Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma.

Biomarker-driven therapeutic clinical trials require the implementation of standardized, evidence-based practices for sample collection. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target for which window-of-opportunity clinical trials could facilitate the identification of promising new agents. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown. To address this, we used a murine model for isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) and human tumor tissue, including IDH-wildtype GBM and IDH-mutant diffuse glioma. First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of 6 phosphoproteins that are readouts of PI3K/AK/mTOR activity (phosphorylated-proline-rich Akt substrate of 40 kDa (p-PRAS40, T246), -mechanistic target of rapamycin (p-mTOR; S2448); -AKT (p-AKT, S473); -ribosomal protein S6 (p-RPS6, S240/244 and S235/236), and -eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1, T37/46). With CITs ≥ 2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. A similar pattern was observed using patient tumor samples from the operating room with p-4EBP1 more sensitive to delayed fixation than p-RPS6 (S240/244). Many clinical trials utilize unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on the detection of p-RPS6 (S240/244), p-4EBP1, and p-AKT. After 5 months, storage at -80°C was required to preserve the expression of p-4EBP1 and p-AKT, whereas p-RPS6 (240/244) expression was not stable regardless of storage temperature. Biomarker heterogeneity impacts optimal tumor sampling. Quantification of p-RPS6 (240/244) expression in multiple regionally distinct human tumor samples from 8 patients revealed significant intratumoral heterogeneity. Thus, the accurate assessment of PI3K/AKT/mTOR signaling in diffuse glioma must overcome intratumoral heterogeneity and multiple preanalytic factors, including time-to-formalin fixation, slide storage conditions, and phosphoprotein of interest.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Whole tumor analysis reveals early origin of the TERT promoter mutation and intercellular heterogeneity in TERT expression.

BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (R = 0.85), IDH1 mutation in OD (R = 0.87), and with tumor purity (R = 0.91 for GBM; R = 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (R = 0.52 for GBM; R = 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.

Artificial Intelligence-Powered Assessment of Pathologic Response to Neoadjuvant Atezolizumab in Patients With NSCLC: Results From the LCMC3 Study.

INTRODUCTION: Pathologic response (PathR) by histopathologic assessment of resected specimens may be an early clinical end point associated with long-term outcomes with neoadjuvant therapy. Digital pathology may improve the efficiency and precision of PathR assessment. LCMC3 (NCT02927301) evaluated neoadjuvant atezolizumab in patients with resectable NSCLC and reported a 20% major PathR rate. METHODS: We determined PathR in primary tumor resection specimens using guidelines-based visual techniques and developed a convolutional neural network model using the same criteria to digitally measure the percent viable tumor on whole-slide images. Concordance was evaluated between visual determination of percent viable tumor (n = 151) performed by one of the 47 local pathologists and three central pathologists. RESULTS: For concordance among visual determination of percent viable tumor, the interclass correlation coefficient was 0.87 (95% confidence interval [CI]: 0.84-0.90). Agreement for visually assessed 10% or less viable tumor (major PathR [MPR]) in the primary tumor was 92.1% (Fleiss kappa = 0.83). Digitally assessed percent viable tumor (n = 136) correlated with visual assessment (Pearson r = 0.73; digital/visual slope = 0.28). Digitally assessed MPR predicted visually assessed MPR with outstanding discrimination (area under receiver operating characteristic curve, 0.98) and was associated with longer disease-free survival (hazard ratio [HR] = 0.30; 95% CI: 0.09-0.97, p = 0.033) and overall survival (HR = 0.14, 95% CI: 0.02-1.06, p = 0.027) versus no MPR. Digitally assessed PathR strongly correlated with visual measurements. CONCLUSIONS: Artificial intelligence-powered digital pathology exhibits promise in assisting pathologic assessments in neoadjuvant NSCLC clinical trials. The development of artificial intelligence-powered approaches in clinical settings may aid pathologists in clinical operations, including routine PathR assessments, and subsequently support improved patient care and long-term outcomes.

Neoadjuvant atezolizumab for resectable non-small cell lung cancer: an open-label, single-arm phase II trial.

In an ongoing, open-label, single-arm phase II study ( NCT02927301 ), 181 patients with untreated, resectable, stage IB-IIIB non-small cell lung cancer received two doses of neoadjuvant atezolizumab monotherapy. The primary end point was major pathological response (MPR; ≤10% viable malignant cells) in resected tumors without EGFR or ALK alterations. Of the 143 patients in the primary end point analysis, the MPR was 20% (95% confidence interval, 14-28%). With a minimum duration of follow-up of 3 years, the 3-year survival rate of 80% was encouraging. The most common adverse events during the neoadjuvant phase were fatigue (39%, 71 of 181) and procedural pain (29%, 53 of 181), along with expected immune-related toxicities; there were no unexpected safety signals. In exploratory analyses, MPR was predicted using the pre-treatment peripheral blood immunophenotype based on 14 immune cell subsets. Immune cell subsets predictive of MPR in the peripheral blood were also identified in the tumor microenvironment and were associated with MPR. This study of neoadjuvant atezolizumab in a large cohort of patients with resectable non-small cell lung cancer was safe and met its primary end point of MPR ≥ 15%. Data from this single-arm, non-randomized trial suggest that profiles of innate immune cells in pre-treatment peripheral blood may predict pathological response after neoadjuvant atezolizumab, but additional studies are needed to determine whether these profiles can inform patient selection and new therapeutic approaches.

PI3K/AKT/mTOR signaling pathway activity in IDH-mutant diffuse glioma and clinical implications.

BACKGROUND: IDH-mutant diffuse gliomas are heterogeneous, and improved methods for optimal patient therapeutic stratification are needed. PI3K/AKT/mTOR signaling activity can drive disease progression and potential therapeutic inhibitors of the pathway are available. Yet, the prevalence of PI3K/AKT/mTOR signaling pathway activity in IDH-mutant glioma is unclear and few robust strategies to assess activity in clinical samples exist. METHODS: PI3K/AKT/mTOR signaling pathway activity was evaluated in a retrospective cohort of 132 IDH-mutant diffuse glioma (91 astrocytoma and 41 oligodendroglioma, 1p/19q-codeleted) through quantitative multiplex immunoprofiling using phospho-specific antibodies for PI3K/AKT/mTOR pathway members, PRAS40, RPS6, and 4EBP1, and tumor-specific anti-IDH1 R132H. Expression levels were correlated with genomic evaluation of pathway intrinsic genes and univariate and multivariate Cox proportional hazard regression models were used to evaluate the relationship with outcome. RESULTS: Tumor-specific expression of p-PRAS40, p-RPS6, and p-4EBP1 was common in IDH-mutant diffuse glioma and increased with CNS WHO grade from 2 to 3. Genomic analysis predicted pathway activity in 21.7% (13/60) while protein evaluation identified active PI3K/AKT/mTOR signaling in 56.6% (34/60). Comparison of expression in male versus female patients suggested sexual dimorphism. Of particular interest, when adjusting for clinical prognostic factors, the level of phosphorylation of RPS6 was strongly associated with PFS (P < .005). Phosphorylation levels of both PRAS40 and RPS6 showed an association with PFS in univariate analysis. CONCLUSIONS: Our study emphasizes the value of proteomic assessment of signaling pathway activity in tumors as a means to identify relevant oncogenic pathways and potentially as a biomarker for identifying aggressive disease.

Temozolomide-induced hypermutation is associated with distant recurrence and reduced survival after high-grade transformation of low-grade IDH-mutant gliomas.

BACKGROUND: Chemotherapy improves overall survival after surgery and radiotherapy for newly diagnosed high-risk IDH-mutant low-grade gliomas (LGGs), but a proportion of patients treated with temozolomide (TMZ) will develop recurrent tumors with TMZ-induced hypermutation. We aimed to determine the prevalence of TMZ-induced hypermutation at recurrence and prognostic implications. METHODS: We sequenced recurrent tumors from 82 patients with initially low-grade IDH-mutant gliomas who underwent reoperation and correlated hypermutation status with grade at recurrence and subsequent clinical outcomes. RESULTS: Hypermutation was associated with high-grade disease at the time of reoperation (OR 12.0 95% CI 2.5-115.5, P = .002) and was identified at transformation in 57% of recurrent LGGs previously exposed to TMZ. After anaplastic (grade III) transformation, hypermutation was associated with shorter survival on univariate and multivariate analysis (HR 3.4, 95% CI 1.2-9.9, P = .024), controlling for tumor grade, subtype, age, and prior radiotherapy. The effect of hypermutation on survival after transformation was validated in an independent, published dataset. Hypermutated (HM) tumors were more likely to develop discontiguous foci of disease in the brain and spine (P = .003). To estimate the overall incidence of high-grade transformation among low-grade IDH-mutant tumors, data from a phase II trial of TMZ for LGG were analyzed. Eight-year transformation-free survival was 53.8% (95% CI 42.8-69.2), and 61% of analyzed transformed cases were HM. CONCLUSIONS: TMZ-induced hypermutation is a common event in transformed LGG previously treated with TMZ and is associated with worse prognosis and development of discontiguous disease after recurrence. These findings impact tumor classification at recurrence, prognostication, and clinical trial design.

Multiplatform genomic profiling and magnetic resonance imaging identify mechanisms underlying intratumor heterogeneity in meningioma.

Meningiomas are the most common primary intracranial tumors, but the molecular drivers of meningioma tumorigenesis are poorly understood. We hypothesized that investigating intratumor heterogeneity in meningiomas would elucidate biologic drivers and reveal new targets for molecular therapy. To test this hypothesis, here we perform multiplatform molecular profiling of 86 spatially-distinct samples from 13 human meningiomas. Our data reveal that regional alterations in chromosome structure underlie clonal transcriptomic, epigenomic, and histopathologic signatures in meningioma. Stereotactic co-registration of sample coordinates to preoperative magnetic resonance images further suggest that high apparent diffusion coefficient (ADC) distinguishes meningioma regions with proliferating cells enriched for developmental gene expression programs. To understand the function of these genes in meningioma, we develop a human cerebral organoid model of meningioma and validate the high ADC marker genes CDH2 and PTPRZ1 as potential targets for meningioma therapy using live imaging, single cell RNA sequencing, CRISPR interference, and pharmacology.

Patient-derived cells from recurrent tumors that model the evolution of IDH-mutant glioma.

BACKGROUND: IDH-mutant lower-grade gliomas (LGGs) evolve under the selective pressure of therapy, but well-characterized patient-derived cells (PDCs) modeling evolutionary stages are lacking. IDH-mutant LGGs may develop therapeutic resistance associated with chemotherapy-driven hypermutation and malignant progression. The aim of this study was to establish and characterize PDCs, single-cell-derived PDCs (scPDCs), and xenografts (PDX) of IDH1-mutant recurrences representing distinct stages of tumor evolution. METHODS: We derived and validated cell cultures from IDH1-mutant recurrences of astrocytoma and oligodendroglioma. We used exome sequencing and phylogenetic reconstruction to examine the evolutionary stage represented by PDCs, scPDCs, and PDX relative to corresponding spatiotemporal tumor tissue and germline DNA. PDCs were also characterized for growth and tumor immortality phenotypes, and PDX were examined histologically. RESULTS: The integrated astrocytoma phylogeny revealed 2 independent founder clonal expansions of hypermutated (HM) cells in tumor tissue that are faithfully represented by independent PDCs. The oligodendroglioma phylogeny showed more than 4000 temozolomide-associated mutations shared among tumor samples, PDCs, scPDCs, and PDX, suggesting a shared monoclonal origin. The PDCs from both subtypes exhibited hallmarks of tumorigenesis, retention of subtype-defining genomic features, production of 2-hydroxyglutarate, and subtype-specific telomere maintenance mechanisms that confer tumor cell immortality. The oligodendroglioma PDCs formed infiltrative intracranial tumors with characteristic histology. CONCLUSIONS: These PDCs, scPDCs, and PDX are unique and versatile community resources that model the heterogeneous clonal origins and functions of recurrent IDH1-mutant LGGs. The integrated phylogenies advance our knowledge of the complex evolution and immense mutational load of IDH1-mutant HM glioma.

Multiplatform Molecular Profiling Reveals Epigenomic Intratumor Heterogeneity in Ependymoma.

Ependymomas exist within distinct genetic subgroups, but the molecular diversity within individual ependymomas is unknown. We perform multiplatform molecular profiling of 6 spatially distinct samples from an ependymoma with C11orf95-RELA fusion. DNA methylation and RNA sequencing distinguish clusters of samples according to neuronal development gene expression programs that could also be delineated by differences in magnetic resonance blood perfusion. Exome sequencing and phylogenetic analysis reveal epigenomic intratumor heterogeneity and suggest that chromosomal structural alterations may precede accumulation of single-nucleotide variants during ependymoma tumorigenesis. In sum, these findings shed light on the oncogenesis and intratumor heterogeneity of ependymoma.

Aberrant gene expression by Sertoli cells in infertile men with Sertoli cell-only syndrome.

Sertoli cell-only (SCO) syndrome is a severe form of human male infertility seemingly characterized by the lack all spermatogenic cells. However, tubules of some SCO testes contain small patches of active spermatogenesis and thus spermatogonial stem cells. We hypothesized that these stem cells cannot replicate and seed spermatogenesis in barren areas of tubule because as-of-yet unrecognized deficits in Sertoli cell gene expression disable most stem cell niches. Performing the first thorough comparison of the transcriptomes of human testes exhibiting complete spermatogenesis with the transcriptomes of testes with SCO syndrome, we defined transcripts that are both predominantly expressed by Sertoli cells and expressed at aberrant levels in SCO testes. Some of these transcripts encode proteins required for the proper assembly of adherent and gap junctions at sites of contact with other cells, including spermatogonial stem cells (SSCs). Other transcripts encode GDNF, FGF8 and BMP4, known regulators of mouse SSCs. Thus, most SCO Sertoli cells can neither organize junctions at normal sites of cell-cell contact nor stimulate SSCs with adequate levels of growth factors. We propose that the critical deficits in Sertoli cell gene expression we have identified contribute to the inability of spermatogonial stem cells within small patches of spermatogenesis in some SCO testes to seed spermatogenesis to adjacent areas of tubule that are barren of spermatogenesis. Furthermore, we predict that one or more of these deficits in gene expression are primary causes of human SCO syndrome.

A20 and ABIN1 Suppression of a Keratinocyte Inflammatory Program with a Shared Single-Cell Expression Signature in Diverse Human Rashes.

Genetic variation in the NF-κB inhibitors, ABIN1 and A20, increase risk for psoriasis. While critical for hematopoietic immune cell function, these genes are believed to additionally inhibit psoriasis by dampening inflammatory signaling in keratinocytes. We dissected ABIN1 and A20's regulatory role in human keratinocyte inflammation using an RNA sequencing-based comparative genomic approach. Here we show subsets of the IL-17 and tumor necrosis factor-α signaling pathways are robustly restricted by A20 overexpression. In contrast, ABIN1 overexpression inhibits these genes more modestly for IL-17, and weakly for tumor necrosis factor-α. Our genome-scale analysis also indicates that inflammatory program suppression appears to be the major transcriptional influence of A20/ABIN1 overexpression, without obvious influence on keratinocyte viability genes. Our findings thus enable dissection of the differing anti-inflammatory mechanisms of two distinct psoriasis modifiers, which may be directly exploited for therapeutic purposes. Importantly, we report that IL-17-induced targets of A20 show similar aberrant epidermal layer-specific transcriptional upregulation in keratinocytes from diseases as diverse as psoriasis, atopic dermatitis, and erythrokeratodermia variabilis, suggesting a contributory role for epidermal inflammation in a broad spectrum of rashes.

Transcriptome profiling of the developing male germ line identifies the miR-29 family as a global regulator during meiosis.

MicroRNAs are essential for spermatogenesis. However, the stage-specific requirements for particular miRNAs in the male mammalian germ line remain largely uncharacterized. The miR-34 family is, to date, the only miRNA proven to be necessary for the production of sperm in mammals, though its germline roles are poorly understood. Here, we generate and analyze paired small RNA and mRNA profiles across different stages of germline development in male mice, focusing on time points shortly before and during meiotic prophase I. We show that in addition to miR-34, miR-29 also mediates widespread repression of mRNA targets during meiotic prophase I in the male mouse germline. Furthermore, we demonstrate that predicted miR-29 target mRNAs in meiotic cells are largely distinct from those of miR-34, indicating that miR-29 performs a regulatory function independent of miR-34. Prior to this work, no germline role has been attributed to miR-29. To begin to understand roles for miR-29 in the germ line, we identify targets of miR-29 undergoing post transcriptional downregulation during meiotic prophase I, which likely correspond to the direct targets of miR-29. Interestingly, candidate direct targets of miR-29 are enriched in transcripts encoding extracellular matrix components. Our results implicate the miR-29 family as an important regulatory factor during male meiosis.

The roles of microRNAs and siRNAs in mammalian spermatogenesis.

MicroRNAs and siRNAs, both of which are AGO-bound small RNAs, are essential for mammalian spermatogenesis. Although their precise germline roles remain largely uncharacterized, recent discoveries suggest that they function in mechanisms beyond microRNA-mediated post-transcriptional control, playing roles in DNA repair and transcriptional regulation within the nucleus. Here, we discuss the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline. We integrate genetic, clinical and genomics data, and draw upon findings from non-mammalian models, to examine potential roles for AGO-bound small RNAs during spermatogenesis. Finally, we evaluate the emerging and differing roles for AGOs and AGO-bound small RNAs in the male and female germlines, suggesting potential reasons for these sexual dimorphisms.

Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males.

Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3'UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1-ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.

AGO4 regulates entry into meiosis and influences silencing of sex chromosomes in the male mouse germline.

The four mammalian Argonaute family members are thought to share redundant functions in the microRNA pathway, yet only AGO2 possesses the catalytic "slicer" function required for RNAi. Whether AGO1, AGO3, or AGO4 possesses specialized functions remains unclear. Here we show that AGO4 localizes to spermatocyte nuclei during meiotic prophase I, specifically at sites of asynapsis and the transcriptionally silenced XY subdomain, the sex body. We generated Ago4 knockout mice and show that Ago4(-/-) spermatogonia initiate meiosis early, resulting from premature induction of retinoic acid-response genes. During prophase I, the sex body assembles incorrectly in Ago4(-/-) mice, leading to disrupted meiotic sex chromosome inactivation (MSCI). This is associated with a dramatic loss of microRNAs, >20% of which arises from the X chromosome. Thus, AGO4 regulates meiotic entry and MSCI in mammalian germ cells, implicating small RNA pathways in these processes.