Machine learning-based radiomic evaluation of treatment response prediction in glioblastoma.

AIM: To investigate machine learning based models combining clinical, radiomic, and molecular information to distinguish between early true progression (tPD) and pseudoprogression (psPD) in patients with glioblastoma. MATERIALS AND METHODS: A retrospective analysis was undertaken of 76 patients (46 tPD, 30 psPD) with early enhancing disease following chemoradiotherapy for glioblastoma. Outcome was determined on follow-up until 6 months post-chemoradiotherapy. Models comprised clinical characteristics, O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, and 307 quantitative imaging features extracted from enhancing disease and perilesional oedema masks on early post-chemoradiotherapy contrast-enhanced T1-weighted imaging, T2-weighted imaging (T2WI), and apparent diffusion coefficient (ADC) maps. Feature selection was performed within bootstrapped cross-validated recursive feature elimination with a random forest algorithm. Naive Bayes five-fold cross-validation was used to validate the final model. RESULTS: Top selected features included age, MGMT promoter methylation status, two shape-based features from the enhancing disease mask, three radiomic features from the enhancing disease mask on ADC, and one radiomic feature from the perilesional oedema mask on T2WI. The final model had an area under the receiver operating characteristics curve (AUC) of 0.80, sensitivity 78.2%, specificity 66.7%, and accuracy of 73.7%. CONCLUSION: Incorporating a machine learning-based approach using quantitative radiomic features from standard-of-care magnetic resonance imaging (MRI), in combination with clinical characteristics and MGMT promoter methylation status has a complementary effect and improves model performance for early prediction of glioblastoma treatment response.

Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs) are inhibitory extracellular matrix molecules that are upregulated after CNS injury. Degradation of CSPGs using the enzyme chondroitinase ABC (ChABC) can promote functional recovery after spinal cord injury. However, the mechanisms underlying this recovery are not clear. Here we investigated the effects of ChABC treatment on promoting plasticity within the spinal cord. We found robust sprouting of both injured (corticospinal) and intact (serotonergic) descending projections as well as uninjured primary afferents after a cervical dorsal column injury and ChABC treatment. Sprouting fibers were observed in aberrant locations in degenerating white matter proximal to the injury in regions where CSPGs had been degraded. Corticospinal and serotonergic sprouting fibers were also observed in spinal gray matter at and below the level of the lesion, indicating increased innervation in the terminal regions of descending projections important for locomotion. Spinal-injured animals treated with a vehicle solution showed no significant sprouting. Interestingly, ChABC treatment in uninjured animals did not induce sprouting in any system. Thus, both denervation and CSPG degradation were required to promote sprouting within the spinal cord. We also examined potential detrimental effects of ChABC-induced plasticity. However, although primary afferent sprouting was observed after lumbar dorsal column lesions and ChABC treatment, there was no increased connectivity of nociceptive neurons or development of mechanical allodynia or thermal hyperalgesia. Thus, CSPG digestion promotes robust sprouting of spinal projections in degenerating and denervated areas of the spinal cord; compensatory sprouting of descending systems could be a key mechanism underlying functional recovery.

Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells.

PURPOSE: Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice. METHODS: The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography. RESULTS: Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed. CONCLUSIONS: Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.

Altered expression of secreted frizzled-related protein-2 in retinitis pigmentosa retinas.

PURPOSE: Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive death of the photoreceptors due to apoptosis. To identify changes in gene expression associated with the degenerative state in RP retinas, expression profiling of apoptosis-related genes was performed using a gridded array technique. METHODS: Total RNAs from RP and control retinas were used to generate radiolabeled cDNA probes to screen gridded membrane arrays of 205 apoptosis-related genes. Reverse transcription-polymerase chain reaction was used to generate probes corresponding to differentially expressed genes for Northern blot analysis and for mRNA in situ hybridization studies of retinal cryosections. Fluorescence immunocytochemistry was performed on retinal sections using available antibodies. RESULTS: By expression profiling, we identified upregulated expression of the mRNA for secreted Frizzled-related protein-2 (SFRP2) in RP retina in comparison with control. By Northern blot analysis, SFRP2 mRNA levels were 2- to 20-fold higher in RP samples than in controls. The localization of SFRP2 mRNA by in situ hybridization varied according to the degree of degeneration, from stratified in relatively well-preserved retinas to diffuse in the highly degenerative state. By immunofluorescence, SFRP2 protein in RP retinas was found mainly to colocalize with the cell adhesion and signal transducing protein beta-catenin. CONCLUSIONS: SFRPs can regulate apoptosis in vitro and appear to interact with the Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. Increased SFRP2 expression in RP retinas suggests that an altered pattern of Wnt signal transduction may be a step in the degenerative process linking causal mutations with eventual photoreceptor demise.

Modulated expression of secreted frizzled-related proteins in human retinal degeneration.

Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive loss of photoreceptors, apparently by apoptosis, and our recent report of increased secreted Frizzled-related protein-2 (SFRP2) in RP retinas suggests altered Wnt signalling may be a component of the degenerative process. The present study shows that levels of SFRPI, SFRP3 and SFRP5 mRNAs are also abnormal in RP, giving rise to idiosyncratic expression patterns. In highly degenerative retinas, the SFRP proteins localize mainly to the inner limiting membrane, but in a well-preserved retina SFRPI and SFRP5 are notably localized to the surviving photoreceptors. Together with increased c-jun mRNA expression in all cases examined, these results support the notion that disruptions of Wnt network signalling are involved retinal neurodegeneration.

Identification by array screening of altered nm23-M2/PuF mRNA expression in mouse retinal degeneration.

In the rd/rd mouse model of inherited retinal degeneration, the majority of photoreceptors die apoptotically between postnatal age (P)10 and 20 days, during which period the inner retina appears morphologically unaffected. To examine mRNA changes associated with the degeneration, we performed differential screening of 588 arrayed murine cDNAs using probes reverse-transcribed from P8 predegenerative and control mouse retinal RNAs. We detected altered expression of the gene encoding nm23-M2, a member of the family of nucleoside diphosphate kinases implicated in diverse processes including metastasis suppression and transcriptional regulation. Retinal nm23 mRNA levels increased during degeneration while control levels decreased over age-matched time-points. In situ hybridization showed the high level of expression at P20 in rd/rd was concentrated in the retinal ganglion cells. Previous studies have indicated upregulation of the stress-response related gene alphaB-crystallin in the rd/rd inner retina, and increased nm23 levels may be a component of this response to photoreceptor loss and altered retinal architecture.

Retinal expression of gamma-crystallins in the mouse.

PURPOSE: High levels of expression of a form of gamma-crystallin mRNA in mouse retina have been identified. Because the six murine gamma-crystallins have generally been regarded as specific to the lens, the expression of these crystallins at the mRNA and protein levels in the retina were evaluated in more detail. METHODS: Expression of gammaE/F-crystallin mRNA was examined by northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis applied to murine retinal and lens total RNAs. For gammaA-D-crystallin mRNAs, a multiplex RT-PCR was used on total cDNAs. The detection of total gamma-crystallin protein in the retina was performed using an antibody to bovine lens gamma-crystallins, applied to protein extracts in immunoblot analysis and to cryostat sections of ocular tissues in immunofluorescence studies. RESULTS: By RT-PCR, we confirmed expression of both gammaE-and gammaF-crystallin as well as all four (gammaA-gammaD) remaining crystallins at the mRNA level in the mouse retina. Gamma-crystallin proteins were also detectable in murine retina by immunoblot analysis, although at a lower level than in the lens. By immunocytochemistry, gamma-crystallins were localized particularly to the inner retina, outer plexiform layer, and the photoreceptors during postnatal development. CONCLUSIONS: Our findings of gamma-crystallin mRNA and protein expression in the retina indicate that none of the major crystallin classes is uniquely expressed in the lens. The expression of gamma-crystallins in the developing murine retina suggests a role analogous to the anti-stress properties established for the small heat-shock protein alphaB-crystallin, perhaps in response to varying exposure to light.

Expression patterns of neurturin and its receptor components in developing and degenerative mouse retina.

PURPOSE: Neurturin (NTN) and its receptor components (GFRalpha2 and Ret) play an important role in the survival of different populations of neurons in the central and peripheral nervous systems. To gain insight into their possible functions throughout normal retinal development and during retinal neuronal apoptosis, the retinal distribution of expression of NTN and GFRalpha2 mRNAs and Ret protein were compared in control and retinal degeneration (rd) mice. METHODS: Eyes from control and rd animals were fixed in paraformaldehyde before sectioning. For in situ hybridization, retinal sections were hybridized with 35S-radiolabeled sense and antisense riboprobes for murine NTN and GFRalpha2 and were autoradiographed. Ret localization was detected by immunofluorescence. RESULTS: Neurturin mRNA expression was modulated through normal postnatal retinal development and was localized primarily to the inner retina and photoreceptor outer segments. GFRalpha2 mRNA displayed a diffuse developmental pattern of expression, but in the mature normal retina, NTN and GFRalpha2 mRNAs were more closely colocalized. Ret protein was localized particularly at the outer segments of photoreceptors, inner retina, and ganglion cell layers, but there were no prominent differences among genotypes. Increased NTN mRNA expression was detected in the retinal pigment epithelium and neural retina in concert with photoreceptor degeneration in rd mouse. In contrast, the level of GFRalpha2 mRNA was lower in rd compared with that in normal retina. CONCLUSIONS: These results suggest that NTN and its receptor are involved in retinal postnatal development and maintenance and that alterations in their transcription patterns are associated with inherited retinal degeneration.

Expression of Pax-6 mRNA in the retinal degeneration (rd) mouse.

The homeobox gene Pax-6 is expressed during eye development in both the retina and lens, and Pax-6 mutations cause ocular abnormalities including retinal defects. We investigated the pattern of Pax-6 gene expression in the rd/rd mouse model of inherited retinal degeneration in comparison with nondegenerative controls, using Northern blot, reverse-transcription (RT)-PCR and in situ hybridization analysis. We observed an increased level of Pax-6 mRNA expression in the degenerative state, which appeared to affect equally the major Pax-6 exon 5a transcriptional splice variants as detected by RT-PCR. By in situ hybridization, Pax-6 mRNA was localized to the inner nuclear and ganglion cell layers of nondegenerative retina, but showed a more diffuse signal pattern in the rd/rd retina. This modulation of Pax-6 mRNA levels and localization is suggestive of activation of expression in retinal glial cells and may reflect reorganization of cellular interactions in response to the degenerative processes.

Expression of alphaB-crystallin in a mouse model of inherited retinal degeneration.

AlphaB-crystallin, which is abundantly expressed in the lens but also in a diversity of other tissues, functions as a stress-inducible molecular chaperone and is increased in brain neurodegenerative diseases. We compared retinal alphaB-crystallin expression in a model of inherited retinal degeneration, the rd mouse, and controls. Northern and in situ hybridization analysis showed alphaB-crystallin mRNA to have an altered spatio-temporal pattern with increased levels localized to glial cells in the degenerative state. Immunocytochemistry confirmed increased expression at Müller cells and astrocytes, together with transiently increased localization to the degenerating photoreceptors. These findings suggest that increased alphaB-crystallin expression is associated with glial cell reaction to neuronal damage in the retina, and may comprise part of the retina's overall defensive response to the stress of apoptotic photoreceptor cell death.

Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration.

Knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. Towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (AAV) vector to deliver and express the correct form of the cGMP phosphodiesterase-beta (PDE-beta) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessive mutation in the endogenous gene. A truncated murine opsin promoter was used to drive expression of the PDE-beta cDNA. Following intraocular injection of AAV. PDE-beta, increased retinal expression of immunoreactive PDE protein was observed, including within photoreceptor cell bodies. Compared with age-matched controls, treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light as measured by in vitro electroretinography. These findings provide evidence that rescue of functional photoreceptor neurons can be achieved by somatic gene therapy.