Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Introduction: Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Methods: Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Results: Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Conclusion: Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder characterized by high levels of cholestanol in the blood and accumulation of cholestanol in multiple tissues, especially the brain, often presents in parkinsonism. However, it remains unknown whether cholestanol plays a role in the pathogenesis of sporadic Parkinson's disease (PD). Here, we show that the levels of serum cholestanol in patients with sporadic PD are higher than those in control participants. Cholestanol activates the protease asparagine endopeptidase (AEP) and induces the fragmentation of α-synuclein (α-syn) and facilitates its aggregation. Furthermore, cholestanol promotes the spreading of α-syn pathology in a mouse model induced by intrastriatal injection of α-syn fibrils. KO of AEP or administration of an AEP inhibitor ameliorates α-syn pathology, degeneration of the nigrostriatal dopaminergic pathway, and PD-like motor symptoms. These results not only indicate that cholestanol contributes to the aggregation and spreading of α-syn by activating AEP but also reveal an opportunity for treating PD with AEP inhibitors.

CXCR4 antagonists disrupt leukaemia-meningeal cell adhesion and attenuate chemoresistance.

The effective prophylaxis and treatment of central nervous system (CNS) involvement in acute lymphoblastic leukaemia (ALL) remains a significant clinical challenge. Developing novel and more effective CNS-directed therapies has been hampered, in part, by our limited understanding of the leukaemia niche in the CNS relative to the bone marrow. Accordingly, defining the molecular and cellular components critical for the establishment and maintenance of the CNS leukaemia niche may lead to new therapeutic opportunities. In prior work we showed that direct intercellular interactions between leukaemia and meningeal cells enhance leukaemia chemoresistance in the CNS. Herein, we show that the CXCR4/CXCL12 chemokine axis contributes to leukaemia-meningeal cell adhesion. Importantly, clinically tested CXCR4 antagonists, which are likely to cross the blood-brain and blood-cerebral spinal fluid barriers and penetrate the CNS, effectively disrupted leukaemia-meningeal cell adhesion. Moreover, by disrupting these intercellular interactions, CXCR4 antagonists attenuated leukaemia chemoresistance in leukaemia-meningeal cell co-culture experiments and enhanced the efficacy of cytarabine in targeting leukaemia cells in the meninges in vivo. This work identifies the CXCR4/CXCL12 axis as an important regulator of intercellular interactions within the CNS leukaemia niche and supports further testing of the therapeutic efficacy of CXCR4 antagonists in overcoming CNS niche-mediated chemoresistance.

Initial Clinical Experience With ClearPoint SmartFrame Array-Aided Stereotactic Procedures.

BACKGROUND: The ClearPoint SmartFrame Array (ClearPoint Neuro, Inc., Solana Beach, CA) system consists of a magnetic resonance imaging compatible frame supported by a customized neuro navigation software. This system received U.S. Food and Drug Administrationclearance for clinical use in January 2021. Our objective was to report initial safety data and user experience of SmartFrame Array-supported stereotactic procedures. METHODS: We prospectively followed the first 10 consecutive patients who underwent stereotactic procedures supported by SmartFrame Array. Clinical and procedural data were recorded and compared to data obtained from prior cases with SmartFrame XG. RESULTS: Ten patients underwent stereotactic needle biopsy, stereotactic laser ablation (SLA), or combined biopsy/SLA procedures. For needle biopsies (n = 9), the average maximal diameter of the contrast-enhancing target lesion was 9.9 ± 2.8 mm. The radial error of stereotaxis was less than 2 mm. Definitive diagnosis was achieved in all cases. For procedures involving SLA (n = 5), 100% of the contrast-enhancing lesion was ablated. All patients were discharged home by postoperative day 2. There were no 30-day readmissions, morbidity, or mortality. The average stereotaxis time for the SmartFrame Array-aided single trajectory procedure was 80 ± 9.5 minutes, which compared favorably to that required for the earlier generation SmartFrame XG frame (111.5 ± 16.5 minutes; P < 0.01). The unique Array design supported stereotactic procedures that cannot be easily achieved with the previous SmartFrame XG frame. CONCLUSIONS: The SmartFrame Array system offers a more rigid and compact build to enhance procedural efficiency while maintaining accuracy and safety. The design supports multi-trajectory stereotaxis, allowing novel clinical applications.

GammaTile® brachytherapy in the treatment of recurrent glioblastomas.

BACKGROUND: GammaTile® (GT) is a recent U.S. Food and Drug Administration (FDA) cleared brachytherapy platform. Here, we report clinical outcomes for recurrent glioblastoma patients after GT treatment following maximal safe resection. METHODS: We prospectively followed twenty-two consecutive Isocitrate Dehydrogenase (IDH) wild-type glioblastoma patients (6 O6-Methylguanine-DNA methyltransferase methylated (MGMTm); sixteen MGMT unmethylated (MGMTu)) who underwent maximal safe resection of recurrent tumor followed by GT placement. RESULTS: The cohort consisted of 14 second and eight third recurrences. In terms of procedural safety, there was one 30-day re-admission (4.5%) for an incisional cerebrospinal fluid leak, which resolved with lumbar drainage. No other wound complications were observed. Six patients (27.2%) declined in Karnofsky Performance Score (KPS) after surgery due to worsening existing deficits. One patient suffered a new-onset seizure postsurgery (4.5%). There was one (4.5%) 30-day mortality from intracranial hemorrhage secondary to heparinization for an ischemic limb. The mean follow-up was 733 days (range 279-1775) from the time of initial diagnosis. Six-month local control (LC6) and twelve-month local control (LC12) were 86 and 81%, respectively. Median progression-free survival (PFS) was comparable for MGMTu and MGMTm patients (~8.0 months). Median overall survival (OS) was 20.0 months for the MGMTu patients and 37.4 months for MGMTm patients. These outcomes compared favorably to data in the published literature and an independent glioblastoma cohort of comparable patients without GT treatment. CONCLUSIONS: This clinical experience supports GT brachytherapy as a treatment option in a multi-modality treatment strategy for recurrent glioblastomas.

Molecular Profile of a Pituitary Rhabdomyosarcoma Arising From a Pituitary Macroadenoma: A Case Report.

Pituitary sarcoma arising in association with pituitary adenoma is an uncommon finding. Most cases of secondary sarcoma have been noted to arise with a median interval of 10.5 years post radiation. In this case report, we describe a 77-year-old man with an incidental discovery of a pituitary macroadenoma on magnetic resonance imaging (MRI) and underwent radiotherapy. Three years after radiation treatment, there was an acute change in clinical symptoms and increase in tumor size and mass effect on the optic chiasm which prompted surgical resection. A pituitary adenoma along with a separate spindle-cell sarcomatous component was identified in histology. Immunohistochemical stain for muscle markers confirmed a development of pituitary rhabdomyosarcoma (RMS). Molecular profiling of the tumor identified mutations in TP53, ATRX, LZTR1, and NF1. Despite its rarity, characterization of pituitary RMS with immunohistochemistry and molecular studies may provide an insight to its pathophysiological relationship with pituitary adenoma.

IDH1 and IDH2 Mutations in Colorectal Cancers.

OBJECTIVES: To elucidate clinicopathologic and molecular characteristics of IDH1 and IDH2 (IDH1/2) mutations in colorectal cancers (CRCs). METHODS: We evaluated IDH1/2 mutations in 1,623 CRCs using a next-generation sequencing assay. RESULTS: IDH1/2 mutations, predominantly IDH1 p.R132C, were detected in 15 (0.9%) CRCs and in 5 (3.0%) of 167 BRAF p.V600E-mutated CRCs. Three IDH1/2-mutated CRCs were associated with inflammatory bowel disease. They were significantly associated with old age, mucinous or signet ring cell adenocarcinoma, and high-grade histomorphology. Concordance of variant allele frequency between IDH1/2 mutants and other trunk drivers in CRCs and presence of IDH1/2 mutation in the adenoma and early adenocarcinoma indicated IDH1/2 mutations could be trunk drivers suitable for targeted therapy. CONCLUSIONS: IDH1/2 mutations in CRCs were uncommon but enriched in BRAF p.V600E-mutated CRCs and perhaps colitis-associated CRCs. Further studies on IDH1/2-mutated CRCs are needed to clarify their clinicopathologic features and implications for targeted therapy.

Association of acute disseminated encephalomyelitis (ADEM) and COVID-19 in a pediatric patient.

Cases of acute disseminated encephalomyelitis (ADEM) and its hyperacute form, acute hemorrhagic leukoencephalitis (AHLE), have been reported in coronavirus disease 2019 (COVID-19) patients as rare, but most severe neurological complications. However, histopathologic evaluations of ADEM/AHLE pathology in COVID patients are extremely limited, so far having only been reported in a few adult autopsy cases. Here we compare the findings with an ADEM-like pathology in a pediatric patient taken through a biopsy procedure. Understanding the neuropathology may shed informative light on the autoimmune process affecting COVID-19 patients and provide critical information to guide the clinical management.

Conditional reprogramming culture conditions facilitate growth of lower-grade glioma models.

BACKGROUND: The conditional reprogramming cell culture method was developed to facilitate growth of senescence-prone normal and neoplastic epithelial cells, and involves co-culture with irradiated fibroblasts and the addition of a small molecule Rho kinase (ROCK) inhibitor. The aim of this study was to determine whether this approach would facilitate the culture of compact low-grade gliomas. METHODS: We attempted to culture 4 pilocytic astrocytomas, 2 gangliogliomas, 2 myxopapillary ependymomas, 2 anaplastic gliomas, 2 difficult-to-classify low-grade neuroepithelial tumors, a desmoplastic infantile ganglioglioma, and an anaplastic pleomorphic xanthoastrocytoma using a modified conditional reprogramming cell culture approach. RESULTS: Conditional reprogramming resulted in robust increases in growth for a majority of these tumors, with fibroblast conditioned media and ROCK inhibition both required. Switching cultures to standard serum containing media, or serum-free neurosphere conditions, with or without ROCK inhibition, resulted in decreased proliferation and induction of senescence markers. Rho kinase inhibition and conditioned media both promoted Akt and Erk1/2 activation. Several cultures, including one derived from a NF1-associated pilocytic astrocytoma (JHH-NF1-PA1) and one from a BRAF p.V600E mutant anaplastic pleomorphic xanthoastrocytoma (JHH-PXA1), exhibited growth sufficient for preclinical testing in vitro. In addition, JHH-NF1-PA1 cells survived and migrated in larval zebrafish orthotopic xenografts, while JHH-PXA1 formed orthotopic xenografts in mice histopathologically similar to the tumor from which it was derived. CONCLUSIONS: These studies highlight the potential for the conditional reprogramming cell culture method to promote the growth of glial and glioneuronal tumors in vitro, in some cases enabling the establishment of long-term culture and in vivo models.

Low-Grade Gemistocytic Morphology in H3 G34R-Mutant Gliomas and Concurrent K27M Mutation: Clinicopathologic Findings.

Mutations in histone H3 are key molecular drivers of pediatric and young adult high-grade gliomas. Histone H3 G34R mutations occur in hemispheric high-grade gliomas and H3 K27M mutations occur in aggressive, though histologically diverse, midline gliomas. Here, we report 2 rare cases of histologically low-grade gliomas with gemistocytic morphology and sequencing-confirmed histone H3 G34R mutations. One case is a histologically low-grade gemistocytic astrocytoma with a G34R-mutation in H3F3A. The second case is a histologically low-grade gemistocytic astrocytoma with co-occurring K27M and G34R mutations in HIST1H3B. Review of prior histone H3-mutant gliomas sequenced at our institution shows a divergent clinical and immunohistochemical pattern in the 2 cases. The first case is similar to prior histone H3 G34R-mutant tumors, while the second case most closely resembles prior histone H3 K27M-mutant gliomas. These represent novel cases of sequencing-confirmed histone H3 G34R-mutant gliomas with low-grade histology and add to the known rare cases of G34R-mutant tumors with gemistocytic morphology. Although K27M and G34R mutations are thought to be mutually exclusive, we document combined K27M and G34R mutations in HIST1H3B and present evidence suggesting the K27M-mutation drove tumor phenotype in this dual mutant glioma.

Localized Hypertrophic Neuropathy as a Neoplastic Manifestation of KRAS-Mediated RASopathy.

Localized hypertrophic neuropathy is a rare Schwann cell proliferation that usually affects single nerves from the extremities, and it is of unclear etiology in its pure form. RASopathies are a defined group of genetic diseases with overlapping clinical features, usually secondary to germline mutations in genes encoding either components or regulators of the RAS/MAPK pathway. Herein, we report an 11-year-old boy presenting with café au lait spots and right leg length discrepancy. A fascicular nerve biopsy of the tibial nerve demonstrated a Schwann cell proliferation with prominent onion-bulb formation, satisfying criteria for localized hypertrophic neuropathy. Molecular genetic analysis demonstrated identical KRAS mutations (c38_40dupGCG) in the peripheral nerve lesion and melanocytes from café au lait spots, but not in blood, supporting a diagnosis of a KRAS-mediated rasopathy with mosaicism. Immunohistochemical staining in the peripheral nerve lesion demonstrated strong pERK staining consistent with downstream MAPK pathway activation. This report suggests that at least a subset of localized hypertrophic neuropathies are bonafide, well-differentiated Schwann cell neoplasms developing through oncogenic RAS signaling, which provides new insights into the controversial entity historically known as localized hypertrophic neuropathy.

TSC2 alterations in anaplastic ependymoma progression to ependymosarcoma.

Ependymosarcomas are rare, biphasic tumors composed of ependymal and sarcomatous components. Due to their rarity, their biologic basis is not well understood. We report the case of a 38-year-old male with anaplastic ependymoma (WHO grade III) that progressed to ependymosarcoma in less than 2 years after multiple resections, chemoradiotherapy, and anti-PD1 immunotherapy. Next-generation sequencing was performed on both high-grade anaplastic ependymoma and ependymosarcoma samples to detect small base changes, insertions, and deletions in exons and splice junctions from a panel of over 400 genes. We identify genetic variants in the tumor suppressors RB1, TP53, and TSC2 in these samples and discuss the potential significance of an additional TSC2 genetic variant in the progression to ependymosarcoma.

Pediatric Idiopathic Basal Ganglia Calcification and Spherocytosis With Chromosome 8p11 Deletion.

Idiopathic basal ganglia calcification (IBGC), also known as Fahr disease, is a rare neurodegenerative disorder characterized by the accumulation of extensive parenchymal and vascular calcifications in the basal ganglia, with variable calcifications elsewhere in the brain. Typically, IBGC presents with neurologic and psychiatric symptoms in middle-aged adults. Recent genetic studies have identified alterations in 4 genes causing IBGC, including alterations in SLC20A2 on chromosome 8p11.2. Currently, there are no clinical descriptions of patients with IBGC occurring within the context of a complex genetic syndrome. Here, we present a case of pediatric 8p11 deletion with IBGC, hereditary spherocytosis, vitreoretinopathy, and focal cortical dysplasia. We review multiple cases of IBGC with pediatric onset due to SLC20A2 deletion in the literature, and raise the consideration of IBGC in the evaluation of pediatric patients with 8p11.2 deletion syndromes.

Pathologic and molecular aspects of anaplasia in circumscribed gliomas and glioneuronal tumors.

Many breakthroughs have been made in the past decade regarding our knowledge of the biological basis of the diffuse gliomas, the most common primary central nervous system (CNS) tumors. These tumors as a group are aggressive, associated with high mortality, and have a predilection for adults. However, a subset of CNS glial and glioneuronal tumors are characterized by a more circumscribed pattern of growth and occur more commonly in children and young adults. They tend to be indolent, but our understanding of anaplastic changes in these tumors continues to improve as diagnostic classifications evolve in the era of molecular pathology and more integrated and easily accessible clinical databases. The presence of anaplasia in pleomorphic xanthoastrocytomas and gangliogliomas is assigned a WHO grade III under the current classification, while the significance of anaplasia in pilocytic astrocytomas remains controversial. Recent data highlight the association of the latter with aggressive clinical behavior, as well as the presence of molecular genetic features of both pilocytic and diffuse gliomas, with the recognition that the precise terminology remains to be defined. We review the current concepts and advances regarding histopathology and molecular understanding of pilocytic astrocytomas, pleomorphic xanthoastrocytomas, and gangliogliomas, with a focus on their anaplastic counterparts.