Methodology Development for Three-Dimensional MR-Guided Near Infrared Spectroscopy of Breast Tumors

Combined Magnetic Resonance (MR) and Near Infrared Spectroscopy (NIRS) has been proposed as a unique method to quantify hemodynamics, water content, and cellular size and packing density of breast tumors, as these tissue constituents can be quantified with increased resolution and overlaid on the structural features identified by the MR. However, the choices in how to reconstruct and visualize this information can have a dramatic impact on the feasibility of implementing this modality in the clinic. This is especially true in 3 dimensions, as there is often limited optical sampling of the breast tissue, and methods need to accurately reflect the tissue composition. In this paper, the implementation and display of fully 3D MR image-guided NIRS is outlined and demonstrated using in vivo data from three healthy women and a volunteer undergoing neoadjuvant chemotherapy. Additionally, a display feature presented here scales the transparency of the optical images to the sensitivity of the measurements, providing a logical way to incorporate partial volume sets of optical images onto the MR volume. These concepts are demonstrated with 3D data sets using Volview software online

Image-Guided Raman Spectroscopic Recovery of Canine Cortical Bone Contrast in Situ

Raman scattering provides valuable biochemical and molecular markers for studying bone tissue composition with use in predicting fracture risk in osteoporosis. Raman tomography can image through a few centimeters of tissue but is limited by low spatial resolution. X-ray computed tomography (CT) imaging can provide high-resolution image-guidance of the Raman spectroscopic characterization, which enhances the quantitative recovery of the Raman signals, and this technique provides additional information to standard imaging methods. This hypothesis was tested in data measured from Teflon tissue phantoms and from a canine limb. Image-guided Raman spectroscopy (IG-RS) of the canine limb using CT images of the tissue to guide the recovery recovered a contrast of 145:1 between the cortical bone and background. Considerably less contrast was found without the CT image to guide recovery. This study presents the first known IG-RS results from tissue and indicates that intrinsically high contrasts (on the order of a hundred fold) are available

Real-time PCR-based analysis of BRAF V600E mutation in low and intermediate grade lymphomas confirms frequent occurrence in hairy cell leukaemia

Hairy cell leukaemia (HCL) is a rare type of B-cell non-Hodgkin lymphoma (B-NHL), which is not known to be associated with any characteristic recurrent karyotypic abnormality. A recent study that used massively parallel whole exome sequencing identified an activating V600E mutation in BRAF, which appeared specific for HCL. Here, we confirm the specificity of BRAF V600E for HCL among low and intermediate grade B-NHL and describe a real-time polymerase chain reaction method for detecting this mutation in cases with low tumour burden. The V600E mutation does not appear to be associated with microsatellite instability, unlike the case in colorectal cancer. Thus, in conjunction with prior data, our results suggest incorporation of BRAF V600E mutation analysis in the diagnostic workup of HCL cases. Additionally, targeting the Ras-Raf-Mek-Erk-Map kinase pathway should be investigated as a potential therapeutic strategy for patients with this disease

Cytogenetic abnormalities in reactive lymphoid hyperplasia: byproducts of the germinal centre reaction or indicators of lymphoma?

Non-random karyotypic abnormalities associated with non-Hodgkin lymphomas (NHLs) have been described in cases of reactive lymphoid hyperplasia (RLH). However, the frequency and types of cytogenetic aberrations detected and their clinical relevance are unknown. To address these questions, we undertook a retrospective analysis of a large series of RLH diagnosed at our institute over 8 years. Cytogenetic abnormalities were identified in 20 of 116 (17%) cases with informative karyotypes, comprising 14 (70%) structural and 11 (55%) numerical changes. Clonal (n 1⁄4 14, 70%) and non-clonal (n 1⁄4 6, 30%) abnormalities were observed. Aberrations of chromosome 14 were the most frequent (n1⁄48, 42%, 7 represented IgH translocations), followed by chromosome 3 (n1⁄44, 3 represented BCL6 translocations), and chromosome 12 (n1⁄44). Abnormal karyotypes were most often associated with florid follicular hyperplasia. Isolated lymphoid organ (lymph node, tonsil or spleen) enlargement (12/20, 60%) was more common, no specific etiology was identified in 10/20 (50%) cases and only 1 of 18 patients with clinical follow-up (range 2–107 months, median 60 months) developed lymphoma. In our experience, cytogenetic abnormalities involving loci associated with B-cell NHL are not infrequently detected in RLH. Their occurrence portends low risk for lymphomagenesis, however longer follow-up is prudent to further evaluate the natural history of such cases

Cytogenetic analysis of 130 renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations

The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (49%) cases, which could be categorized into three classes of mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes with characteristic 11q13 rearrangement in balanced translocations with 10 or more different chromosome partners in all cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO (N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases showed diverse types of abnormalities with the involvement of two or more chromosomes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify classes 1 and 2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement, loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mitelman database. Correlation of clinical data with all the three classes of ROs showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit three principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO

Non-Invasive Raman Tomographic Imaging of Canine Bone Tissue

Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images. With recent advances, diffuse tomography shows promise for in vivo clinical imaging.1, 2 In principle, algorithms developed for fluorescence imaging in tissue can be applied to Raman signals. Although the Raman effect is weaker than fluorescence, the scattered signal is detectable, and thus tomography is achievable. Here we demonstrate the first diffuse tomography reconstructions based on Raman scatter. Raman mapping and imaging are well-established techniques for examining material surfaces.3 Subsurface mapping of simple planar objects was reported recently4, 5 using fiber optic probes with spatially separated injection and collection fibers.6 Noninvasive measurements of bone Raman spectra were demonstrated at depths of5mm role= presentation \u3e5mm below the skin.5 Bone is promising for Raman tomography because the spectra are rich in compositional information,7 which reflects bone maturity and health. Spectroscopically measured bone composition changes have been correlated with aging8 and susceptibility to osteoporotic fracture.9 The Raman spectrum of bone mineral is easily distinguished from the spectra of proteins and other organic tissue constituents, facilitating recovery of even weak signals by multivariate techniques. Assessments of bone quantity and quality are essential to detect and monitor fracture risk and fracture healing with disease or injury. Common sites for fracture with osteoporosis are the spine, proximal femur, and distal radius. Stress fractures are most frequently seen in the weight-bearing sites of the tibia and metatarsals. Fracture risk depends on bone geometry, architecture, and material properties, as well as the nature of applied load (magnitude, rate, and direction). As a result, noninvasive imaging and nondestructive analysis methods have been developed to assess many of these bone attributes that are increasingly important to clinical practice and basic research in orthopedics.10 Current clinical in vivo methods include dual-energy x-ray absorptiometry (DXA), quantitative computed tomography (QCT), magnetic resonance imaging (MRI), ultrasound, and most recently, high-resolution peripheral QCT. Ex vivo analyses of bone specimens from patients or animals have also utilized these and other techniques. In this study, we couple micro-computed tomography (micro-CT) and diffuse optical tomography with Raman spectroscopy to recover spatial and composition information from bone tissue ex vivo. We demonstrate the first reconstruction-based recovery of Raman signals through thick tissues to yield molecular information about subsurface bone tissue. Reconstructions from transcutaneous Raman measurements are challenging, because layers of skin, muscle, fat, and connective tissue lie over the bone sites of interest. These layers have different optical properties and thus variably scatter and polarize the injected light. We chose a canine model because of specimen availability and a bone size similar to human bone. We selected the tibia, a site that is clinically important and has relatively few overlying soft tissues. Measurements were made on the medial surface, where the only additional optical barrier is the crural extensor retinaculum ligament. The canine hind limb was harvested from an animal euthanized in an approved (UCUCA) University of Michigan study. The section of the limb distal to the knee was excised and scanned using in vivo micro-CT (eXplore Locus RS, GE Healthcare, Ontario, Canada). The tibia was scanned at80kV role= presentation \u3e80kV and 450μA role= presentation \u3e450μA with an exposure time of 100ms role= presentation \u3e100ms using a 360-deg scan technique. The image was reconstructed at a 93-μm role= presentation \u3e93-μm voxel resolution [Fig. 1a ]

Integrative genomics analysis of chromosome 5p gain in cervical cancer reveals target over-expressed genes, including Drosha

Background: Copy number gains and amplifications are characteristic feature of cervical cancer (CC) genomes for which the underlying mechanisms are unclear. These changes may possess oncogenic properties by deregulating tumor-related genes. Gain of short arm of chromosome 5 (5p) is the most frequent karyotypic change in CC. Methods: To examine the role of 5p gain, we performed a combination of single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and gene expression analyses on invasive cancer and in various stages of CC progression. Results: The SNP and FISH analyses revealed copy number increase (CNI) of 5p in 63% of invasive CC, which arises at later stages of precancerous lesions in CC development. We integrated chromosome 5 genomic copy number and gene expression data to identify key target over expressed genes as a consequence of 5p gain. One of the candidates identified was Drosha (RNASEN), a gene that is required in the first step of microRNA (miRNA) processing in the nucleus. Other 5p genes identified as targets of CNI play a role in DNA repair and cell cycle regulation (BASP1, TARS, PAIP1, BRD9, RAD1, SKP2, and POLS), signal transduction (OSMR), and mitochondrial oxidative phosphorylation (NNT, SDHA, and NDUFS6), suggesting that disruption of pathways involving these genes may contribute to CC progression. Conclusion: Taken together, we demonstrate the power of integrating genomics data with expression data in deciphering tumor-related targets of CNI. Identification of 5p gene targets in CC denotes an important step towards biomarker development and forms a framework for testing as molecular therapeutic targets

Identification of Copy Number Gain and Overexpressed Genes on Chromosome Arm 20q by an Integrative Genomic Approach in Cervical Cancer: Potential Role in Progression

Recurrent karyotypic abnormalities are a characteristic feature of cervical cancer (CC) cells, which may result in deregulated expression of important genes that contribute to tumor initiation and progression. To examine the role of gain of the long arm of chromosome 20 (20q), one of the common chromosomal gains in CC, we evaluated CC at various stages of progression using single nucleotide polymorphism (SNP) array, gene expression profiling, and fluorescence in situ hybridization (FISH) anal- yses. This analysis revealed copy number increase (CNI) of 20q in >50% of invasive CC and identified two focal amplicons at 20q11.2 and 20q13.13 in a subset of tumors. We further demonstrate that the acquisition of 20q gain occurs at an early stage in CC development and the high-grade squamous intraepithelial lesions (HSIL) that exhibit 20q CNI are associated (P 5 0.05) with persistence or progression to invasive cancer. We identified a total of 26 overexpressed genes as consequence of 20q gain (N 5 14), as targets of amplicon 1 (N 5 9; two genes also commonly expressed with 20q gain) and amplicon 2 (N 5 6; one gene also commonly expressed with 20q gain). These include a number of functionally important genes in cell cycle regulation (E2F1, TPX2, KIF3B, PIGT, and B4GALT5), nuclear function (CSEL1), viral replication (PSMA7 and LAMA5), methylation and chromatin remodeling (ASXL1, AHCY, and C20orf20), and transcription regulation (TCEA2). Our findings implicate a role for these genes in CC tumorigenesis, represent an important step toward the development of clinically significant biomarkers, and form a framework for testing as molecular therapeutic targets

Promoter Hypermethylation of FANCF: Disruption of Fanconi Anemia-BRCA Pathway in Cervical Cancer

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methy- lation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hyper- sensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimen- tal therapeutic protocols

Child and Parent Physical Activity, Sleep, and Screen Time During COVID-19 and Associations With Mental Health:Implications for Future Psycho-Cardiological Disease?

The COVID-19 pandemic has afforded the opportunity for some to improve lifestyle behaviours, while for others it has presented key challenges. Adverse changes in global lifestyle behaviours, including physical activity, sleep, and screen time can affect proximal mental health and in turn distal cardiovascular outcomes. We investigated differences in physical activity, sleep, and screen time in parents and children during early stages of the COVID-19 pandemic in Australia compared to pre-COVID-19 national data; and estimated associations between these movement behaviours with parent and child mental health. Cross-sectional baseline data from the COVID-19 Pandemic Adjustment Study (CPAS; N = 2,365) were compared to nationally representative pre-pandemic data from the Longitudinal Study of Australian Children (LSAC; N = 9,438). Participants were parents of children aged ≤ 18 years, residing in Australia. Parents provided self-report measures of mental health, physical activity and sleep quality, and reported on child mental health, physical activity and screen time. Children in CPAS had significantly more sleep problems and more weekend screen time. Their parents had significantly poorer sleep quality, despite increased weekly physical activity. Children's sleep problems were significantly associated with increased mental health problems, after accounting for socioeconomic status, physical activity, and screen time. Poorer parent sleep quality and lower levels of physical activity were significantly associated with poorer mental health. Monitoring this cohort over time will be important to examine whether changes in movement behaviour are enduring or naturally improve with the easing of restrictions; and whether these changes have lasting effects on either parent or child mental health, and in turn, future risk for CVD