A Sequential Targeting Strategy Interrupts AKT-Driven Subclone-Mediated Progression in Glioblastoma.

PURPOSE: Therapy resistance and fatal disease progression in glioblastoma are thought to result from the dynamics of intra-tumor heterogeneity. This study aimed at identifying and molecularly targeting tumor cells that can survive, adapt, and subclonally expand under primary therapy. EXPERIMENTAL DESIGN: To identify candidate markers and to experimentally access dynamics of subclonal progression in glioblastoma, we established a discovery cohort of paired vital cell samples obtained before and after primary therapy. We further used two independent validation cohorts of paired clinical tissues to test our findings. Follow-up preclinical treatment strategies were evaluated in patient-derived xenografts. RESULTS: We describe, in clinical samples, an archetype of rare ALDH1A1+ tumor cells that enrich and acquire AKT-mediated drug resistance in response to standard-of-care temozolomide (TMZ). Importantly, we observe that drug resistance of ALDH1A1+ cells is not intrinsic, but rather an adaptive mechanism emerging exclusively after TMZ treatment. In patient cells and xenograft models of disease, we recapitulate the enrichment of ALDH1A1+ cells under the influence of TMZ. We demonstrate that their subclonal progression is AKT-driven and can be interfered with by well-timed sequential rather than simultaneous antitumor combination strategy. CONCLUSIONS: Drug-resistant ALDH1A1+/pAKT+ subclones accumulate in patient tissues upon adaptation to TMZ therapy. These subclones may therefore represent a dynamic target in glioblastoma. Our study proposes the combination of TMZ and AKT inhibitors in a sequential treatment schedule as a rationale for future clinical investigation.

Spinal ectopic recurrence of craniopharyngioma in a pediatric patient.

Craniopharyngiomas are rare, benign lesions that can be treated with surgery, radiation therapy, or a combination of these modalities. They have a propensity for local recurrence, but there have also been rare cases reported of ectopic recurrence. Here, we present the case of a 15-year-old girl with a recurrence of craniopharyngioma in the spine, which is the second-ever reported case of recurrence outside of the brain in a pediatric patient, and review the 19 reported cases of ectopic recurrence in pediatric patients due to cerebrospinal fluid dissemination.

ATRT-SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

Aggressive Childhood-onset Papillary Craniopharyngioma Managed With Vemurafenib, a BRAF Inhibitor.

The papillary subtype of craniopharyngioma (CP) rarely occurs in children and commonly presents as a suprasellar lesion. Patients with papillary CPs frequently harbor the BRAF-V600E mutation, and treatment with a BRAF inhibitor results in tumor shrinkage in several patients. Herein, we report a patient with childhood-onset papillary CP treated with vemurafenib for 40 months after multiple surgeries. At age 10, he presented with growth failure secondary to an intrasellar cystic lesion. He had 3 transsphenoidal surgeries before age 12 and a 4th surgery 25 years later for massive tumor recurrence. Pathology showed a papillary CP with positive BRAF-V600E mutation. Rapid tumor regrowth 4 months after surgery led to treatment with vemurafenib that resulted in tumor reduction within 6 weeks. Gradual tumor regrowth occurred after a dose reduction of vemurafenib because of elevated liver enzymes. He had further surgeries and within 7 weeks after stopping vemurafenib, there was massive tumor recurrence. He resumed treatment with vemurafenib before radiation therapy and similar tumor shrinkage occurred within 16 days. In this patient with childhood-onset papillary CP that was refractory to multiple surgeries, the use of vemurafenib resulted in significant tumor shrinkage that allowed for the completion of radiation therapy and tumor control.

Effects of CAPTEM (Capecitabine and Temozolomide) on a Corticotroph Carcinoma and an Aggressive Corticotroph Tumor.

Corticotroph carcinomas and aggressive corticotroph tumors can be resistant to conventional therapy, including surgery, radiotherapy, and medical treatment. Recent evidence suggests that temozolomide (an oral alkylating agent) administered with capecitabine (pro-drug of 5-fluorouracil) may improve progression-free survival in patients with high-risk corticotroph tumors and carcinomas. This led to the use of capecitabine and temozolomide (CAPTEM) in two patients, one with a corticotroph carcinoma and the other with an aggressive corticotroph tumor, as well the in vitro analysis of capecitabine and 5-fluorouracil on cell growth and hormone production. Both patients had previous surgical and radiation therapy. The first patient developed leptomeningeal spread 2 years after his radiation treatment. He had 12 cycles of CAPTEM, which resulted in tumor control associated with clinical and radiological improvement. Twenty-seven months later, CAPTEM was restarted for disease recurrence with ongoing tumor response. The second patient had a rapid tumor regrowth 2 years after his third surgical resection. He was treated with 12 cycles of CAPTEM, which led to tumor shrinkage with no tumor regrowth 22 months after cessation of therapy. Experiments using mouse ACTH-producing pituitary tumor AtT20 cells demonstrated that treatment with 5-fluorouracil in combination with temozolomide had an additive effect in reducing cell viability and ACTH production in the culture medium. Our patients and experimental data in AtT20 cells support CAPTEM as a potential treatment option for aggressive corticotroph tumors and carcinomas. However, a prospective clinical trial is required to determine whether CAPTEM is superior to temozolomide in the treatment of these tumors.

An update to the Monro-Kellie doctrine to reflect tissue compliance after severe ischemic and hemorrhagic stroke.

High intracranial pressure (ICP) can impede cerebral blood flow resulting in secondary injury or death following severe stroke. Compensatory mechanisms include reduced cerebral blood and cerebrospinal fluid volumes, but these often fail to prevent raised ICP. Serendipitous observations in intracerebral hemorrhage (ICH) suggest that neurons far removed from a hematoma may shrink as an ICP compliance mechanism. Here, we sought to critically test this observation. We tracked the timing of distal tissue shrinkage (e.g. CA1) after collagenase-induced striatal ICH in rat; cell volume and density alterations (42% volume reduction, 34% density increase; p < 0.0001) were highest day one post-stroke, and rebounded over a week across brain regions. Similar effects were seen in the filament model of middle cerebral artery occlusion (22% volume reduction, 22% density increase; p ≤ 0.007), but not with the Vannucci-Rice model of hypoxic-ischemic encephalopathy (2.5% volume increase, 14% density increase; p ≥ 0.05). Concerningly, this 'tissue compliance' appears to cause sub-lethal damage, as revealed by electron microscopy after ICH. Our data challenge the long-held assumption that 'healthy' brain tissue outside the injured area maintains its volume. Given the magnitude of these effects, we posit that 'tissue compliance' is an important mechanism invoked after severe strokes.

Reflection-mode virtual histology using photoacoustic remote sensing microscopy.

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are both labor intensive and contribute to creating significant delays in clinical feedback for treatment decisions that can extend to 2-3 weeks for standard paraffin-embedded tissue preparation and interpretation, especially if ancillary testing is needed. Here, we present the first comprehensive study on the broad application of a novel label-free reflection-mode imaging modality known as photoacoustic remote sensing (PARS) for visualizing salient subcellular structures from various common histopathological tissue preparations and for use in unprocessed freshly resected tissues. The PARS modality permits non-contact visualizations of intrinsic endogenous optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. The technique was examined both as a rapid assessment tool that is capable of managing large samples (> 1 cm2) in under 10 min, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment.

Improving maximal safe brain tumor resection with photoacoustic remote sensing microscopy.

Malignant brain tumors are among the deadliest neoplasms with the lowest survival rates of any cancer type. In considering surgical tumor resection, suboptimal extent of resection is linked to poor clinical outcomes and lower overall survival rates. Currently available tools for intraoperative histopathological assessment require an average of 20 min processing and are of limited diagnostic quality for guiding surgeries. Consequently, there is an unaddressed need for a rapid imaging technique to guide maximal resection of brain tumors. Working towards this goal, presented here is an all optical non-contact label-free reflection mode photoacoustic remote sensing (PARS) microscope. By using a tunable excitation laser, PARS takes advantage of the endogenous optical absorption peaks of DNA and cytoplasm to achieve virtual contrast analogous to standard hematoxylin and eosin (H&E) staining. In conjunction, a fast 266 nm excitation is used to generate large grossing scans and rapidly assess small fields in real-time with hematoxylin-like contrast. Images obtained using this technique show comparable quality and contrast to the current standard for histopathological assessment of brain tissues. Using the proposed method, rapid, high-throughput, histological-like imaging was achieved in unstained brain tissues, indicating PARS' utility for intraoperative guidance to improve extent of surgical resection.