Stephen P. Slone

Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Tartrate-Resistant Acid Phosphatase as an Immunohistochemical Marker for Inflammatory Macrophages

Human serum contains 2 isoforms of type-5 tartrate-resistant acid phosphatase (TRACP): 5a and 5b. TRACP-5b is osteoclastic. Our goal was to determine if serum TRACP-5a could originate from inflammatory macrophages (MPhi). We stained 246 paraffin-embedded tissue samples for TRACP using monoclonal antibody 9C5 (mab9C5) to isoforms 5a and 5b and a novel mab220 specific to isoform 5a. CD68 and lysozyme were also stained. MPhi of chronic and granulomatous inflammation and in tissues that undergo strong antigenic stimulation were strongly positive for TRACP, more so with mab220 than with mab9C5. Noninflammatory MPhi in lymph node sinuses or germinal centers and red pulp MPhi of spleen were weak or negative for TRACP. Marginal zone lymphocytes and sebaceous glands of skin were weakly positive for TRACP. Tissue mast cells displayed strong TRACP staining. Neuroendocrine cells of gastrointestinal tissues were strongly immunoreactive with mab9C5 but negative with mab220. Restricted expression of TRACP primarily in inflammatory MPhi supports our hypothesis that circulating TRACP-5a could be a biomarker of chronic inflammatory disease activity.

Sinus Histiocytosis With Massive Lymphadenopathy and Langerhans Cell Histiocytosis Express the Cellular Adhesion Molecule CD31

Context.-We investigated expression of the adhesion molecule CD31 in sinus histiocytosis with massive lymphadenopathy (SHML) and Langerhans cell histiocytosis (LCH) because (1) SHML and LCH cells express a variety of cellular adhesion molecules and (2) SHML has been characterized as a reactive histiocytic proliferation, and tissue macrophages (histiocytes) are known to express CD31. Objective.-The purpose of this study was to determine whether SHML and LCH cells express CD31 and whether dual staining with CD31 and S100 facilitates diagnosis of these disease states. Methods.-Formalin-fixed, paraffin-embedded archival tissues were immunohistochemically stained via the labeled streptavidin-biotin method using antibodies against CD31 and S100 protein after heat-induced epitope retrieval. Archival tissues included SHML (n = 2), LCH (n = 10), malignant melanoma (n = 5), sinus hyperplasia (n = 4), granulomas (n = 4), granular cell tumor (n = 6), and normal skin (n = 4). Results.-Normal Langerhans cells in the epidermis were CD31(-)/S100(+); neoplastic Langerhans cells in LCH were CD31(+)/S100(+). Histiocytes in granulomas and in sinus hyperplasia were CD31(+)/S100(-); abnormal histiocytes in SHML were CD31(+)/S100(+). S100(+) tumors (malignant melanoma and granular cell tumor) were CD31(-). Conclusions.-The spectrum of cell types that express CD31 is expanded to include SHML and LCH. We speculate that up-regulation of CD31 in neoplastic Langerhans cells contributes to the migratory capability of LCH cells. CD31 may be a useful nonlysosomal marker of macrophages and their neoplastic counterparts (true histiocytic sarcomas). An immunohistochemical staining panel that includes CD31 and S100 facilitates the diagnosis of SHML and LCH.

Hepatoid adenocarcinoma of the lung: report of five cases and review of the literature

The current diagnostic criteria for hepatoid adenocarcinoma of lung include typical acinar or papillary adenocarcinoma and a component resembling hepatocellular carcinoma and expressing α-fetoprotein (AFP). Distinguishing hepatoid adenocarcinoma of lung from hepatocellular carcinoma metastatic to lung is difficult in patients with both lung and liver masses and in patients at risk for lung and liver cancer because of smoking and viral hepatitis, respectively. We studied morphologic features of hepatoid adenocarcinoma of lung and established an immunohistochemical panel to facilitate distinction of hepatoid adenocarcinoma of lung from hepatocellular carcinoma metastatic to lung. Five cases of hepatoid adenocarcinoma of lung were stained with hematoxylin and eosin and mucicarmine for histomorphologic evaluation. The 14-marker immunohistochemical profile was established for hepatoid adenocarcinoma of lung and compared with that of hepatocellular carcinoma. Two cases of hepatoid adenocarcinoma of lung had signet-ring cell components. Three cases were pure hepatoid adenocarcinoma without components of acinar or papillary adenocarcinoma, signet-ring cells or neuroendocrine carcinoma. Like hepatocellular carcinoma, hepatoid adenocarcinoma of lung expresses CK8 (5/5), CK18 (5/5), AFP (3/5) and HepPar1 (5/5), shows cytoplasmic staining with TTF-1 (5/5) and does not express CK14 (0/5). Unlike hepatocellular carcinoma, it expresses CK5/6 (1/5), CK7 (3/5), CK19 (4/5), CK20 (1/5), HEA125 (5/5), MOC31 (5/5), monoclonal CEA (3/5) and napsin A (1/5). An immunohistochemical panel that includes a variety of cytokeratins, monoclonal CEA and EpCAM markers (HEA125 and MOC31) facilitates distinction of hepatoid adenocarcinoma of lung from hepatocellular carcinoma metastatic to lung, especially when correlated with clinical and radiologic findings. We propose modification of the current diagnostic criteria for hepatoid adenocarcinoma of lung. Tumor composition can be either pure hepatoid adenocarcinoma or hepatoid adenocarcinoma with components of typical acinar or papillary adenocarcinoma, signet-ring cells or neuroendocrine carcinoma. AFP expression is not requisite for diagnosis as long as other markers of hepatic differentiation are expressed.

IL-4 production by CD8+ lymphomatoid papulosis, type C, attracts background eosinophils

There are two subsets of CD8+ T cells: Tc1 and Tc2. INF‐γ production by Tc1 cells causes granulomatous inflammation. IL‐4 production by Tc2 cells attracts eosinophils. A 76‐year‐Caucasian female presented with CD8+ lymphomatoid papulosis (LyP), type C. We hypothesized that the LyP cells belonged to the Tc2 subset because of abundant background eosinophils. Hematoxylin and eosin and immunohistochemical stains were carried out on tissue sections from a skin punch biopsy. Antibodies for immunohistochemical stains included CD3, CD4, CD5, CD7, CD8, CD30, CD56, ALK‐1, clusterin and IL‐4. There was involvement of the dermis by a dense lymphoid infiltrate composed of large atypical cells and numerous eosinophils. The LyP cells expressed CD5, CD8, CD30 and IL‐4. Keratinocytes showed a membranous pattern of immunoreactivity for IL‐4. IL‐4 production by CD8+ LyP, type C indicates that it belongs to the Tc2 subset. The cytokine milieu produced by the LyP cells attracted eosinophils. The IL‐13R complex on keratinocytes bound IL‐4 and produced a membranous staining pattern. Although CD8+ LyP is rare, we believe that this CD30+ lymphoproliferative disorder should be included in the World Health Organization‐European Organization for Research and Treatment of Cancer classification of cutaneous T‐cell lymphomas.

Tartrate-resistant acid phosphatase 5a in sarcoidosis: Further evidence for a novel macrophage biomarker in chronic inflammation

BACKGROUND/PURPOSE Tartrate-resistant acid phosphatase (TRACP) 5a is expressed strongly in inflammatory macrophages (MΦ). Serum TRACP5a is elevated in rheumatoid arthritis patients with extra-articular manifestations of rheumatoid nodules, in a percentage of patients with end-stage chronic kidney disease, and may be a risk marker for acute myocardial infarction. This proof-of-concept study was undertaken in patients with sarcoidosis to further substantiate our hypothesis that TRACP5a protein is a biomarker for macrophages in other chronic inflammatory diseases. METHODS Immunohistochemical staining for TRACP5a and CD68 was performed in tissues of 19 patients with sarcoidosis. We also measured circulating TRACP5a protein and other inflammation biomarkers including interkeukin-6, angiotensin-converting enzyme, and C-reactive protein in 13 patients. Twenty healthy age-matched nonsmoking individuals were used as the reference group. RESULTS All sarcoidosis tissues showed strong staining for TRACP5a and CD68 in the non-caseating granulomatous lesions and localized specifically to MΦ, multinucleate giant cells, and epithelioid MΦ. Serum TRACP5a protein was elevated significantly in active sarcoidosis patients compared with the control group, and levels fluctuated with disease activity in one patient studied longitudinally. CONCLUSION TRACP5a protein is expressed abundantly in the granulomatous tissues and may be elevated in a significant proportion of sarcoidosis patients. These findings further support our hypothesis that serum TRACP5a is derived from systemic inflammatory MΦ and thereby may be a biomarker of inflammation for sarcoidosis and also reflect its disease activity.

Effect of freeze-dried berries on the development of reflux-induced esophageal adenocarcinoma.

The incidence of esophageal adenocarcinoma in humans is increasing more rapidly than any other malignancy in the United States. Animal studies have demonstrated the efficacy of freeze-dried berry supplementation on carcinogen-induced esophageal squamous cell carcinoma in rats; however, no such studies have been done in esophagoduodenal anastomosis (EDA), an animal model for reflux-induced esophageal adenocarcinoma (EAC) development. Eight-week-old male Sprague-Dawley rats were randomized into 3 groups: EDA + control diet (EDA-CD; n = 10); EDA + 2.5% black raspberry diet (EDA-BRB; n = 11) and EDA + 2.5% blueberry diet (EDA-BB; n = 12). After 2 wk of feeding the respective diets, the rats underwent EDA surgery to induce gastroesophageal reflux and then continued the diet. Measurement of feed intake suggested that all EDA-operated animals had lower feed intake starting at 10 wk after surgery and this was significant close to termination at 24 wk. There were no significant differences in either reflux esophagitis (RE), intestinal metaplasia (IM) (70% in CD, 64% in BRB, and 66% in BB; P = 0.1) or EAC incidence (30% for CD, 34% for BRB, and 25% for BB; P = 0.2) with supplementation. Berry diets did not alter COX-2 levels, but BB diet significantly reduced MnSOD levels (1.23 ± 0.2) compared to control diet (2.05 ± 0.14; P < 0.05). We conclude that a dietary supplementation of freeze-dried BRB and BB at 2.5% (w/w) was not effective in the prevention of reflux-induced esophageal adenocarcinoma in this EDA animal model.

Applications and performance of monoclonal antibodies to human tartrate resistant acid phosphatase

BACKGROUND Tartrate-resistant acid phosphatase (TRACP) is an enzyme common to cells of the mononuclear phagocyte system and a clinically relevant biomarker for osteoclasts and inflammatory macrophages. The purpose was to assess applications and performance of six anti-TRACP monoclonal antibodies. METHODS Mab9C5, 14G6, 162, 203, 220, and 89 were used as capture and detection antibodies in quantitative immunoassay, and for western blot (WB), immunoprecipitation, and immunohistochemistry of paraffin sections containing chronic inflammatory infiltrates. The clinical performance of mab14G6 for immunoassay of serum TRACP5b activity was compared to two commercial kit methods. RESULTS Mab9C5 is useful for WB and immunohistochemistry methods only. Mab14G6, 162, and 203 are useful for quantitative immunoassay and immunoprecipitation, however, mab203 causes inactivation of enzymatic activity. Mab220 and 89 are specific for TRACP5a and useful in all applications. Mab14G6 has similar clinical sensitivity and specificity as two commercial methods. CONCLUSIONS TRACP is an important marker in osteoimmunology. Specific antibodies with unique specificity for TRACP isoforms and defined applications will be valuable for clinical evaluation of bone metabolic, inflammatory and autoimmune diseases and will aid in basic research of TRACP biochemistry and biology.

Glomus tumor of the ovary masquerading as granulosa cell tumor: case report.

A solid right adnexal mass in a 73-year-old woman bled profusely with mobilization mimicking a granulosa cell tumor. There was almost complete replacement of the ovary by a circumscribed, 4.0 cm tumor with a hemorrhagic, solid cut surface. Morphologic and phenotypic correlation supported a diagnosis of glomus tumor. Large gaping vessels and small sinusoidal-type vessels formed an anastomotic vascular network with an inner endothelial lining (CD31+/CD34+) and an outer layer of glomocytes (actin+/desmin-/inhibin-). The hemangiopericytoma-like vasculature accounted for bleeding during surgery.

Multiple isolated extramedullary relapse of acute promyelocytic leukemia after allogeneic hematopoietic stem cell transplant: a case report and review of literature

Isolated extramedullary disease (EMD) is uncommon, especially in acute promyelocytic leukemia (APL) after allogeneic hematopoietic stem cell transplantation (HSCT). We review the literature and present a 32 year old woman with APL who developed multiple EMDs after allogeneic HSCT within the calvarium, and later found to have various isolated lesions including femur, humerus and thoraco lumbar vertebrae. She was treated with local radiotherapy (XRT) to EMD lesions, all-trans retinoic acid, arsenic trioxide and donor lymphocyte infusion at different time points in her clinical course, without success. Out of reported cases in clinical setting as ours, average onset of isolated EMD is 25 months and median survival 14 months. Effective treatment of isolated EMD after HSCT is not yet clear, but ATO in combination with local XRT, tamibarotene and second HSCT have shown good results in some reported cases, but accumulation of more cases is needed to elucidate optimal therapy in such setting.