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Metabolic heterogeneity between neoplastic cells and surrounding stroma has been described in several epithelial malignancies; however, the metabolic phenotypes of neoplastic lymphocytes and neighboring stroma in diffuse large B-cell lymphoma (DLBCL) is unknown. We investigated the metabolic phenotypes of human DLBCL tumors by using immunohistochemical markers of glycolytic and mitochondrial oxidative phosphorylation (OXPHOS) metabolism. The lactate importer MCT4 is a marker of glycolysis, whereas the lactate importer MCT1 and TOMM20 are markers of OXPHOS metabolism. Staining patterns were assessed in 33 DLBCL samples as well as 18 control samples (non-neoplastic lymph nodes). TOMM20 and MCT1 were highly expressed in neoplastic lymphocytes, indicating an OXPHOS phenotype, whereas non-neoplastic lymphocytes in the control samples did not express these markers. Stromal cells in DLBCL samples strongly expressed MCT4, displaying a glycolytic phenotype, a feature not seen in stromal elements of non-neoplastic lymphatic tissue. Furthermore, the differential expression of lactate exporters (MCT4) on tumor-associated stroma and lactate importers (MCT1) on neoplastic lymphocytes support the hypothesis that neoplastic cells are metabolically linked to the stroma likely via mutually beneficial reprogramming. MCT4 is a marker of tumor-associated stroma in neoplastic tissue. Our findings suggest that disruption of neoplastic-stromal cell metabolic heterogeneity including MCT1 and MCT4 blockade should be studied to determine if it could represent a novel treatment target in DLBCL.

Objectives/Hypothesis In many cancers, varying regions within the tumor are often phenotypically heterogeneous, including their metabolic phenotype. Further, tumor regions can be metabolically compartmentalized, with metabolites transferred between compartments. When present, this metabolic coupling can promote aggressive behavior. Tumor metabolism in papillary thyroid cancer (PTC) is poorly characterized. Study Design Immunohistochemical staining of tissue samples. Methods Papillary thyroid cancer specimens from 46 patients with (n = 19) and without advanced disease (n = 27) were compared to noncancerous thyroid tissue (NCT) and benign thyroid specimens (n = 6 follicular adenoma [FA] and n = 5 nodular goiter [NG]). Advanced disease was defined as the presence of lateral neck lymphadenopathy. Immunohistochemistry was performed for translocase of outer mitochondrial membrane 20 (TOMM20), a marker of oxidative phosphorylation, and monocarboxylate transporter 4 (MCT4), a marker of glycolysis. Results Papillary thyroid cancer and FA thyrocytes had high staining for TOMM20 compared to NCT and nodular goiter (NG) (P < 0.01). High MCT4 staining in fibroblasts was more common in PTC with advanced disease than in any other tissue type studied (P < 0.01). High MCT4 staining was found in all 19 cases of PTC with advanced disease, in 11 of 19 samples with low-stage disease, in one of five samples of FA, in one of 34 NCT, and in 0 of six NG samples. Low fibroblast MCT4 staining in PTC correlated with the absence of clinical adenopathy (P = 0.028); the absence of extrathyroidal extension (P = 0.004); low American Thyroid Association risk (P = 0.001); low AGES (age, grade, extent, size) score (P = 0.004); and low age, metastasis, extent of disease, size risk (P = 0.002). Conclusion This study suggests that multiple metabolic compartments exist in PTC, and low fibroblast MCT4 may be a biomarker of indolent disease. Level of Evidence N/A. Laryngoscope, 2015

Two major trends that have been affecting the provision of oncology care in the United States are a shift from volume-based to value-based care and a push toward patient-centered healthcare. However, these two trends are not always completely aligned with each other. Value-based payment models, including clinical pathways, are one strategy being implemented by oncology stakeholders to help encourage the uptake of value-based oncology care. If structured with the patient in mind, they can improve quality of care for patients with cancer, decrease inappropriate care while enabling appropriate personalization of care, and constrain rising prices by demanding a stronger link between cost and value. If not structured appropriately, they can limit patient choice, impede access to innovative treatments, and encourage one-size-fits-all oncology care. Clin Cancer Res; 23(16); 4545–9. ©2017 AACR.

Background Twenty percent of patients with classical Hodgkin Lymphoma (cHL) have aggressive disease defined as relapsed or refractory disease to initial therapy. At present we cannot identify these patients pre-treatment. The microenvironment is very important in cHL because non-cancer cells constitute the majority of the cells in these tumors. Non-cancer intra-tumoral cells, such as tumor-associated macrophages (TAMs) have been shown to promote tumor growth in cHL via crosstalk with the cancer cells. Metabolic heterogeneity is defined as high mitochondrial metabolism in some tumor cells and glycolysis in others. We hypothesized that there are metabolic differences between cancer cells and non-cancer tumor cells, such as TAMs and tumor-infiltrating lymphocytes in cHL and that greater metabolic differences between cancer cells and TAMs are associated with poor outcomes. Methods A case-control study was conducted with 22 tissue samples of cHL at diagnosis from a single institution. The case samples were from 11 patients with aggressive cHL who had relapsed after standard treatment with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or were refractory to this treatment. The control samples were from 11 patients with cHL who achieved a remission and never relapsed after ABVD. Reactive non-cancerous lymph nodes from four subjects served as additional controls. Samples were stained by immunohistochemistry for three metabolic markers: translocase of the outer mitochondrial membrane 20 (TOMM20), monocarboxylate transporter 1 (MCT1), and monocarboxylate transporter 4 (MCT4). TOMM20 is a marker of mitochondrial oxidative phosphorylation (OXPHOS) metabolism. Monocarboxylate transporter 1 (MCT1) is the main importer of lactate into cells and is a marker of OXPHOS. Monocarboxylate transporter 4 (MCT4) is the main lactate exporter out of cells and is a marker of glycolysis. The immunoreactivity for TOMM20, MCT1, and MCT4 was scored based on staining intensity and percentage of positive cells, as follows: 0 for no detectable staining in > 50% of cells; 1+ for faint to moderate staining in > 50% of cells, and 2+ for high or strong staining in > 50% of cells. Results TOMM20, MCT1, and MCT4 expression was significantly different in Hodgkin and Reed Sternberg (HRS) cells, which are the cancerous cells in cHL compared with TAMs and tumor-associated lymphocytes. HRS have high expression of TOMM20 and MCT1, while TAMs have absent expression of TOMM20 and MCT1 in all but two cases. Tumor-infiltrating lymphocytes have low TOMM20 expression and absent MCT1 expression. Conversely, high MCT4 expression was found in TAMs, but absent in HRS cells in all but one case. Tumor-infiltrating lymphocytes had absent MCT4 expression. Reactive lymph nodes in contrast to cHL tumors had low TOMM20, MCT1, and MCT4 expression in lymphocytes and macrophages. High TOMM20 and MCT1 expression in cancer cells with high MCT4 expression in TAMs is a signature of high metabolic heterogeneity between cancer cells and the tumor microenvironment. A high metabolic heterogeneity signature was associated with relapsed or refractory cHL with a hazard ratio of 5.87 (1.16–29.71; two-sided P < .05) compared with the low metabolic heterogeneity signature. Conclusion Aggressive cHL exhibits features of metabolic heterogeneity with high mitochondrial metabolism in cancer cells and high glycolysis in TAMs, which is not seen in reactive lymph nodes. Future studies will need to confirm the value of these markers as prognostic and predictive biomarkers in clinical practice. Treatment intensity may be tailored in the future to the metabolic profile of the tumor microenvironment and drugs that target metabolic heterogeneity may be valuable in this disease.

Objective The tumor microenvironment frequently displays abnormal cellular metabolism, which contributes to aggressive behavior. Metformin inhibits mitochondrial oxidative phosphorylation, altering metabolism. Though the mechanism is unclear, epidemiologic studies show an association between metformin use and improved outcomes in head and neck squamous cell carcinoma (HNSCC). We sought to determine if metformin alters metabolism and apoptosis in HNSCC tumors. Study Design Window of opportunity trial of metformin between diagnostic biopsy and resection. Participants were patients with newly diagnosed HNSCC. Fifty patients were enrolled, and 39 completed a full-treatment course. Metformin was titrated to standard diabetic dose (2,000 mg/day) for a course of 9 or more days prior to surgery. Methods Immunohistochemistry (IHC) for the metabolic markers caveolin-1 (CAV1), B-galactosidase (GALB), and monocarboxylate transporter 4 (MCT4), as well as the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay and Ki-67 IHC, were performed in pre- and postmetformin specimens. Exploratory mass spectroscopy imaging (MSI) to assess lactate levels also was performed in three subjects. Results Metformin was well tolerated. The average treatment course was 13.6 days. Posttreatment specimens showed a significant increase in stromal CAV1 (P < 0.001) and GALB (P < 0.005), as well as tumor cell apoptosis by TUNEL assay (P < 0.001). There was no significant change in stromal MCT4 expression or proliferation measured by Ki67. Lactate levels in carcinoma cells were increased 2.4-fold postmetformin (P < 0.05), as measured by MSI. Conclusion Metformin increases markers of reduced catabolism and increases senescence in stromal cells as well as carcinoma cell apoptosis. This study demonstrates that metformin modulates metabolism in the HNSCC microenvironment. Level of Evidence 4. Laryngoscope, 127:1808–1815, 2017

In many cancers, varying regions within the tumor are often phenotypically heterogeneous, including their metabolic phenotype. Further, tumor regions can be metabolically compartmentalized, with metabolites transferred between compartments. When present, this metabolic coupling can promote aggressive behavior. Tumor metabolism in papillary thyroid cancer (PTC) is poorly characterized.Immunohistochemical staining of tissue samples.Papillary thyroid cancer specimens from 46 patients with (n = 19) and without advanced disease (n = 27) were compared to noncancerous thyroid tissue (NCT) and benign thyroid specimens (n = 6 follicular adenoma [FA] and n = 5 nodular goiter [NG]). Advanced disease was defined as the presence of lateral neck lymphadenopathy. Immunohistochemistry was performed for translocase of outer mitochondrial membrane 20 (TOMM20), a marker of oxidative phosphorylation, and monocarboxylate transporter 4 (MCT4), a marker of glycolysis.Papillary thyroid cancer and FA thyrocytes had high staining for TOMM20 compared to NCT and nodular goiter (NG) (P0.01). High MCT4 staining in fibroblasts was more common in PTC with advanced disease than in any other tissue type studied (P0.01). High MCT4 staining was found in all 19 cases of PTC with advanced disease, in 11 of 19 samples with low-stage disease, in one of five samples of FA, in one of 34 NCT, and in 0 of six NG samples. Low fibroblast MCT4 staining in PTC correlated with the absence of clinical adenopathy (P = 0.028); the absence of extrathyroidal extension (P = 0.004); low American Thyroid Association risk (P = 0.001); low AGES (age, grade, extent, size) score (P = 0.004); and low age, metastasis, extent of disease, size risk (P = 0.002).This study suggests that multiple metabolic compartments exist in PTC, and low fibroblast MCT4 may be a biomarker of indolent disease.N/A. Laryngoscope, 126:2410-2418, 2016.

The tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) is comprised of cancer-associated fibroblasts (CAFs), immune cells, and other supporting cells. Genetic changes in the carcinoma cells, such as alterations to TP53, NOTCH1, and specific gene expression profiles, contribute to derangements in cancer and microenvironment cells such as increased ROS, overproduction of cytokines, and epithelial to mesenchymal transition (EMT). CAFs are among the most critical elements of the TME contributing to proliferation, invasion, and metastasis. The adaptive immune response is suppressed in HNSCC through overexpression of cytokines, triggered apoptosis of T cells, and alterations in antigen processing machinery. Overexpression of critical cytokines, such as transforming growth factor-β (TGF-β), contributes to EMT, immune suppression, and evolution of CAFs. Inflammation and hypoxia are driving forces in angiogenesis and altered metabolism. HNSCC utilizes glycolytic and oxidative metabolism to fuel tumorigenesis via coupled mechanisms between cancer cell regions and cells of the TME. Increased understanding of the TME in HNSCC illustrates that the long-held notion of “condemned mucosa” reflects a process that extends beyond the epithelial cells to the entire tissue comprised of each of these elements.

Introduction It has previously been suggested that the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) has prognostic significance. Furthermore, gene expression profiling in DLBCL patients has identified separate subsets with glycolytic and mitochondrial (oxidative phosphorylative) metabolic signatures. Glycolytic metabolism forms the basis for FDG PET scans, widely used in staging and response assessment in DLBCL. While many assume that the tumor as a whole is primarily glycolytic, the metabolic patterns of cancer cells (C) and surrounding cancer-associated stromal cells (CAS) remain relatively unknown. We investigated the in situ metabolic patterns of C and CAS cells as well as tumor-associated macrophages (TAM) in DLBCL. Methods All patients diagnosed with DLBCL at Thomas Jefferson University from 2009-12 with available diagnostic tissue (N=28) were studied. Two immunohistochemical (IHC) biomarkers of metabolism were used as stains. Monocarboxylate transporter 4 (MCT4) shuttles glycolysis-derived lactate out of the cell and is a biomarker for glycolysis. Translocase of the outer mitochondrial membrane 20 (TOMM20) is a biomarker for mitochondrial mass, i.e. the oxidative phosphorylation phenotype. The horseradish immunoperoxidase method was used to stain 5 μm thick, paraffin-embedded tissue samples, using TOMM20 and MCT4 antibodies (Santa Cruz). Immunostains were graded on a scale of 0 to 2+ based on the intensity and percentage of immunoreactivity in C, CAS, and TAM cells. Available clinical data collected by chart review included SUVmax (SUVm) on PET/CT scan, R-IPI, treatment regimen, complete response (CR) rates and Ki-67. Fisher's exact test was used to test for association among categorical variables. Results Cancer cells in 27/28 samples (96%) stained 2+ for TOMM20, with one sample staining 1+. MCT4 expression was completely absent in C cells in all samples. Conversely, TOMM20 was either not expressed (46%) or had 1+ expression (54%) in CAS and TAM. MCT4 expression was 2+ in 46% of CAS and 75% of TAM, and 1+ in the CAS and TAM of the remaining samples. Reactive lymphocytes (RL) had no MCT4 expression and uniform 1+ TOMM20 expression and can be regarded as internal controls. Levels of MCT4 expression in CAS correlated with MCT4 expression in TAM in the same patient (p=0.0069). 16/28 patients (57%) attained CR with anthracycline/rituximab based frontline therapy. MCT4 staining in CAS/TAM was associated with better CR rates with increasing intensity of scoring. (1+/1+ < 1+/2+ < 2+/2+; p=0.032). Table 1. S.No Stage R-IPI TOMM20 MCT4 SUVm CR C CAS TAM RL C CAS TAM RL 1 4 P 2+ 1+ 1+ 1+ 0 2+ 2+ 0 19.82 Y 2 4 * 2+ 0 0 1+ 0 1+ 2+ 0 * N 3 4 G 2+ 1+ 1+ 1+ 0 2+ 2+ 0 19.00 N 4 * * 2+ 1+ 1+ 1+ 0 2+ 2+ 0 * Y 5 4 G 2+ 0 0 1+ 0 1+ 2+ 0 3.50 Y 6 4 P 2+ 0 0 1+ 0 1+ 2+ 0 18.80 N 7 4 P 2+ 1+ 1+ 1+ 0 2+ 2+ 0 * Y 8 4 P 2+ 1+ 1+ 1+ 0 2+ 2+ 0 16.51 N 9 4 * 2+ 0 0 1+ 0 1+ 2+ 0 42.19 Y 10 * * 2+ 1+ 1+ 1+ 0 1+ 1+ 0 * N 11 4 G 1+ 0 0 0 0 1+ 1+ 0 * Y 12 2 VG 2+ 1+ 1+ 1+ 0 2+ 2+ 0 30.72 Y 13 4 P 2+ 0 0 1+ 0 1+ 1+ 0 * N 14 4 P 2+ 1+ 1+ 1+ 0 2+ 2+ 0 32.00 N 15 4 * 2+ 0 0 1+ 0 1+ 2+ 0 17.31 Y 16 4 G 2+ 0 0 1+ 0 1+ 2+ 0 7.90 Y 17 2 * 2+ 1+ 1+ 1+ 0 2+ 2+ 0 16.52 Y 18 4 P 2+ 0 0 1+ 0 2+ 2+ 0 38.90 Y 19 4 G 2+ 1+ 1+ 1+ 0 1+ 1+ 0 31.00 N 20 4 G 2+ 0 0 1+ 0 1+ 1+ 0 27.00 N 21 4 * 2+ 0 0 1+ 0 1+ 2+ 0 12.68 Y 22 4 * 2+ 1+ 1+ 1+ 0 2+ 2+ 0 * Y 23 4 P 2+ 1+ 1+ 1+ 0 1+ 1+ 0 6.99 N 24 4 P 2+ 1+ 1+ 1+ 0 2+ 2+ 0 26.91 Y 25 1 * 2+ 0 0 1+ 0 1+ 1+ 0 20.86 N 26 4 P 2+ 0 0 1+ 0 2+ 2+ 0 9.80 Y 27 4 G 2+ 1+ 1+ 1+ 0 2+ 2+ 0 23.50 Y 28 4 P 2+ 1+ 1+ 1+ 0 1+ 2+ 0 * N P=poor G=good VG=very good *=NA Conclusions Cancer cells in DLBCL do not express MCT4, but instead strongly express TOMM20, suggesting an oxidative phosphorylative rather than a glycolytic metabolic phenotype. Conversely, CAS and TAM always express MCT4, suggesting a glycolytic phenotype. Glycolysis and lactate export in CAS/TAM may be spatially related with mitochondrial metabolism in cancer cells, indicating tumor-stromal metabolic coupling. Glycolytic stroma in DLBCL may contribute to the PET-avidity in these tumors. Increasing MCT4 staining in CAS and TAM was associated with better CR rates, perhaps indicating tumors with increased chemosensitivity. Further investigation of the underlying mechanisms are needed to develop a better understanding of tumor-stromal interaction and its possible role in treatment of DLBCL. Figure 1. H&E (40x). Tissue sample (study patient) showing cancer cells admixed with stromal elements Figure 1. H&E (40x). Tissue sample (study patient) showing cancer cells admixed with stromal elements Figure 2. MCT4 stain (40x) demonstrating exclusive staining of stromal cells, sparing cancer cells Figure 2. MCT4 stain (40x) demonstrating exclusive staining of stromal cells, sparing cancer cells Figure 3. TOMM-20 stain (40x) demonstrating 2+ staining of cancer cells, sparing stromal cells Figure 3. TOMM-20 stain (40x) demonstrating 2+ staining of cancer cells, sparing stromal cells Disclosures Pro: Seattle Genetics: Consultancy, Other: Travel expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses.

Background Hodgkin lymphoma (HL) is mainly composed of reactive cells. Lymphocytes, macrophages, eosinophils, mast cells, plasma cells, and other stromal cells constitute these supportive or reactive cells. In-situ glycolytic and oxidative phosphorylation metabolism of reactive cells and cancer cells is unknown in HL. We interrogated HL specimens (N=24) to examine the metabolic compartments in-situ in this disease. Introduction High glycolytic activity is seen in the majority of HL on the basis of high uptake of 2-[18] fluoro-2-deoxy-glucose (FDG) on positron emission tomography (PET). The advent of FDG-PET in HL has revealed that these tumors as a unit are glycolytic and staging and assessment of response to treatment has improved. However, it is unknown if cancer cells and reactive cells share a metabolic phenotype. Purpose To study mitochondrial and glycolytic metabolism in HL cancer and reactive cells. Methods/Materials Two immunohistochemical biomarkers of metabolism were employed on HL tumor sections. Translocase of the Outer Mitochondrial Membrane 20 (TOMM20) protein expression is a biomarker of functional mitochondrial mass and oxidative phosphorylation. Monocarboxylate transporter 4 (MCT4) is a biomarker of glycolysis and lactate export. We selected 24 consecutive cases of classical HL based on morphology and immunohistochemical (IHC) staining for CD30, CD15 and CD45. We recorded FDG-PET standard uptake values (SUV) where available for all tumors studied. Immunohistochemical stains were performed on 5-micron thick, formalin-fixed, paraffin-embedded tissue sections using the horseradish peroxidase method. The primary antibodies used were TOMM20 and MCT4 (Santa Cruz). The immunostaining was graded on a scale of 0 to 2+ according to the intensity and percentage of immunoreactive cancer and reactive cells. Results Reed Sternberg and Hodgkin cells (RS/H) had high TOMM20 expression and low MCT4. In fact, the highest TOMM20 expression out of all cells analyzed was found in the RS/H cells in 20 of the 24 samples. In the other 4 samples, cancer and reactive lymphocytes (RL) had similar expression of TOMM20. The tumor stroma or stromal cells in proximity to cancer cells which includes histiocytes (TS/H) had low TOMM20 expression and high MCT4 expression in all 24 samples analyzed. Higher than baseline FDG-PET uptake is a measure of glycolysis and was found in all tumors where FDG-PET was performed. High MCT4 expression was not found in normal stroma or stroma at a distance from cancer cells. Histiocytes always had low TOMM20 expression but MCT4 expression was high in proximity to cancer cells and low at a distance from cancer cells. Conclusion Glycolysis and lactate export occurs in cancer-associated stroma (TS/H) that is spatially linked to mitochondrial metabolism in cancer cells (RS/H) in HL. This suggests that the most FDG-PET avid cells within HL are the reactive cells and not the cancer cells. Also, normalization of FDG-PET uptake when assessing response to treatment suggests reversal to a metabolically normal stroma. Representative images, 60x: Disclosures: No relevant conflicts of interest to declare.

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

A 72-year-old female with metastatic breast cancer developed oligoblastic granulocytic leukemia 6 months after initiation of chemotherapy. Cytogenetic examination of the bone marrow cells revealed a balanced t(X;19)(q12;q13.3) as the sole abnormality in 50% of the metaphases. The remaining cells showed a normal female karyotype. The der(19) chromosome displayed consistent folding in the Xq13–q23 region in all metaphases, indicating involvement of the inactive X chromosome in translocation.