Substantial Volume Changes and Plan Adaptations During Preoperative Radiation Therapy in Extremity Soft Tissue Sarcoma Patients

Purpose

Many authors suggest that extremity soft tissue sarcomas (ESTS) do not change significantly in size during preoperative radiation therapy (RT). This cone beam computed tomography study investigates the justification to deliver the entire course with 1 initial RT plan by observing anatomic changes during RT.

Sialendoscopy plus laser lithotripsy in sialolithiasis of the submandibular gland in 64 patients: A simple and safe procedure

ObjectiveTo demonstrate the safety and efficiency of holmium laser-assisted lithotripsy during sialendoscopy of the submandibular gland using a retrospective, interventional consecutive case series.MethodsWe performed 374 sialendoscopies between 2008 and 2015 and evaluated all patients regarding clinical symptoms, clinical findings, therapy and outcome. We performed 109 procedures of holmium laser-assisted lithotripsy in 64 patients whose sialoliths measured 5 mm or more in diameter. In addition to retrospective case note reviews, we performed telephone interviews of all patients in January 2017.ResultsWe performed 374 consecutive submandibular gland sialendoscopy procedures in 276 patients between 2008 to 2015. Sialolithiasis had either previously been diagnosed, or symptoms highly suggestive of sialolithiasis of the submandibular gland presented in 197 patients.Holmium laser-assisted Laser lithotripsy was performed in 109 cases (64.9%). Smaller mobile concrement was removed directly either by forceps or wire basket, or following marsupialisation of the submandibular duct. This was the case in 88 patients (29.1%). Three patients (0.8%) required surgical removal of the submandibular gland due to early abscess. The majority of patients (n = 374 procedures; 90.1%) remained symptom-free after two or more years following intervention. In the remaining procedures (n = 37 procedures; 9.9%), patients reported discreet postprandial problems but did not seek medical attention. In total, we managed to preserve the submandibular gland and avoid open surgery in 99% of patients through endoscopic management of submandibular concrement and duct stenosis.ConclusionHolmium laser-assisted lithotripsy is a simple, safe, and effective procedure for treating patients with sialolithiasis of the submandibular gland. Removal of the gland is rarely required, and removing the gland without prior sialendoscopy is no longer recommended. It should be offered to all patients with submandibular gland sialolithiasis, or such patients should be referred to the appropriate centre for sialendoscopy before submandibulectomy is considered.     

HDAC inhibitors restore BRAF‐inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non‐genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient‐ and in vivo‐derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase‐dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro‐apoptotic BIM or NOXA together with reduced expression of anti‐apoptotic proteins. Independent of these changes, panobinostat reduced c‐Myc and pre‐treatment of cells with siRNA against c‐Myc reduced BRAFi/HDACi drug‐induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK‐independent pathways.     

Heat-induced BRCA2 degradation in human tumours provides rationale for hyperthermia-PARP-inhibitor combination therapies

Purpose: Hyperthermia (40–44?°C) effectively sensitises tumours to radiotherapy by locally altering tumour biology. One of the effects of heat at the cellular level is inhibition of DNA repair by homologous recombination via degradation of the BRCA2-protein. This suggests that hyperthermia can expand the group of patients that benefit from PARP-inhibitors, a drug exploiting homologous recombination deficiency. Here, we explore whether the molecular mechanisms that cause heat-mediated degradation of BRCA2 are conserved in cell lines from various origins and, most importantly, whether, BRCA2 protein levels can be attenuated by heat in freshly biopted human tumours.

Experimental design: Cells from four established cell lines and from freshly biopsied material of cervical (15), head- and neck (9) or bladder tumours (27) were heated to 42?°C for 60?min ex vivo. In vivo hyperthermia was studied by taking two biopsies of the same breast or cervical tumour: one before and one after treatment. BRCA2 protein levels were measured by immunoblotting.

Results: We found decreased BRCA2-levels after hyperthermia in all established cell lines and in 91% of all tumours treated ex vivo. For tumours treated with hyperthermia in vivo, technical issues and intra-tumour heterogeneity prevented obtaining interpretable results.

Conclusions: This study demonstrates that heat-mediated degradation of BRCA2 occurs in tumour material directly derived from patients. Although BRCA2-degradation may not be a practical biomarker for heat deposition in situ, it does suggest that application of hyperthermia could be an effective method to expand the patient group that could benefit from PARP-inhibitors.     

Confocal endomicroscopy in diagnosis of intestinal chronic graft‐versus‐host disease

Graft‐versus‐host disease (GvHD) is a major complication of allogeneic stem cell transplantation. High‐resolution in vivo histology of the intestine by confocal endomicroscopy (CEM) detects acute GvHD (aGvHD) with high sensitivity. This pilot study aims to evaluate the diagnostic value of CEM for intestinal chronic GvHD (cGvHD). The study included 20 patients with gastrointestinal symptoms and confirmed cGvHD in other organs as well as 20 patients with clinically suspected acute GvHD for control. Confocal endomicroscopy was performed as gastroscopy followed by sigmoidoscopy after intravenous injection of fluorescein (10%) and topical application of acriflavine (0.05%). Histopathology from H&E‐stained biopsy samples throughout the intestinal tract complemented the survey. All histological features of intestinal cGvHD were predominantly mild to moderate. Stroma fibrosis detected by standard histology (16/20 patients) was not seen by CEM. Apoptosis assessed by histology in 12/20 patients was concordant with CEM (8/12 patients). Confocal endomicroscopy revealed esophageal manifestation of cGvHD in 3 patients. For each biopsy site, CEM correlated with intestinal histology (r = 0.64). Classical histology from intestinal biopsy samples taken under CEM monitoring confirmed the final diagnosis of cGvHD. The sensitivity of CEM with 40% in cGvHD was significantly lower compared to 70% in patients with aGvHD. Confocal endomicroscopy detected acute features of cGvHD and contributed to the diagnosis of esophageal cGvHD but failed to display stroma fibrosis in vivo. Although CEM represents a useful noninvasive tool in routine diagnostic of intestinal aGvHD, the method is not sufficient to fully establish the diagnosis of cGvHD within the intestinal tract. Confocal endomicroscopy allowed acquisition of targeted biopsies in patients suspected of having cGvHD.     

Functional characterization of the lectin pathway of complement in human serum

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum.Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms.We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.     

Long term iNOS expression in thoracic lymph nodes of silicotic rats

Beside the lung, thoracic lymph nodes are most affected during silicosis. The mechanisms leading to enlargement of the lymph nodes and partial activation of lymph node cells are still unclear. The present study demonstrates an increase in iNOS mRNA expression in the lung draining lymph nodes of rats at 1, 2, and 8 months following silica exposure. Histopathological analysis revealed that iNOS protein was exclusively expressed by macrophages located within the granulomatous areas of the enlarged lymph nodes. In contrast, no differences in mRNA expression and number of iNOS-positive cells were found in the lungs of silica-exposed and non-exposed rats. In vitro experiments showed that silica particles alone did not induce NO release in primary alveolar macrophages (AMs) or the alveolar macrophage cell line NR8383. However, the addition of interferon (IFN)-gamma led to a significant nitric oxide production by primary AMs. NR8383 cells responded only when a combination of IFN-gamma and silica particles was applied. These results indicate that the macrophage activator IFN-gamma, which has already been shown to be expressed at elevated levels by lymphocytes of the silicotic lymph nodes, may be responsible for the long-lasting iNOS expression in thoracic lymph nodes. Our observations support the hypothesis that the mutual activation of lymphocytes and macrophages is a central process in the development of chronic silicosis.