Sandra Camelo-Piragua

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering microscopy provides a rapid, label-free means of detecting tumor infiltration of brain tissue ex vivo and in vivo. Virtual Histology During brain tumor surgery, precision is key. Removing healthy tissue can cause neurologic deficits; leaving behind tumor tissue can allow cancer to spread and treatment to fail. To help the surgeon clearly see tumor versus normal tissue, Ji and colleagues developed a stimulated Raman scattering (SRS) microscopy method and demonstrated its ability to identify malignant human brain tissue. In SRS microscopy, laser beams are directed at the tissue sample to generate a series of output signals called “Raman spectra.” These spectra depend on the molecular composition of the tissue. Ji et al. implanted human brain cancer (glioblastoma) cells into mice, allowed them to infiltrate and grow into tumors, and then removed slices for SRS imaging. From the resulting spectra, the authors were able to differentiate the two major components of brain tissue—lipid-rich white matter and protein-rich cortex—as well as tumors, which are full of proteins. Intraoperatively, using an imaging window into mouse brains, the authors found that SRS microscopy could locate tumor infiltration in areas that appeared normal by eye, which suggests that this tool could be applied during surgery. Imaging fresh tissue slices ex vivo could also complement or perhaps replace standard hematoxylin and eosin (H&E) staining in the clinic because it avoids artifacts inherent in imaging frozen or fixed tissues. To this end, Ji and colleagues showed that SRS microscopy could identify hypercellular tumor regions in fresh surgical specimens from a patient with glioblastoma. Certain diagnostic features were present in these specimens and readily identified by SRS, including pseudopalisading necrosis and microvascular proliferation. The next step will be to apply SRS microscopy to a large collection of human specimens to see whether this technology may be useful in quickly distinguishing glioblastoma from healthy tissue, both outside and inside the operating room. Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy

Quantitative SRS microscopy can detect human brain tumor infiltration with high sensitivity and specificity, even in tissues appearing grossly normal. Image-based classifier calls out cancer cells Ji and colleagues used a microscopy technique called stimulated Raman scattering, or SRS, to image cancer cells in human brain tissue. SRS produces different signals for proteins and lipids, which can then be assigned a color (blue and green, respectively), allowing the authors to differentiate brain cortex from tumor from white matter. Biopsies from adult and pediatric patients with glioblastoma revealed not only distinctive features with SRS microscopy but also the presence of infiltrating cells in tissues that appeared otherwise normal with traditional staining. Such infiltrating cells are important to catch early because leaving them behind after surgery nearly always leads to cancer recurrence. To make this SRS microscopy approach amenable to routine use in neuropathology, the authors also created an objective classifier that integrated different image characteristics, such as the protein/lipid ratio, axonal density, and degree of cellularity, into one output, on a scale of 0 to 1, that would alert the pathologist to tumor infiltration. The classifier was built using more than 1400 images from patients with glioblastoma and epilepsy, and could distinguish between tumor-infiltrated and nontumor regions with >99% accuracy, regardless of tumor grade or histologic subtype. This label-free imaging technology could therefore be used to complement existing neurosurgical workflows, allowing for rapid and objective characterization of brain tissues and, in turn, clinical decision-making. Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

Mutant IDH1-specific immunohistochemistry distinguishes diffuse astrocytoma from astrocytosis

One of the most vexing issues in diagnostic neuropathology relates to the distinction of diffuse astrocytomas (and other diffuse gliomas) from astrocytosis (gliosis) on biopsies, particularly small biopsies. This challenging differential diagnosis arises in two general situations: (1) low cellularity edges of infiltrating astrocytomas versus mild astrocytosis from a nearby reactive condition; and (2) florid astrocytosis (e.g., near a vascular malformation) versus more cellular astrocytomas. The “holy grail” sought in such diagnostic dilemmas is a tumor-specific marker. To date, the most widely used marker for this purpose has been p53 detection by immunohistochemistry; since mutant p53 has a longer half-life than wild-type p53, it can be more readily detected immunohistochemically than wild-type protein [3, 9]. However, p53 immunohistochemistry is not an entirely accurate marker since: (1) it may show light labeling of non-neoplastic cells; (2) not all TP53 gene mutations result in immunohistochemically detectable p53; and (3) some reactive conditions (notably progressive multifocal leukoencephalopathy) may be strongly positive [8]. Another immunohistochemical marker of tumor cells is the vIII mutant of the epidermal growth factor receptor (EGFR) protein. However, this marker is not of diagnostic utility in the above differential diagnosis, since EGFRvIII is primarily expressed in glioblastomas rather than lower-grade astrocytomas. Moreover, antibodies are not widely available or readily optimized for standard immunohistochemistry, again limiting its differential diagnostic utility. Recently, isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations have been demonstrated in a variety of diffuse gliomas, with IDH1 mutations occurring commonly in lower-grade gliomas [1, 2, 10, 12]. Notably, nearly all IDH1 mutations are the same, with CGT–CAT transition causing a specific amino acid change from arginine to histidine at codon 132 (R132H). As a result, the detection of IDH1 mutations may be a specific means to aid in differentiating between glioma and gliosis. Indeed, one recent paper utilized a PCR-based assay to show that IDH mutations are found in astrocytomas but not in reactive conditions [6]. Of 57 non-neoplastic conditions, none showed IDH1/2 mutations. In contrast, 67.3% of grade II and grade III diffuse gliomas did; in addition, in a small subset of gliomas, IDH mutations were demonstrated in the infiltrative edge of the tumor, an area represented in the “near miss” scenario in stereotactic biopsy. Despite the promise of IDH mutations as a tumor-specific marker, not all institutions have ready access to mutation detection methods, and DNA extraction followed by sequencing may be problematic in very small biopsies. Furthermore, IDH1 immunohistochemistry, using an antibody specific to the common R132H mutant form of IDH1, may be more sensitive than sequencing to detect tumors with mutations [4]. Using R132H mutant IDH1 and p53 immunohistochemistry, we therefore studied 21 samples of WHO grade II diffuse astrocytoma and 20 samples of reactive conditions (10 resections for epilepsy, 7 infarcts, 2 evacuated hematomas and 1 traumatic brain injury), all surgical biopsy specimens. The mean patient age for low-grade astrocytomas and reactive cases was 33.4 and 32.5 years, respectively. Immunohistochemical staining for IDH1 was done on BenchMark XT automated tissue staining systems (Ventana Medical Systems, Inc., Tucson, AZ) using validated protocols. Endogenous peroxidase activity was blocked by H2O2 and antigen retrieved using CC1 reagent (Ventana Medical Systems). After washing, tissue sections were incubated with mouse monoclonal anti-R132H-IDH1 antibody culture supernatant, followed by incubation with UltraView HRP-conjugated multimer antibody reagent (Ventana Medical Systems). Antigen detection was performed using UltraView diaminobenzidine chromogen step (Ventana Medical Systems). Tissues were counterstained with hematoxylin and scored independently by two investigators (SC-P, MJ). Immunohistochemical staining for p53 was performed using a mouse monoclonal antibody (Santa Cruz, CA; # SC47698) using standard protocol. Positive granular cytoplasmic staining of tumor cells for mutant IDH1 was found in 9 out of 21 (42.9%) WHO grade II astrocytomas, but was entirely absent in all 20 reactive samples (Fig. 1). Positive nuclear staining of tumor cells with p53 was found in 10 out of 21 (47.6%) astrocytomas; of 20 reactive cases, none showed nuclear staining in astrocytes, but one showed positive nuclear signal in macrophages (CD68-positive). Five tumors showed co-expression of mutant IDH1 and p53. When used together, mutant IDH1 and p53 demonstrated the presence of tumor in 14 out of 21 (66.7%) cases. Fig. 1 R132H mutant IDH1 immunohistochemistry in WHO grade II astrocytoma and astrocytosis. Strong granular cytoplasmic mutant IDH1 staining in cellular area of astrocytoma (a HE b mutant IDH1) and in infiltrating tumor cells (c HE d mutant ... To date, detection of IDH mutations in low-grade astrocytomas (WHO grade II) ranges from 59 to 88% [1, 5, 7, 11, 12]; as expected, the use of an antibody specific only for the R132H mutant IDH1 resulted in a slightly lower detection rate. However, in positive cases, tumor cells demonstrated staining both in the densely cellular areas of the tumor, as well as in the less cellular infiltrating tumor edges. This latter finding is important since the less cellular areas of tumors can be the most difficult to differentiate from non-neoplastic conditions in a stereotactic biopsy and may not yield sufficient tumor DNA after extraction to allow mutant IDH1 detection by sequencing. We therefore demonstrate, for the first time, that use of immunohistochemistry with an antibody specific for the common mutant form of IDH1 is a powerful and easy adjunct to practical neuropathological diagnosis. The antibody is likely to find its place quickly alongside that of p53 in such cases. Indeed, our data further suggests that when p53 is used concomitantly with mutant IDH1, the ability of immunohistochemistry to confirm the morphologic impression of glioma is enhanced. These findings also illustrate the increasing rapidity with which molecular assays are being converted to immunohistochemical stains. In the diagnosis of atypical teratoid/rhabdoid tumor, the transition from fluorescence in situ hybridization for chromosome 22q loss, to INI1 gene sequencing, to INI1 immunohistochemistry took well over 10 years; today, INI1 immunohistochemistry represents the commonly and widely used method. In the case of IDH1, scarcely more than one year has passed between discovery of mutations in diffuse gliomas and the implementation of diagnostic IDH1 immunohistochemistry—attesting to the quickening pace of diagnostic change.

Isocitrate dehydrogenase 1 analysis differentiates gangliogliomas from infiltrative gliomas.

Recent work has identified novel point mutations in isocitrate dehydrogenase 1 (IDH1) in the majority of the World Health Organization grades II and III infiltrative gliomas and secondary grade IV glioblastomas. Gangliogliomas consist of neoplastic ganglion and glial cells and, in contrast to infiltrative gliomas, are generally indolent. Yet distinguishing between a ganglioglioma and an infiltrative glioma with admixed gray matter can be difficult, perhaps accounting for some “gangliogliomas” that ultimately show aggressive behavior. In this multi‐institutional study, 98 cases originally diagnosed as ganglioglioma were analyzed for IDH1 mutations, 86 of which had follow‐up data available. Eight cases (8.2%) were positive for R132H IDH1 mutations; six had silent IDH2 mutations and two had nonsense IDH2 mutations. The presence of mutant IDH1 in gangliogliomas correlated with a greater risk of recurrence (P = 0.0007) and malignant transformation and/or death (P < 0.0001) compared with tumors that were IDH1 wild type. Furthermore, the age of patients with IDH1‐mutant gangliogliomas was higher than those without mutations (25.5 vs. 46.1 years, P = 0.0033). IDH1/2 testing of tumors suspected of being gangliogliomas may therefore be advisable, particularly in the adult population.

A Sensitive and Specific Diagnostic Panel to Distinguish Diffuse Astrocytoma from Astrocytosis: Chromosome 7 Gain with Mutant Isocitrate Dehydrogenase 1 and p53

One of the major challenges of surgical neuropathology is the distinction of diffuse astrocytoma (World Health Organization grade II) from astrocytosis. The most commonly used ancillary tool to solve thisproblem is p53 immunohistochemistry (IHC), but this is neither sensitive nor specific. Isocitrate dehydrogenase 1 (IDH1) mutations arecommon in lower-grade gliomas, with most causing a specific amino acid change (R132H) that can be detected with a monoclonal antibody. IDH2 mutations are rare, but they also occur in gliomas. In addition, gains of chromosome 7 are common in gliomas. In this study, we assessed the status of p53, IDH1/2, and chromosome 7 to determine the most useful panel to distinguish astrocytoma from astrocytosis. We studied biopsy specimens from 21 World Health Organization grade II diffuse astrocytomas and 20 reactive conditions. The single most sensitive test to identify astrocytoma is fluorescence insitu hybridization for chromosome 7 gain (76.2%). The combinationof p53 and mutant IDH1 IHC provides a higher sensitivity (71.4%) than either test alone (47.8%); this combination offers a practical initial approach for the surgical pathologist. The best overall sensitivity (95%) is achieved when fluorescence in situ hybridization for chromosome 7 gain is added to the p53-mutant IDH1 IHC panel.

Mechanisms of Glioma Formation: Iterative Perivascular Glioma Growth and Invasion Leads to Tumor Progression, VEGF-Independent Vascularization, and Resistance to Antiangiogenic Therapy

As glioma cells infiltrate the brain they become associated with various microanatomic brain structures such as blood vessels, white matter tracts, and brain parenchyma. How these distinct invasion patterns coordinate tumor growth and influence clinical outcomes remain poorly understood. We have investigated how perivascular growth affects glioma growth patterning and response to antiangiogenic therapy within the highly vascularized brain. Orthotopically implanted rodent and human glioma cells are shown to commonly invade and proliferate within brain perivascular space. This form of brain tumor growth and invasion is also shown to characterize de novo generated endogenous mouse brain tumors, biopsies of primary human glioblastoma (GBM), and peripheral cancer metastasis to the human brain. Perivascularly invading brain tumors become vascularized by normal brain microvessels as individual glioma cells use perivascular space as a conduit for tumor invasion. Agent-based computational modeling recapitulated biological perivascular glioma growth without the need for neoangiogenesis. We tested the requirement for neoangiogenesis in perivascular glioma by treating animals with angiogenesis inhibitors bevacizumab and DC101. These inhibitors induced the expected vessel normalization, yet failed to reduce tumor growth or improve survival of mice bearing orthotopic or endogenous gliomas while exacerbating brain tumor invasion. Our results provide compelling experimental evidence in support of the recently described failure of clinically used antiangiogenics to extend the overall survival of human GBM patients.

Extensive Survey of STAT6 Expression in a Large Series of Mesenchymal Tumors

OBJECTIVES Expression of strong nuclear STAT6 is thought to be a specific marker for solitary fibrous tumors (SFTs). Little is known about subtle expression patterns in other mesenchymal lesions. METHODS We performed immunohistochemical studies against the C-terminus of STAT6 in tissue microarrays and whole sections, comprising 2366 mesenchymal lesions. RESULTS Strong nuclear STAT6 was expressed in 285 of 2,021 tumors, including 206 of 240 SFTs, 49 of 408 well-differentiated/dedifferentiated liposarcomas, eight of 65 unclassified sarcomas, and 14 of 184 desmoid tumors, among others. Expression in SFTs was predominately limited to the nucleus. Other positive tumors typically expressed both nuclear and cytoplasmic STAT6. Complete absence of STAT6 was most common in pleomorphic liposarcoma and alveolar soft part sarcoma (60% and 72% cases negative, respectively). CONCLUSIONS Strong nuclear STAT6 is largely specific for SFTs. Physiologic low-level cytoplasmic/nuclear expression is common in mesenchymal neoplasia and is of uncertain significance.

Rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated Raman scattering microscopy

Conventional methods for intraoperative histopathologic diagnosis are labour- and time-intensive, and may delay decision-making during brain-tumour surgery. Stimulated Raman scattering (SRS) microscopy, a label-free optical process, has been shown to rapidly detect brain-tumour infiltration in fresh, unprocessed human tissues. Here, we demonstrate the first application of SRS microscopy in the operating room by using a portable fibre-laser-based microscope and unprocessed specimens from 101 neurosurgical patients. We also introduce an image-processing method – stimulated Raman histology (SRH) – which leverages SRS images to create virtual haematoxylin-and-eosin-stained slides, revealing essential diagnostic features. In a simulation of intraoperative pathologic consultation in 30 patients, we found a remarkable concordance of SRH and conventional histology for predicting diagnosis (Cohens kappa, κ > 0.89), with accuracy exceeding 92%. We also built and validated a multilayer perceptron based on quantified SRH image attributes that predicts brain-tumour subtype with 90% accuracy. Our findings provide insight into how SRH can now be used to improve the surgical care of brain tumour patients.

A novel mutation expands the genetic and clinical spectrum of MYH7-related myopathies

MYH7 mutations are an established cause of Laing distal myopathy, myosin storage myopathy, and cardiomyopathy, as well as additional myopathy subtypes. We report a novel MYH7 mutation (p.Leu1597Arg) that arose de novo in two unrelated probands. Proband 1 has a myopathy characterized by distal weakness and prominent contractures and histopathology typical of multi-minicore disease. Proband 2 has an axial myopathy and histopathology consistent with congenital fiber type disproportion. These cases highlight the broad spectrum of clinical and histological patterns associated with MYH7 mutations, and provide further evidence that MYH7 is likely responsible for a greater proportion of congenital myopathies than currently appreciated.

Prognostic and Predictive Biomarkers in Adult and Pediatric Gliomas: Toward Personalized Treatment

It is increasingly clear that both adult and pediatric glial tumor entities represent collections of neoplastic lesions, each with individual pathological molecular events and treatment responses. In this review, we discuss the current prognostic biomarkers validated for clinical use or with future clinical validity for gliomas. Accurate prognostication is crucial for managing patients as treatments may be associated with high morbidity and the benefits of high risk interventions must be judged by the treating clinicians. We also review biomarkers with predictive validity, which may become clinically relevant with the development of targeted therapies for adult and pediatric gliomas.