Madalina Tuluc

Cancer metabolism, stemness and tumor recurrence: MCT1 and MCT4 are functional biomarkers of metabolic symbiosis in head and neck cancer

Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67−/TOMM20−/COX−/MCT1−); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67−/TOMM20−/COX−/MCT1−). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target “three-compartment tumor metabolism” in head and neck cancers. It is remarkable that two “non-proliferating” populations of cells (Ki-67−/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial “fuels” for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial “stem cell” layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target “metabolic symbiosis.”

Salivary duct carcinoma: the predominance of apocrine morphology, prevalence of histologic variants, and androgen receptor expression.

Salivary duct carcinoma (SDC) is a prototypic aggressive salivary gland carcinoma. Our aim is to determine the prevalence of histologic variants (micropapillary, basal-like) and androgen receptor (AR) expression in a large multi-institutional series of SDC. AR status was determined by immunohistochemistry (IHC). Most SDCs were characterized by an apocrine phenotype and AR expression. Cases with a nonapocrine phenotype and AR-negative status were studied by additional IHC and fluorescence in situ hybridization for ETV6 or MYB/NFIB. The diagnosis of SDC was confirmed in 187 of 199 (94%) cases. Variant morphologies were identified in 12 cases: micropapillary (n=6), sarcomatoid (n=3), mucinous (n=2), and basal-like (n=1). AR IHC was performed in 183 cases, of which 179 (97.8%) showed AR expression. On the basis of morphologic appearance and results of additional studies, 12 cases were reclassified as squamous cell carcinoma (SCC) (n=4), epithelial-myoepithelial carcinoma with high-grade transformation (HGT) (n=2), myoepithelial carcinoma (n=2), mammary analogue secretory carcinoma, high grade (ETV6 translocated; n=1), adenoid cystic carcinoma with HGT (n=1), acinic cell carcinoma with HGT (n=1), and adenosquamous carcinoma (n=1). AR-negative SDC is extremely rare, and the majority of such cases are more accurately classified as other entities. HGTs of other salivary carcinomas and squamous cell carcinoma are the most common mimics of SDC. SDCs with variant morphologies still show at least a minor component of conventional apocrine appearance. Thus, apocrine morphology defines SDC.

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment: RAS and NFκB target stromal MCT4

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.

Postradiotherapy neck dissection for head and neck squamous cell carcinoma: pattern of pathologic residual carcinoma and prognosis.

For patients with head and neck cancer who were treated using primary radiotherapeutic approaches, the pattern of pathologic residual carcinoma in the neck dissection specimen and its effect on clinical outcome remains unknown.

The Variable Presentations of Anaplastic Spindle Cell Squamous Carcinoma Associated with Tall Cell Variant of Papillary Thyroid Carcinoma

BACKGROUND In 1976, Hawk and Hazard described the tall cell variant (TCV) of papillary thyroid carcinoma (PTC). While the lesions they described had cytologic features of papillary carcinoma, they showed more aggressive behavior with a greater propensity for extrathyroid extension and lymphovascular invasion than classic PTC. In 1991, Bronner and LiVolsi described a series of patients with TCV that progressed to spindle cell squamous carcinoma (SCSC), a unique form of anaplastic thyroid carcinoma. This study describes the variable clinical and pathologic presentations in 31 patients with anaplastic SCSC arising in association with TCV. METHODS The surgical pathology archives as well as the personal consultation files of one of the authors (V.A.L.) were reviewed to retrieve cases of SCSC arising in association with TCV. The available clinical as well as pathologic information on all patients was reviewed. RESULTS A total of 31 patients with SCSC arising in association with TCV were retrieved from our files. The average age at primary presentation was 67 (range 32-92) with a female-to-male ratio of 2:1. Three clinical scenarios for SCSC associated with TCV were identified. These were type I, consisting of TCV with SCSC at the time of presentation (18 patients); type II, consisting of SCSC arising as a recurrence in patients with a known history of TCV (5 patients); and type III, consisting of SCSC presenting as a primary laryngeal squamous cell carcinoma in a patient with or without a known history of TCV (8 patients). The type III cases were of most concern since they often were confused with primary laryngeal squamous cell carcinoma and most often were diagnosed after laryngectomy. CONCLUSIONS SCSC of the thyroid is almost exclusively associated with TCV and can have variable clinical presentations. SCSC is most commonly seen associated with a primary diagnosis of TCV. SCSC may be seen, however, in patients with recurrent PTC and most importantly may present in a fashion similar to primary laryngeal SCSC. Therefore, caution should be exercised in evaluating laryngeal squamous lesions in patients with known history of TCV and without known risks factors for head and neck carcinogenesis.

Prognostic Indications of Elevated MCT4 and CD147 across Cancer Types: A Meta-Analysis

Background. Metabolism in the tumor microenvironment can play a critical role in tumorigenesis and tumor aggression. Metabolic coupling may occur between tumor compartments; this phenomenon can be prognostically significant and may be conserved across tumor types. Monocarboxylate transporters (MCTs) play an integral role in cellular metabolism via lactate transport and have been implicated in metabolic synergy in tumors. The transporters MCT1 and MCT4 are regulated via expression of their chaperone, CD147. Methods. We conducted a meta-analysis of existing publications on the relationship between MCT1, MCT4, and CD147 expression and overall survival and disease-free survival in cancer, using hazard ratios derived via multivariate Cox regression analyses. Results. Increased MCT4 expressions in the tumor microenvironment, cancer cells, or stromal cells were all associated with decreased overall survival and decreased disease-free survival (p < 0.001 for all analyses). Increased CD147 expression in cancer cells was associated with decreased overall survival and disease-free survival (p < 0.0001 for both analyses). Few studies were available on MCT1 expression; MCT1 expression was not clearly associated with overall or disease-free survival. Conclusion. MCT4 and CD147 expression correlate with worse prognosis across many cancer types. These results warrant further investigation of these associations.

TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer.

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer.

Current Management of Locally Advanced Head and Neck Cancer: The Combination of Chemotherapy With Locoregional Treatments

This review will discuss the evolution of the role of chemotherapy in the treatment of locally advanced head and neck cancer (HNC), over the last few decades. Studies were identified by searching PubMed electronic databases. Surgery followed by radiotherapy (RT) or definitive RT are potentially curative approaches for locally advanced HNC. While chemotherapy itself is not curative, it can improve cure rates when given as an adjunct to RT. The benefit of combining chemotherapy with RT is related to the timing of the chemotherapy. Several prospective randomized trials have demonstrated that concurrent delivery of chemotherapy and RT (CRT) is the most promising approach, given that locoregional recurrence is the leading pattern of failure for patients with locally advanced HNC. Induction chemotherapy before CRT has not been shown to be superior to CRT alone and the added toxicity may negatively impact the compliance with CRT. Sequential chemotherapy administration, in the form of induction chemotherapy followed by RT or CRT, has been successful as a strategy for organ preservation in patients with potentially resectable laryngeal and hypopharyngeal cancer. Systemic chemotherapy delivered concurrently with RT is used as a standard treatment for locally advanced HNC.

Clinical experience transitioning from IMRT to VMAT for head and neck cancer.

To quantify clinical differences for volumetric modulated arc therapy (VMAT) versus intensity modulated radiation therapy (IMRT) in terms of dosimetric endpoints and planning and delivery time, twenty head and neck cancer patients have been considered for VMAT using Nucletron Oncentra MasterPlan delivered via an Elekta linear accelerator. Differences in planning time between IMRT and VMAT were estimated accounting for both optimization and calculation. The average delivery time per patient was obtained retrospectively using the record and verify software. For the dosimetric comparison, all contoured organs at risk (OARs) and planning target volumes (PTVs) were evaluated. Of the 20 cases considered, 14 had VMAT plans approved. Six VMAT plans were rejected due to unacceptable dose to OARs. In terms of optimization time, there was minimal difference between the two modalities. The dose calculation time was significantly longer for VMAT, 4 minutes per 358 degree arc versus 2 minutes for an entire IMRT plan. The overall delivery time was reduced by 9.2 ± 3.9 minutes for VMAT (51.4 ± 15.6%). For the dosimetric comparison of the 14 clinically acceptable plans, there was almost no statistical difference between the VMAT and IMRT. There was also a reduction in monitor units of approximately 32% from IMRT to VMAT with both modalities demonstrating comparable quality assurance results. VMAT provides comparable coverage of target volumes while sparing OARs for the majority of head and neck cases. In cases where high dose modulation was required for OARs, a clinically acceptable plan was only achievable with IMRT. Due to the long calculation times, VMAT plans can cause delays during planning but marked improvements in delivery time reduce patient treatment times and the risk of intra-fraction motion.

Fibroblast growth factor-2 (FGF2) and syndecan-1 (SDC1) are potential biomarkers for putative circulating CD15+/CD30+ cells in poor outcome Hodgkin lymphoma patients.

BackgroundHigh risk, unfavorable classical Hodgkin lymphoma (cHL) includes those patients with primary refractory or early relapse, and progressive disease. To improve the availability of biomarkers for this group of patients, we investigated both tumor biopsies and peripheral blood leukocytes (PBL) of untreated (chemo-naïve, CN) Nodular Sclerosis Classic Hodgkin Lymphoma (NS-cHL) patients for consistent biomarkers that can predict the outcome prior to frontline treatment.Methods and materialsBioinformatics data mining was used to generate 151 candidate biomarkers, which were screened against a library of 10 HL cell lines. Expression of FGF2 and SDC1 by CD30+ cells from HL patient samples representing good and poor outcomes were analyzed by qRT-PCR, immunohistochemical (IHC), and immunofluorescence analyses.ResultsTo identify predictive HL-specific biomarkers, potential marker genes selected using bioinformatics approaches were screened against HL cell lines and HL patient samples. Fibroblast Growth Factor-2 (FGF2) and Syndecan-1 (SDC1) were overexpressed in all HL cell lines, and the overexpression was HL-specific when compared to 116 non-Hodgkin lymphoma tissues. In the analysis of stratified NS-cHL patient samples, expression of FGF2 and SDC1 were 245 fold and 91 fold higher, respectively, in the poor outcome (PO) group than in the good outcome (GO) group. The PO group exhibited higher expression of the HL marker CD30, the macrophage marker CD68, and metastatic markers TGFβ1 and MMP9 compared to the GO group. This expression signature was confirmed by qualitative immunohistochemical and immunofluorescent data. A Kaplan-Meier analysis indicated that samples in which the CD30+ cells carried an FGF2+/SDC1+ immunophenotype showed shortened survival. Analysis of chemo-naive HL blood samples suggested that in the PO group a subset of CD30+ HL cells had entered the circulation. These cells significantly overexpressed FGF2 and SDC1 compared to the GO group. The PO group showed significant down-regulation of markers for monocytes, T-cells, and B-cells. These expression signatures were eliminated in heavily pretreated patients.ConclusionThe results suggest that small subsets of circulating CD30+/CD15+ cells expressing FGF2 and SDC1 represent biomarkers that identify NS-cHL patients who will experience a poor outcome (primary refractory and early relapsing).