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PDF File - (58K) - Secondary screen identified 168 GSC -effective compounds with selective inhibition profile

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

PDF File - (32K) - GSC-enriched GBM samples form tumors after transplantation into immunodeficient mice

PDF File - (874K) - A - Representative images demonstrating tumor formation success in an animal transplanted with serum (I) and sphere (II) derived GBM146 Ki67 and Human Nuclei (HuNu) immunostaining shows that virtually all transplanted cells died off in the animal transplanted with serum derived cells while an infiltrating tumor mass is visible in the animal transplanted with sphere derived cells of the same GBM sample (i - injection site, V- ventricle, SVZ - subventricular zone). B - Immunohistological sections for Human Nuclei (HuNu) and Ki67 demonstrating good survival of transplanted cells in animals that were sacrificed on the same day as well as 48h after transplantation (IT- injection tract, ST-striatum).

PDF File - (67K) - Effects of OLDA, PALDA and NADA are not mediated via cannabinoid, vanilloid or dopamine receptors A - Exposure of GSC -enriched GBM 157 cells to CB1, CB2, TRPV1 receptor agonists or their combination, does not reproduce the phenotype seen after exposure to OLDA, PALDA or NADA. Graph showing ATP level expressed as % of DMSO treated controls. B - Exposure of OLDA, PALDA or NADA-pretreated GSC -enriched GBM 157 cells (at IC50 concentration) to antagonists of CB, TRPV and D receptors does not rescue the phenotype caused by OLDA, PALDA or NADA (B, C and D, respectively). The graphs are showing ATP levels expressed as % of the DMSO treated controls (100%). SE shown.

PDF File - (61K) - OLDA, Emetine, 5256360 and 5560509 decrease clonal sphere formation in low passage GBM312 Graphs show a concentration-dependent reduction of clonal neurosphere size, neurosphere number as well as total cell mass. Error bars represent SEM.

PDF File - (39K) - Limiting dilution transplantation of GBM157 cells treated with OLDA, 5256360 or 5560509 shows decreased tumor formation frequency

PDF File - (523K) - OLDA, Emetine, 5256360 and 5560509 inhibit proliferation of sphere cultured GBM312 cells, using CFSE (carboxyfluorescein diacetate, succinimidyl ester) cell tracing experiments Proliferative inhibition is graphically represented by an increase in fluorescence or a shift to the right, away from that of the control. A proliferation index (PI) is a numerical value generated for each sample that measures the total increase in cell number divided by the original parent number. PI's are expressed as a percent of control: Emetine (0.2�M)- 18.6%, OLDA (4�M)- 16.0%, Compound 33 (2�M)- 31%, and Compound 62 (2�M)- 24.3%.

PDF File - (40K) - Growth kinetics of serum and sphere derived cells in screening (sphere) media reveals only minor differences in the degree of growth/proliferation between sphere and serum derived cultures of the same cell type Graph shows results of an ATP-based proliferation assay as measured 3 days after cell plating. Error bars depict SEM.

PDF File - (55K) - Sensitivity of GBM cells to OLDA, PALDA and NADA changes as a function of time, for which the cells were cultured in GSC -enriching media GBM107 cells were exposed to GSC -enriching media for increasing amounts of time and were then screened for sensitivity to dopaminergic cannabinoid compounds. While screening in serum-containing media requires a higher compound concentration due to the protective effect of serum in general, serum-derived cells become more and more sensitive towards GSC -specific compounds over time after transfer into sphere media (A, B, C). Such a difference is not present using non-GSC selective chemotherapeutics (D) or non-GBM control cells (E, F). ('ser in ser': serum derived cells screened in serum media; 'ser in sph': serum derived cells screened in sphere media; '1d sph': serum derived cells were cultured in sphere media for one day before the screen, etc.)

BackgroundGlioblastoma is a deadly brain tumor with median patient survival of 14.6 months. At the core of this malignancy are rare, highly heterogenous malignant stem-like tumor initiating cells. Aberrant signaling across the EGFR-PTEN-AKT-mTOR signal transduction pathways are common oncogenic drivers in these cells. Though gene-level clustering has determined the importance of the EGFR signaling pathway as a treatment indicator, multiparameter protein-level analyses are necessary to discern functional attributes of signal propagation. Multiparameter single cell analyses is emerging as particularly useful in identifying such attributes.MethodsSingle cell targeted proteomic analysis of EGFR-PTEN-AKT-mTOR proteins profiled heterogeneity in a panel of fifteen patient derived gliomaspheres. A microfluidic cell array ‘chip’ tool served as a low cost methodology to derive high quality quantitative single cell analytical outputs. Chip design specifications produced extremely high signal-to-noise ratios and brought experimental efficiencies of cell control and minimal cell use to accommodate experimentation with these rare and often slow-growing cell populations. Quantitative imaging software generated datasets to observe similarities and differences within and between cells and patients. Bioinformatic self-organizing maps (SOMs) and hierarchical clustering stratified patients into malignancy and responder groups which were validated by phenotypic and statistical analyses.ResultsFifteen patient dissociated gliomaspheres produced 59,464 data points from 14,866 cells. Forty-nine molecularly defined signaling phenotypes were identified across samples. Bioinformatics resolved two clusters diverging on EGFR expression (p = 0.0003) and AKT/TORC1 activation (p = 0.08 and p = 0.09 respectively). TCGA status of a subset showed genetic heterogeneity with proneural, classical and mesenchymal subtypes represented in both clusters. Phenotypic validation measures indicated drug responsive phenotypes to EGFR blocking were found in the EGFR expressing cluster. EGFR expression in the subset of drug-treated lines was statistically significant (pConclusionsQuantitative single cell heterogeneity profiling resolves signaling diversity into meaningful non-obvious phenotypic groups suggesting EGFR is decoupled from AKT/TORC1 signalling while identifying potentially valuable targets for personalized therapeutic approaches for deadly tumor-initiating cell populations.

Author(s): Laks, Dan R; Oses-Prieto, Juan A; Alvarado, Alvaro G; Nakashima, Jonathan; Chand, Shreya; Azzam, Daniel B; Gholkar, Ankur A; Sperry, Jantzen; Ludwig, Kirsten; Condro, Michael C; Nazarian, Serli; Cardenas, Anjelica; Shih, Michelle YS; Damoiseaux, Robert; France, Bryan; Orozco, Nicholas; Visnyei, Koppany; Crisman, Thomas J; Gao, Fuying; Torres, Jorge Z; Coppola, Giovanni; Burlingame, Alma L; Kornblum, Harley I | Abstract: Background:Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. Methods:To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. Results:An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. Conclusions:These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Peripheral T-cell lymphomas are a heterogeneous group of lymphoid malignancies. Among these, hepatosplenic γδ T-cell lymphoma (HTCL) represents an aggressive and treatment-resistant subgroup for which new avenues of treatment are critically needed. HTCL is characterized by primary extranodal distribution of the malignant cells with typical intrasinusoidal infiltration of the liver, spleen, and bone marrow, which results in hepatosplenomegaly and peripheral blood cytopenias. Another characteristic feature is the expression of γδ T-cell receptors. HTCL exhibits a rapid progressive course and an extremely poor response to currently known therapeutic strategies, with a 5-year overall survival rate of only 7%. In this review, we discuss the clinical, pathologic, and molecular characteristics of this disease, along with the challenges that are associated with its diagnosis and treatment.

Squamous cell carcinoma of the external auditory canal, middle ear and temporal bone is a rare and unusual malignancy. The lack of a unifying classification system in the past, along with the rarity of the disease has made the development of clear treatment guidelines difficult. In this report, we describe a clinical case of a patient with this rare malignancy, discuss the challenges associated with the diagnosis and treatment of the disease, and review the literature for trends while outlining the most beneficial treatment strategy for this patient population.

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.

Renewable neurosphere formation in culture is a defining characteristic of certain brain tumor initiating cells. This retrospective study was designed to assess the relationship among neurosphere formation in cultured human glioma, tumorigenic capacity, and patient clinical outcome. Tumor samples were cultured in neurosphere conditions from 32 patients with glioma, including a subpopulation of 15 patients with primary glioblastoma. A subsample of renewable neurosphere cultures was xenografted into mouse brain to determine if they were tumorigenic. Our study shows that both renewable neurosphere formation and tumorigenic capacity are significantly associated with clinical outcome measures. Renewable neurosphere formation in cultured human glioma significantly predicted an increased hazard of patient death and more rapid tumor progression. These results pertained to both the full population of glioma and the subpopulation of primary glioblastoma. Similarly, there was a significant hazard of progression for patients whose glioma had tumorigenic capacity. Multivariate analysis demonstrated that neurosphere formation remained a significant predictor of clinical outcome independent of Ki67 proliferation index. In addition, multivariate analysis of neurosphere formation, tumor grade and patient age, demonstrated that neurosphere formation was a robust, independent predictor of glioma tumor progression. Although the lengthy duration of this assay may preclude direct clinical application, these results exemplify how neurosphere culture serves as a clinically relevant model for the study of malignant glioma. Furthermore, this study suggests that the ability to propagate brain tumor stem cells in vitro is associated with clinical outcome. Disclosure of potential conflicts of interest is found at the end of this article.

Background and Purpose— In chronic hydrocephalus, a role for tissue hypoxia resulting from cerebrovascular compression is suggested. The purpose of this study was to evaluate whether changes in cerebral blood flow (CBF) in the time course of adult kaolin-induced hydrocephalus correlated with immunohistochemical neuronal responses. Methods— In 46 adult Sprague-Dawley rats, kaolin hydrocephalus was induced and immunostaining of neurofilament protein (NF68), synaptophysin (SYN38), and neuronal nitric oxide synthase (NOS) was performed at 2 (short term), 4 (intermediate term), and 6 and 8 (long term) weeks. Local CBF was measured quantitatively by [ 14 C]iodoantipyrine ([ 14 C]IAP) autoradiography in the short-term stage and in both long-term stages. Results— At 2 weeks, neuronal NOS immunoreactivity was globally increased in cortical areas and within the hippocampus. Four weeks after hydrocephalus induction, a reactive increase of SYN38 and NF68 immunoreactivity in the periventricular cortex was seen. At 6 and 8 weeks, when the ventricular size was decreasing, immunohistochemical changes in the hippocampus became most evident. A maintained toxic NOS reactivity in the CA1 subfield was accompanied by a loss of NF68 staining. In the CA3 subfield, however, focal increases in NF68 and SYN38 immunoreactivity were found. Cortical and hippocampal blood flow showed prolonged decreases of 25% to 55% compared with control animals. At 8 weeks, control levels were reached. Conclusions— The observed temporary CBF decrease appears to correlate with an early global neuronal ischemic response. In addition, it may also account for the delayed selective response of ischemia-vulnerable structures, eg, hippocampus, in chronic adult kaolin-induced hydrocephalus.

Playing Hide and Seek Targeted cancer therapies have shown promising results in patients, but few of these drugs provide long-term benefits because tumor cells rapidly develop drug resistance. Nathanson et al. (p. 72 , published online 5 December) show that glioblastoma cells can become resistant to erlotinib, an epidermal growth factor receptor (EGFR)–targeted drug, by eliminating extrachromosomal copies of the mutant EGFR gene. After a period of drug withdrawal, the mutant EGFR gene reappears on extrachromosomal DNA and the tumor cells become resensitized. The discovery that cancer cells can evade drug therapy by this “hide and seek” mechanism may help to optimize the dosing schedule of erlotinib in glioblastoma patients.

Cutaneous metastases from colorectal carcinomas are rare manifestations of widespread metastatic disease with poor prognosis. Unusual sites of cutaneous metastasis have been reported in the literature [1–13]. To the authors’ knowledge, this case will be the second case of skin metastasis to the distal leg in colon cancer in the literature. In our case, making the correct diagnosis was challenging because the metastatic soft tissue was superinfected. It is important to recognize suspicious skin lesions in cancer patients as a possible metastatic disease to the skin. Biopsy is very important because the gross appearance of these skin lesions is not distinctive.

Humoral hypercalcemia of malignancy is frequently observed in patients with solid tumors. However, few instances have been described involving patients with gynecological malignancies. We report a case of endometrioid carcinoma of the uterine corpus in a patient who initially presented with hypercalcemia. The elevated calcium levels were found to be the result of an increased serum concentration of parathyroid hormone-related peptide (PTHrP). PTHrP is commonly secreted by malignant cells and suppresses PTH. This case demonstrates that endometrial cancer should be considered in the differential diagnosis of patients presenting with symptomatic or asymptomatic hypercalcemia.

We describe the nature and goals of high-throughput screening experiments, focusing on the challenges they present from the view-point of statistical analysis. We suggest graphical displays to facilitate quality control. We describe sources of systematic variation and methods to correct for it. We consider the problem of ranking compounds with respect to their effects on one cell type and we suggest a couple of procedures, depending on the available number of replicates. Finally, we explore the use of a hierarchical framework for hypothesis testing, to study the effects of compounds in multiple cell lines.

At this time, brain tumor stem cells remain a controversial hypothesis while malignant brain tumors continue to present a dire prognosis of severe morbidity and mortality. Yet, brain tumor stem cells may represent an essential cellular target for glioma therapy as they are postulated to be the tumorigenic cells responsible for recurrence. Targeting oncogenic pathways that are essential to the survival and growth of brain tumor stem cells represents a promising area for developing therapeutics. However, due to the multiple oncogenic pathways involved in glioma, it is necessary to determine which pathways are the essential targets for therapy. Furthermore, research still needs to comprehend the morphogenic processes of cell populations involved in tumor formation. Here, we review research and discuss perspectives on models of glioma in order to delineate the current issues in defining brain tumor stem cells as therapeutic targets in models of glioma.

Multipotent neural progenitor cells or neural stem cells (NSC) can be propagated in vitro from a variety of sources and have great potential for neural repair. Although it is well known that NSC divide in response to basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF), cofactors necessary for survival and maintenance of a multipotent potential are still a matter of debate. In the current study, we examined the requirements for NSC proliferation and survival in vitro using the neurosphere culture system. Apotransferrin (TF), along with EGF and FGF-2, was sufficient for the formation of primary neurospheres derived from embryonic rat cortices. The addition of low concentrations of insulin or insulin-like growth factor-1 (IGF-1) enhanced neurosphere size and number and was necessary for continued passaging. Both insulin and IGF-1 acted at low concentrations, suggesting that their effects were mediated by their cognate receptors, both of which were expressed by neurosphere cultures. Sphere-forming progenitors survived for long periods in culture without EGF or FGF-2 when either insulin or IGF-1 was added to the media. Cell cycle analysis determined that surviving progenitors were relatively quiescent during the period without mitogens. Upon the reintroduction of EGF and FGF-2, surviving progenitors gave rise to new spheres that produced largely glial-restricted progeny compared with sister cultures. These data indicate that the neurogenic potential of NSC may be intimately linked to a continuous exposure to mitogens.

Neural progenitor transplantation is a potential treatment for neurodegenerative diseases, including Huntington's disease (HD). In the current study, we tested the potential of rat embryonic neural progenitors expanded in vitro as therapy in the rat quinolinic acid-lesioned striatum, a model that demonstrates some of the pathological features of HD. We used positron emission tomography (PET) to demonstrate that the intrastriatal injection of cultured rat neural progenitors results in improved metabolic function in the striatum and overlying cortex when compared to media-injected controls. Transplanted progenitors were capable of surviving, migrating long distances and differentiating into neurons and glia. The cortices of transplanted animals contained greater numbers of neurons in regions that had shown metabolic improvement. However, histological analysis revealed that only a small fraction of these increased neurons could be accounted for by engrafted cells, indicating that the metabolic sparing was likely the result of a trophic action of the transplanted cells on the host. Behavioral testing of the implanted animals did not reveal improvement in apomorphine-induced rotation. These data demonstrate that progenitor cell implantation results in enhanced metabolic function and sparing of neuron number, but that these functions do not necessarily result in the restoration of complex circuitry.

Rapamycin, a selective inhibitor of TOR (target of rapamycin), has been used in clinical trials for the treatment of glioma (Cloughesy et al., 2008). However, even though some patients respond temporarily to rapamycin treatment, virtually all recur. The mechanisms underlying this resistance to therapy are unknown. Here, we utilized patient-derived glioblastoma (GBM) neurosphere cultures to examine these mechanisms. GBM cell cultures exposed to chronic rapamycin yield a population of rapamycin resistant cells with an increased malignant phenotype upon removal from treatment, enhanced sphere formation in vitro and enhanced tumorigenesis. We used an unbiased, proteomics-based approach to discover phosphopeptides that were upregulated during rapamycin treatment in vitro. Amongst the peptides that we uncovered was Tle4/Groucho, a global co-repressor and known modulator of several key signaling pathways, including the Wnt and Notch pathways. In an expression microarray, Tle4 was found to be significantly upregulated within chronic rapa treated GBM cells. We show that TLE4 is a key modulator of resistance to mammalian target of rapamycin (mTOR) pathway specific inhibition. While TLE4 knockdown has no clear effect on naive cells, this knockdown promotes sensitivity under chronic rapamycin treatment as evidenced by reduced neurosphere formation. TLE4 mediate rapamycin resistance through activation of the quiescent population. Our results indicate that chronic mTOR specific inhibition imparts TLE4 mediated resistance through both ERK activation and inhibition of mTOR. The identification of this role for TLE4 in rapamycin resistance provides a novel target for mTOR pathway-associated chemotherapeutic agents. References: Cloughesy, T.F., Yoshimoto, K., Nghiemphu, P., Brown, K., Dang, J., Zhu, S., Hsueh, T., Chen, Y., Wang, W., Youngkin, D., et al. (2008). Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med 5, e8. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5607. doi:1538-7445.AM2012-5607

Introduction Body contouring injections by non-licensed providers are frequently sought out by a subset of the male-to-female transgender community. Although short-term side effects such as pulmonary embolism and injection site infection are well known, long-term consequences of such practices are less well studied. Case presentation Here we describe the case of a 40-year-old African American male-to-female transgender patient who presented to our institution with hypercalcemia and acute renal failure secondary to body contouring injections with industrial strength silicone by non-licensed providers, a decade prior to her visit. Work-up revealed an extensive granulomatous inflammatory process in the injection area resulting in electrolyte abnormalities and kidney injury. The patient’s lab results and symptoms responded well to long-term corticosteroid treatment and correlated with treatment adherence. Conclusion Affected patients can sometimes present with unusual clinical symptoms many years after silicone injections. In a constantly growing transgender community that often utilizes non-licensed providers for silicone injections, the medical community will likely face an increasing number of patients with long-term side effects of such practices. Therefore, it is imperative for physicians to recognize such cases promptly and initiate potentially life-saving treatment.