Coffin-Siris Syndrome and SMARCB1 Mutation Presenting With Schwannomatosis: A Case Report and Literature Review.

Coffin-Siris syndrome (CSS) is a rare genetic condition associated with mutations in genes responsible for the modulation of gene expression and chromatin remodeling. Patients with CSS commonly present with congenital anomalies, intellectual disabilities, and developmental delays. We describe a case of a 28-year-old woman with a confirmed diagnosis of CSS and SMARCB1 mutation who presents with multiple schwannomas and an intra-abdominal neurofibroma. The patient underwent embolization and resection of an enlarging, symptomatic schwannoma of her left medial upper arm. In detailing the patient's presentation, this case report underscores the association between SMARCB1 mutations, CSS, and tumorigenesis.

Best practices for achieving consensus in HER2-low expression in breast cancer: current perspectives from practising pathologists.

AIMS: Human epidermal growth factor receptor 2 (HER2) expression is an important biomarker in breast cancer (BC). Most BC cases categorised as HER2-negative (HER2-) express low levels of HER2 [immunohistochemistry (IHC) 1+ or IHC 2+/in-situ hybridisation not amplified (ISH-)] and represent a clinically relevant therapeutic category that is amenable to targeted therapy using a recently approved HER2-directed antibody-drug conjugate. A group of practising pathologists, with expertise in breast pathology and BC biomarker testing, outline best practices and guidance for achieving consensus in HER2 IHC scoring for BC. METHODS AND RESULTS: The authors describe current knowledge and challenges of IHC testing and scoring of HER2-low expressing BC and provide best practices and guidance for accurate identification of BCs expressing low levels of HER2. These expert pathologists propose an algorithm for assessing HER2 expression with validated IHC assays and incorporate the 2023 American Society of Clinical Oncology and College of American Pathologist guideline update. The authors also provide guidance on when to seek consensus for HER2 IHC scoring, how to incorporate HER2-low into IHC reporting and present examples of HER2 IHC staining, including challenging cases. CONCLUSIONS: Awareness of BC cases that are negative for HER protein overexpression/gene amplification and the related clinical relevance for targeted therapy highlight the importance of accurate HER2 IHC scoring for optimal treatment selection.

Non-Islet Cell Tumor Hypoglycemia Secondary to a 20 cm Intra-Abdominal Leiomyoma in a Male Patient: A Case Report and Literature Review.

Non-islet cell tumor hypoglycemia (NICTH) is a rare clinical entity associated with large mesenchymal tumors. Its pathogenesis is most commonly mediated by tumor overproduction of "big" insulin-like growth factor-2. Here, we present a 54-year-old male who presented with noninsulin-mediated hypoglycemia and a 20 cm intra-abdominal leiomyoma. His hypoglycemic episodes resolved after the resection of his tumor. To our knowledge, this is the only documented case in the English literature of NICTH associated with leiomyoma in a male patient. NICTH due to a benign leiomyoma should be in the differential diagnosis for any patient with hypoglycemia and an abdominal mass.

Presurgical Upgrade Prediction of DCIS to Invasive Ductal Carcinoma Using Time-dependent Deep Learning Models with DCE MRI.

Purpose To determine whether time-dependent deep learning models can outperform single time point models in predicting preoperative upgrade of ductal carcinoma in situ (DCIS) to invasive malignancy at dynamic contrast-enhanced (DCE) breast MRI without a lesion segmentation prerequisite. Materials and Methods In this exploratory study, 154 cases of biopsy-proven DCIS (25 upgraded at surgery and 129 not upgraded) were selected consecutively from a retrospective cohort of preoperative DCE MRI in women with a mean age of 59 years at time of diagnosis from 2012 to 2022. Binary classification was implemented with convolutional neural network (CNN)-long short-term memory (LSTM) architectures benchmarked against traditional CNNs without manual segmentation of the lesions. Combinatorial performance analysis of ResNet50 versus VGG16-based models was performed with each contrast phase. Binary classification area under the receiver operating characteristic curve (AUC) was reported. Results VGG16-based models consistently provided better holdout test AUCs than did ResNet50 in CNN and CNN-LSTM studies (multiphase test AUC, 0.67 vs 0.59, respectively, for CNN models [P = .04] and 0.73 vs 0.62 for CNN-LSTM models [P = .008]). The time-dependent model (CNN-LSTM) provided a better multiphase test AUC over single time point (CNN) models (0.73 vs 0.67; P = .04). Conclusion Compared with single time point architectures, sequential deep learning algorithms using preoperative DCE MRI improved prediction of DCIS lesions upgraded to invasive malignancy without the need for lesion segmentation. Keywords: MRI, Dynamic Contrast-enhanced, Breast, Convolutional Neural Network (CNN) Supplemental material is available for this article. © RSNA, 2024.

The current state of digital cytology and artificial intelligence (AI): global survey results from the American Society of Cytopathology Digital Cytology Task Force.

INTRODUCTION: The integration of whole slide imaging (WSI) and artificial intelligence (AI) with digital cytology has been growing gradually. Therefore, there is a need to evaluate the current state of digital cytology. This study aimed to determine the current landscape of digital cytology via a survey conducted as part of the American Society of Cytopathology (ASC) Digital Cytology White Paper Task Force. MATERIALS AND METHODS: A survey with 43 questions pertaining to the current practices and experiences of WSI and AI in both surgical pathology and cytology was created. The survey was sent to members of the ASC, the International Academy of Cytology (IAC), and the Papanicolaou Society of Cytopathology (PSC). Responses were recorded and analyzed. RESULTS: In total, 327 individuals participated in the survey, spanning a diverse array of practice settings, roles, and experiences around the globe. The majority of responses indicated there was routine scanning of surgical pathology slides (n = 134; 61%) with fewer respondents scanning cytology slides (n = 150; 46%). The primary challenge for surgical WSI is the need for faster scanning and cost minimization, whereas image quality is the top issue for cytology WSI. AI tools are not widely utilized, with only 16% of participants using AI for surgical pathology samples and 13% for cytology practice. CONCLUSIONS: Utilization of digital pathology is limited in cytology laboratories as compared to surgical pathology. However, as more laboratories are willing to implement digital cytology in the near future, the establishment of practical clinical guidelines is needed.

Habitat escalated adaptive therapy (HEAT): a phase 2 trial utilizing radiomic habitat-directed and genomic-adjusted radiation dose (GARD) optimization for high-grade soft tissue sarcoma.

BACKGROUND: Soft tissue sarcomas (STS), have significant inter- and intra-tumoral heterogeneity, with poor response to standard neoadjuvant radiotherapy (RT). Achieving a favorable pathologic response (FPR ≥ 95%) from RT is associated with improved patient outcome. Genomic adjusted radiation dose (GARD), a radiation-specific metric that quantifies the expected RT treatment effect as a function of tumor dose and genomics, proposed that STS is significantly underdosed. STS have significant radiomic heterogeneity, where radiomic habitats can delineate regions of intra-tumoral hypoxia and radioresistance. We designed a novel clinical trial, Habitat Escalated Adaptive Therapy (HEAT), utilizing radiomic habitats to identify areas of radioresistance within the tumor and targeting them with GARD-optimized doses, to improve FPR in high-grade STS. METHODS: Phase 2 non-randomized single-arm clinical trial includes non-metastatic, resectable high-grade STS patients. Pre-treatment multiparametric MRIs (mpMRI) delineate three distinct intra-tumoral habitats based on apparent diffusion coefficient (ADC) and dynamic contrast enhanced (DCE) sequences. GARD estimates that simultaneous integrated boost (SIB) doses of 70 and 60 Gy in 25 fractions to the highest and intermediate radioresistant habitats, while the remaining volume receives standard 50 Gy, would lead to a > 3 fold FPR increase to 24%. Pre-treatment CT guided biopsies of each habitat along with clip placement will be performed for pathologic evaluation, future genomic studies, and response assessment. An mpMRI taken between weeks two and three of treatment will be used for biological plan adaptation to account for tumor response, in addition to an mpMRI after the completion of radiotherapy in addition to pathologic response, toxicity, radiomic response, disease control, and survival will be evaluated as secondary endpoints. Furthermore, liquid biopsy will be performed with mpMRI for future ancillary studies. DISCUSSION: This is the first clinical trial to test a novel genomic-based RT dose optimization (GARD) and to utilize radiomic habitats to identify and target radioresistance regions, as a strategy to improve the outcome of RT-treated STS patients. Its success could usher in a new phase in radiation oncology, integrating genomic and radiomic insights into clinical practice and trial designs, and may reveal new radiomic and genomic biomarkers, refining personalized treatment strategies for STS. TRIAL REGISTRATION: NCT05301283. TRIAL STATUS: The trial started recruitment on March 17, 2022.

Utility of artificial intelligence in a binary classification of soft tissue tumors.

Soft tissue tumors (STTs) pose diagnostic and therapeutic challenges due to their rarity, complexity, and morphological overlap. Accurate differentiation between benign and malignant STTs is important to set treatment directions, however, this task can be difficult. The integration of machine learning and artificial intelligence (AI) models can potentially be helpful in classifying these tumors. The aim of this study was to investigate AI and machine learning tools in the classification of STT into benign and malignant categories. This study consisted of three components: (1) Evaluation of whole-slide images (WSIs) to classify STT into benign and malignant entities. Five specialized soft tissue pathologists from different medical centers independently reviewed 100 WSIs, representing 100 different cases, with limited clinical information and no additional workup. The results showed an overall concordance rate of 70.4% compared to the reference diagnosis. (2) Identification of cell-specific parameters that can distinguish benign and malignant STT. Using an image analysis software (QuPath) and a cohort of 95 cases, several cell-specific parameters were found to be statistically significant, most notably cell count, nucleus/cell area ratio, nucleus hematoxylin density mean, and cell max caliper. (3) Evaluation of machine learning library (Scikit-learn) in differentiating benign and malignant STTs. A total of 195 STT cases (156 cases in the training group and 39 cases in the validation group) achieved approximately 70% sensitivity and specificity, and an AUC of 0.68. Our limited study suggests that the use of WSI and AI in soft tissue pathology has the potential to enhance diagnostic accuracy and identify parameters that can differentiate between benign and malignant STTs. We envision the integration of AI as a supportive tool to augment the pathologists' diagnostic capabilities.

Emerging Landscape of Targeted Therapy of Breast Cancers With Low Human Epidermal Growth Factor Receptor 2 Protein Expression.

CONTEXT.­: Human epidermal growth factor receptor 2 (HER2) status in breast cancer is currently classified as negative or positive for selecting patients for anti-HER2 targeted therapy. The evolution of the HER2 status has included a new HER2-low category defined as an HER2 immunohistochemistry score of 1+ or 2+ without gene amplification. This new category opens the door to a targetable HER2-low breast cancer population for which new treatments may be effective. OBJECTIVE.­: To review the current literature on the emerging category of breast cancers with low HER2 protein expression, including the clinical, histopathologic, and molecular features, and outline the clinical trials and best practice recommendations for identifying HER2-low-expressing breast cancers by immunohistochemistry. DATA SOURCES.­: We conducted a literature review based on peer-reviewed original articles, review articles, regulatory communications, ongoing and past clinical trials identified through ClinicalTrials.gov, and the authors' practice experience. CONCLUSIONS.­: The availability of new targeted therapy potentially effective for patients with breast cancers with low HER2 protein expression requires multidisciplinary recognition. In particular, pathologists need to recognize and identify this category to allow the optimal selection of patients for targeted therapy.

Stroma-Mediated Breast Cancer Cell Proliferation Indirectly Drives Chemoresistance by Accelerating Tumor Recovery between Chemotherapy Cycles.

The ability of tumors to survive therapy reflects both cell-intrinsic and microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity induced by stroma-produced paracrine factors. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies. In vitro studies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. At the same time, consistent with prior studies, fibroblast-produced secreted factors stimulated treatment-independent enhancement of tumor cell proliferation. Spatial analyses indicated that proximity to stroma is often associated with enhanced tumor cell proliferation in vivo. These observations suggested an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To evaluate this hypothesis, a spatial agent-based model of stroma impact on proliferation/death dynamics was developed that was quantitatively parameterized using inferences from histologic analyses and experimental studies. The model demonstrated that the observed enhancement of tumor cell proliferation within stroma-proximal niches could enable tumors to avoid elimination over multiple chemotherapy cycles. Therefore, this study supports the existence of an indirect mechanism of environment-mediated chemoresistance that might contribute to the negative correlation between stromal content and poor therapy outcomes. SIGNIFICANCE: Integration of experimental research with mathematical modeling reveals an indirect microenvironmental chemoresistance mechanism by which stromal cells stimulate breast cancer cell proliferation and highlights the importance of consideration of proliferation/death dynamics. See related commentary by Wall and Echeverria, p. 3667.

Paracrine enhancement of tumor cell proliferation provides indirect stroma-mediated chemoresistance via acceleration of tumor recovery between chemotherapy cycles.

The ability of tumors to survive therapy reflects both cell-intrinsic and microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity by stroma-produced paracrine factors. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies. Our in vitro studies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. At the same time, consistent with prior studies, we observed treatment-independent enhancement of tumor cell proliferation by fibroblast-produced secreted factors. Using spatial statistics analyses, we found that proximity to stroma is often associated with enhanced tumor cell proliferation in vivo . Based on these observations, we hypothesized an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To evaluate the feasibility of this hypothesis, we developed a spatial agent-based model of stroma impact on proliferation/death dynamics. The model was quantitatively parameterized using inferences from histological analyses and experimental studies. We found that the observed enhancement of tumor cell proliferation within stroma-proximal niches can enable tumors to avoid elimination over multiple chemotherapy cycles. Therefore, our study supports the existence of a novel, indirect mechanism of environment-mediated chemoresistance that might contribute to the negative correlation between stromal content and poor therapy outcomes.

Genomic Patterns of Malignant Peripheral Nerve Sheath Tumor (MPNST) Evolution Correlate with Clinical Outcome and Are Detectable in Cell-Free DNA.

Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis. SIGNIFICANCE: MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups. This article is highlighted in the In This Issue feature, p. 517.

Validation of PRKCB Immunohistochemistry as a Biomarker for the Diagnosis of Ewing Sarcoma.

Background: Ewing sarcoma (ES) can be confirmed by identifying the EWSR1-FLI1 fusion transcript. This study is to investigate whether immunostaining (IHC) of PRKCB-a protein directly regulated by EWSR1-FLI1 is a surrogate maker for diagnosing ES in routine practice. Methods: Microarray gene expression analyses were conducted. RKCB IHC was applied to 69 ES confirmed by morphology and molecular methods, and 41 non-Ewing small round cell tumors. EWSR1 rearrangement, EWSR1-FLI1 fusion or t(11;22)(q24;q12) were identified by fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, or cytogenetic analysis, respectively. Results: Gene array analyses showed significant overexpression of the PRKCB in ES. PRKCB IHC was positive in 19 cases of ES with EWSR1-FLI1 fusion, 3 cases with cytogenetic 11:22 translocation and 59 cases with EWSR1 rearrangement while negative in only one EWSR1 rearranged case. PRKCB IHC is sensitive (98%) and specific (96%) in detecting EWSR1 rearranged ES. Conclusions: PRKCB is a reliable antibody for diagnosing ES in routine practice.

Integrative Diagnostics: The Time Is Now-A Report From the International Society for Strategic Studies in Radiology.

Enormous recent progress in diagnostic testing can enable more accurate diagnosis and improved clinical outcomes. Yet these tests are increasingly challenging and frustrating; the volume and diversity of results may overwhelm the diagnostic acumen of even the most dedicated and experienced clinician. Because they are gathered and processed within the "silo" of each diagnostic discipline, diagnostic data are fragmented, and the electronic health record does little to synthesize new and existing data into usable information. Therefore, despite great promise, diagnoses may still be incorrect, delayed, or never made. Integrative diagnostics represents a vision for the future, wherein diagnostic data, together with clinical data from the electronic health record, are aggregated and contextualized by informatics tools to direct clinical action. Integrative diagnostics has the potential to identify correct therapies more quickly, modify treatment when appropriate, and terminate treatment when not effective, ultimately decreasing morbidity, improving outcomes, and avoiding unnecessary costs. Radiology, laboratory medicine, and pathology already play major roles in medical diagnostics. Our specialties can increase the value of our examinations by taking a holistic approach to their selection, interpretation, and application to the patient's care pathway. We have the means and rationale to incorporate integrative diagnostics into our specialties and guide its implementation in clinical practice.

Effect of Favorable Pathologic Response After Neoadjuvant Radiation Therapy Alone in Soft-tissue Sarcoma.

Purpose: Whether the therapeutic response of soft-tissue sarcoma to neoadjuvant treatment is predictive for clinical outcomes is unclear. Given the rarity of this disease and the confounding effects of chemotherapy, this study analyzes whether a favorable pathologic response (fPR) after neoadjuvant radiation therapy (RT) alone is associated with clinical benefits. Methods and Materials: An institutional review board-approved retrospective review was conducted on a database of patients with primary soft-tissue sarcoma treated at our institution between 1987 and 2015 with neoadjuvant RT alone followed by surgical resection. Time-to-event outcomes estimated with a Kaplan-Meier analysis included overall survival, progression-free survival (PFS), locoregional control, and distant control (DC). Cox regression analyses were performed to determine prognostic variables associated with clinical outcomes. Results: Of the overall cohort of 315 patients, 181 patients (57%) were included in the primary analysis with documented pathologic necrosis (PN) rates (mean: 59%) and a median follow up from diagnosis of 48 months (range, 4-170 months). The median neoadjuvant RT dose was 50 Gy (range, 40-60 Gy), and the majority of patients had negative surgical margins (79%). Only 35 patients (19%) achieved a fPR (PN ≥95%), which was associated with a higher R0 resection rate (94% vs. 75%; P = .013), a significant 5-year PFS benefit (74% vs. 43%; P = .014), and a nonsignificant 5-year DC benefit (76% vs. 62%; P = .12) compared with PN <95%. On multivariable analysis, fPR was an independent predictor for PFS (hazard ratio: 0.47; 95% confidence interval, 0.25-0.90; P = .022). Conclusions: Achieving fPR with neoadjuvant RT alone is associated with a higher R0 resection rate and possible DC benefit, translating into a significant improvement in PFS. Further studies to improve pathologic response rates and prospectively validate this endpoint are warranted.

NCCN Guidelines® Insights: Gastrointestinal Stromal Tumors, Version 2.2022.

Gastrointestinal stromal tumors (GIST) are the most common type of soft tissue sarcoma that occur throughout the gastrointestinal tract. Most of these tumors are caused by oncogenic activating mutations in the KIT or PDGFRA genes. The NCCN Guidelines for GIST provide recommendations for the diagnosis, evaluation, treatment, and follow-up of patients with these tumors. These NCCN Guidelines Insights summarize the panel discussion behind recent important updates to the guidelines, including revised systemic therapy options for unresectable, progressive, or metastatic GIST based on mutational status, and updated recommendations for the management of GIST that develop resistance to specific tyrosine kinase inhibitors.

A Malignant Glomus Tumor of the Liver Harboring MIR143-NOTCH2 Rearrangement: From Diagnosis to Management.

A primary malignant glomus tumor of the liver is extremely rare and diagnostically challenging. We present an exceptional case of such with a diagnosis confirmed by MIR143-NOTCH2 rearrangement. The case was successfully managed with neoadjuvant chemotherapy followed by surgery. This report highlights the utilization of molecular analysis to aid in the diagnosis of rare soft tissue malignancies and supports a multimodality approach to the treatment of large, high-grade malignant glomus tumors.

Assessing ROSE for adequacy of EBUS-TBNA compared with a direct-to-cell block approach as a response to the COVID-19 pandemic.

INTRODUCTION: Rapid on-site evaluation (ROSE) has been used during the endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) procedure as standard practice. Because of the COVID-19 (coronavirus disease 2019) pandemic, our institute had had to discontinue ROSE and adopt a direct-to-cell block approach. In the present study, we aimed to determine whether this change has had significant effects on the cytopathology quality. MATERIALS AND METHODS: A total of 1903 EBUS-TBNA cases from 734 patients were collected (1097 cases with ROSE for 452 patients; 806 cases without ROSE but with direct-to-cell block for 282 patients). The clinical and cytology data were analyzed using SAS, version 9.4, software to render calculated standardized residuals and a fitted multivariate generalized linear model. RESULTS: On average, a biopsy from a patient with ROSE was 0.936 (=exp -0.066) times less likely to be reported as satisfactory compared with a biopsy from a patient without ROSE, although the difference was not statistically significant (P = 0.785). The inadequacy rate of EBUS-TBNA was 6.4% higher on average for cases with ROSE compared with a direct-to-cell block approach. However, this difference was also not statistically significant. The proportions of biopsies reported as diagnostic for malignancy and other were significantly different between the ROSE and no-ROSE groups with a standardized residual of 1.80 (P = 0.036) and -2.27 (P = 0.012), respectively. CONCLUSIONS: Discontinuing ROSE and using a direct-to-cell block approach had no negative effects on cytopathology quality. This practice can be considered acceptable during the COVID-19 pandemic when social distancing and the shortage of staff and supplies have resulted in challenges to delivering quality care to cancer patients whose treatment cannot be postponed.

Soft Tissue Sarcoma, Version 2.2022, NCCN Clinical Practice Guidelines in Oncology.

Soft tissue sarcomas (STS) are rare malignancies of mesenchymal cell origin that display a heterogenous mix of clinical and pathologic characteristics. STS can develop from fat, muscle, nerves, blood vessels, and other connective tissues. The evaluation and treatment of patients with STS requires a multidisciplinary team with demonstrated expertise in the management of these tumors. The complete NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Soft Tissue Sarcoma provide recommendations for the diagnosis, evaluation, and treatment of extremity/superficial trunk/head and neck STS, as well as retroperitoneal/intra-abdominal STS, desmoid tumors, and rhabdomyosarcoma. This portion of the NCCN Guidelines discusses general principles for the diagnosis and treatment of retroperitoneal/intra-abdominal STS, outlines treatment recommendations, and reviews the evidence to support the guidelines recommendations.

Proton export upregulates aerobic glycolysis.

INTRODUCTION: Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the "Warburg Effect." It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. RESULTS: To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. CONCLUSIONS: Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.

Lipogenesis mediated by OGR1 regulates metabolic adaptation to acid stress in cancer cells via autophagy.

Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here, we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype, is a cellular adaption to extracellular acidity. LD marker PLIN2 is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing G-protein-coupled receptor (GPCR) OGR1, which is expressed highly in breast tumors. OGR1 depletion inhibits acid-induced lipid accumulation, while activation by a synthetic agonist triggers LD formation. Inhibition of OGR1 downstream signaling abrogates the lipogenic phenotype, which can be rescued with OGR1 ectopic expression. OGR1-depleted cells show growth inhibition under acidic growth conditions in vitro and tumor formation in vivo. Isotope tracing shows that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1-depleted cells are defective in endoplasmic reticulum stress response and autophagy and hence fail to accumulate LDs affecting survival under acidic stress.