct OPSCC patient selection. Apparently, the pivotal driving forces that determine how a OPSCC responds to treatment, have yet to be elucidated. Therefore, the holistiC early respOnse assessMent for oroPharyngeaL cancer paTiEnts study focuses on a holistic approach to gain insight in novel potential prognostic biomarkers, acquired before and early during treatment, to predict response to treatment in HPV-negative patients with OPSCC. METHODS AND ANALYSIS: This single-centre prospective observational study investigates 60 HPV-negative patients with OPSCC scheduled for primary radiotherapy (RT) with cisplatin or cetuximab, according to current clinical practice. A holistic approach will be used that aims to map the macroscopic (with Intra Voxel Incoherent Motion Diffusion Kurtosis Imaging (IVIM-DKI); before, during, and 3 months after RT), microscopic (with biopsies of the primary tumour acquired before treatment and irradiated ex vivo to assess radiosensitivity), and molecular landscape (with circulating tumour DNA (ctDNA) analysed before, during and 3 months after treatment). The main end point is locoregional control (LRC) 2 years after treatment. The primary objective is to determine whether a relative change in the mean of the diffusion coefficient D (an IVIM-DKI parameter) in the primary tumour early during treatment, improves the performance of a predictive model consisting of tumour volume only, for 2 years LRC after treatment. The secondary objectives investigate the potential of other IVIM-DKI parameters, ex vivo sensitivity characteristics, ctDNA, and combinations thereof as potential novel prognostic markers. ETHICS AND DISSEMINATION: The study was approved by the Medical Ethical Committee of Erasmus Medical Center. The main results of the trial will be presented in international meetings and medical journals. TRIAL REGISTRATION NUMBER: NL8458.

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.

Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2.

Leukemic stem cells (LSCs) reside within bone marrow niches that maintain their relatively quiescent state and convey resistance to conventional treatment. Many of the microenvironmental signals converge on RAC GTPases. Although it has become clear that RAC proteins fulfill important roles in the hematopoietic compartment, little has been revealed about the downstream effectors and molecular mechanisms. We observed that in BCR-ABL-transduced human hematopoietic stem/progenitor cells (HSPCs) depletion of RAC2 but not RAC1 induced a marked and immediate decrease in proliferation, progenitor frequency, cobblestone formation and replating capacity, indicative for reduced self-renewal. Cell cycle analyses showed reduced cell cycle activity in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Moreover, a decrease in mitochondrial membrane potential was observed upon RAC2 downregulation, paralleled by severe mitochondrial ultrastructural malformations as determined by automated electron microscopy. Proteome analysis revealed that RAC2 specifically interacted with a set of mitochondrial proteins including mitochondrial transport proteins SAM50 and Metaxin 1, and interactions were confirmed in independent co-immunoprecipitation studies. Downregulation of SAM50 also impaired the proliferation and replating capacity of BCR-ABL-expressing cells, again associated with a decreased mitochondrial membrane potential. Taken together, these data suggest an important role for RAC2 in maintaining mitochondrial integrity.

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.