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Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA. Mol Cancer Ther; 10(3); 427–36. ©2011 AACR.

Supplementary Figures 1-3, Table 1 from Lipoprotein Lipase Links Dietary Fat to Solid Tumor Cell Proliferation

Supplemental File S1. The siRNA sequences and real-time qPCR primer sequences.

Supplemental File S2. A representative video of a Hop62 cell that undergoes multipolar cell division.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 2029–38. ©2015 AACR.

Supplemental Fig. S3. (A) Schematic diagram showing the relationship between CDK2, CP110, activated KRAS and anaphase catastrophe. (B) Engineered overexpression of CP110 was detected in A549, Hop62, H522 and H460 cells (versus control transfectants) using anti-HA antibody after 24 and 48 hours of transfection. A representative immunoblot for each cell line is displayed.

Supplemental Fig. S5. Representative CP110 immunohistochemistry staining of lung normal (N) versus malignant (T) tissues harvested independently from KRAS wild-type transgenic mice and KRAS mutant transgenic mice.The table displays KRAS mutation information, CP110 scoring and intensity of CP110 immunostaining in 6 lung cancer specimens from cyclin-E transgenic mice and Kras LSL-G12D driven engineered mice respectively.

Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

Xanthomas are histiocytic lesions of the skin, soft tissue, and bone and are generally considered to be reactive in nature. When they arise in the bones of the jaw, they are referred to as central xanthomas. New evidence supports the hypothesis that central xanthomas are a separate and distinct entity from their extragnathic counterparts. Noonan syndrome (NS) is an autosomal dominant disorder that has been associated with giant cell lesions, which also commonly occur in the jaw. We present a case of a 15-year-old boy with NS who presented with a radiolucent lesion of the mandible that on excision was found to be a central xanthoma. Although giant cell lesions have been well described in NS, xanthomas of the jaw have not been reported. We will also discuss the entities that must be excluded before making a diagnosis of central xanthoma, as this can affect both treatment and follow-up.

KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo. To solidify this association, loss of Usp18 in KrasLA2/+/Usp18−/− mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas. Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905–14. ©2017 AACR.

Detection of somatic mutations in non–small cell lung cancers (NSCLCs), especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM) and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gene AmpliSeq Cancer Hotspot Panel v2 from May 2013 to July 2015. Using 10 ng of DNA, up to 11 samples were simultaneously sequenced on the Ion Torrent PGM (316 and 318 chips). We identified variants with the Ion Torrent Variant Caller Plugin, and Golden Helix's SVS software was used for annotation and prediction of the significance of the variants. Three hundred ninety-eight samples were successfully sequenced (12.1% failure rate). In all, 633 variants in 41 genes were detected with a median of 2 (range of 0 to 7) variants per sample. Mutations detected in BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA were considered potentially actionable and were identified in 237 samples, most commonly in KRAS (37.9%), EGFR (11.1%), BRAF (4.8%), and PIK3CA (4.3%). In our patient population, all mutations in EGFR, KRAS, and BRAF were mutually exclusive. The Ion Torrent Ampliseq technology can be utilized on small biopsy and cytology specimens, requires very little input DNA, and can be applied in clinical laboratories for genotyping of NSCLC. This targeted next-generation sequencing approach allows for detection of common and also rare mutations that are clinically actionable in multiple patients simultaneously.

Background. Although genetic profiling of tumors is a potentially powerful tool to predict drug sensitivity and resistance, its routine use has been limited because clinicians are often unfamiliar with interpretation and incorporation of the information into practice. We established a Molecular Tumor Board (MTB) to interpret individual patients’ tumor genetic profiles and provide treatment recommendations. Patients and Methods. DNA from tumor specimens was sequenced in a Clinical Laboratory Improvement Amendments-certified laboratory to identify coding mutations in a 50-gene panel (n = 34) or a 255-gene panel (n = 1). Cases were evaluated by a multidisciplinary MTB that included pathologists, oncologists, hematologists, basic scientists, and genetic counselors. Results. During the first year, 35 cases were evaluated by the MTB, with 32 presented for recommendations on targeted therapies, and 3 referred for potential germline mutations. In 56.3% of cases, MTB recommended treatment with a targeted agent based on evaluation of tumor genetic profile and treatment history. Four patients (12.5%) were subsequently treated with a MTB-recommended targeted therapy; 3 of the 4 patients remain on therapy, 2 of whom experienced clinical benefit lasting >10 months. Conclusion. For the majority of cases evaluated, the MTB was able to provide treatment recommendations based on targetable genetic alterations. The most common reasons that MTB-recommended therapy was not administered stemmed from patient preferences and genetic profiling at either very early or very late stages of disease; lack of drug access was rarely encountered. Increasing awareness of molecular profiling and targeted therapies by both clinicians and patients will improve acceptance and adherence to treatments that could significantly improve outcomes. Implications for Practice: Case evaluation by a multidisciplinary Molecular Tumor Board (MTB) is critical to benefit from individualized genetic data and maximize clinical impact. MTB recommendations shaped treatment options for the majority of cases evaluated. In the few patients treated with MTB-recommended therapy, disease outcomes were positive and support genetically informed treatment.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 2029–38. ©2015 AACR.

The histologic differential diagnosis between intramuscular myxoma and low-grade myxofibrosarcoma can be quite difficult in some cases. To identify a diagnostic immunohistochemical marker, we compared the staining profiles of 19 different antigens, including cell cycle proteins, apoptosis proteins, and proliferative markers, and selected other signaling and structural proteins in these two tumors. Ten cases each of intramuscular myxoma and low-grade myxofibrosarcoma were stained with antibodies directed against apoptosis regulatory proteins (Bcl2, activated caspase-3, phospho-H2A.X, and cleaved PARP), cell cycle regulatory proteins (Rb1, Cyclin-A, CDKN1B, and Cdt1), proliferative markers (KI67, MCM2, phospho-histone H3, and geminin), cell signalling molecules (c-Myc, EGF, EGFR, PLA2G4A, and HSP90), a dendritic cell marker (CD209), and the extracellular matrix proteoglycan decorin. Staining patterns of myxoma and myxofibrosarcoma were compared using Fisher's exact test and the Mann-Whitney test. For each potential diagnostic marker studied, the proportions of cases scored as positive on both dichotomous or ordinal scales were not significantly different between myxoma and myxofibrosarcoma. Myxoma and myxofibrosarcoma share a common immunophenotype for each of the markers studied. Distinction between these tumors is still predominantly based on morphologic criteria.

High-risk subtypes of the human papillomavirus (HPV) are known to drive the pathogenesis of cervical, anogenital, and oropharyngeal squamous cell carcinomas. Recent reports have shown that HPV is also associated with small cell neuroendocrine carcinomas of the cervix and oropharynx. Little is known about HPV as a driver of neuroendocrine tumors at other sites, in particular, small cell lung cancer (SCLC). The aim of this study was to evaluate SCLC for the presence of high-risk HPV to further elucidate the role of HPV in SCLC. Archived formalin-fixed, paraffin-embedded surgical resection specimens from 20 primary SCLC from 19 patients were identified from 2004 to 2013. Two cervical small cell carcinomas were included as controls. Small cell neuroendocrine phenotype was confirmed by review of morphology and prior immunohistochemistry staining. Immunohistochemistry for p16 (INK4a) expression was performed in all cases. DNA was extracted from formalin-fixed, paraffin-embedded specimens and run on the Roche Linear Array HPV Genotyping test and a real-time polymerase chain reaction HPV assay. Pathologic tumor stage was collected from surgical pathology reports. High-risk HPV genotypes were not detected in any of the 20 SCLC specimens, whereas p16 was up-regulated in 14 (70%) of 20. p16 up-regulation can be used as an indicator of disruption of the Rb pathway either by integration of the HPV E7 oncoprotein or other mechanisms. In conclusion, our findings indicate that, unlike some other small cell neuroendocrine carcinomas, the pathogenesis of SCLC does not appear to be associated with high-risk HPV infection, a potentially very useful characteristic when determining primary from metastatic tumors.

CIC-rearranged sarcomas comprise a recognized subset of the so called “undifferentiated small round cell sarcomas” that lack the traditional EWSR1 fusion to one of the ETS family members.1 A smaller percentage have demonstrated BCOR gene rearrangements.2 The CIC gene is most commonly fused with the DUX4 or DUX4L10 genes through t(4;19)(q35;q13) or t(10;19)(q26;q13). An alternative fusion partner, the FOXO4 (Xq13.1) gene, has recently been identified.3 CIC-rearranged sarcomas have a distinct gene signature compared to classic Ewing sarcomas with upregulation of ETS transcription factors and WT1. They are more common in adults, in soft tissue locations as opposed to bone, and they have a mixed response to neo/adjuvant traditional Ewing sarcoma chemotherapy protocols.4 This article is protected by copyright. All rights reserved.

Male germ cell tumors (GCTs) are a model for a curable solid tumor. GCTs can differentiate into mature teratomas. Embryonal carcinomas (ECs) represent the stem cell compartment of GCTs and are the malignant counterpart to embryonic stem (ES) cells. GCTs and EC cells are useful to investigate differentiation therapy and chemotherapy response. This study explored mechanistic interactions between all-trans-retinoic acid (RA), which induces differentiation of EC and ES cells, and the Hedgehog (Hh) pathway, a regulator of self-renewal and proliferation. RA was found to induce mRNA and protein expression of Patched 1 (Ptch1), the Hh ligand receptor and negative regulator of this pathway. PTCH1 is also a target gene of Hh signaling through Smoothened (Smo) activation. Yet, this observed RA-mediated Ptch1 induction was independent of Smo. It occurred despite co-treatment with RA and Smo inhibitors. Retinoid induction of Ptch1 also occurred in other RA-responsive cancer cell lines and in normal ES cells. Notably, this enhanced Ptch1 expression was preceded by induction of the homeobox transcription factor Meis1, a direct RA target. Direct interaction between Meis1 and Ptch1 was confirmed using chromatin immunoprecipitation assays. To establish the translational relevance of this work, Ptch1 expression was shown to be deregulated in human ECs relative to mature teratoma and the normal seminiferous tubule. Taken together, these findings reveal a previously unrecognized mechanism through which RA can inhibit the Hh pathway via Ptch1 induction. Engaging this pathway is a new way to repress the Hh pathway that can be translated into the cancer clinic.

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.

Background Prior studies highlighted cyclin D1 as a key biomarker of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. This study builds on prior work by examining the roles of cyclin D1, cyclin D3, and cyclin E in mediating erlotinib sensitivity or resistance. Methods Expression plasmids for G1 cyclins were independently transfected into NIH 3T3 cells and effects on erlotinib sensitivity were examined. The expression profiles of G1 cyclins were compared in erlotinib-sensitive and erlotinib-resistant lung cancer cell lines. A549 and H358 cells were treated with erlotinib and changes in cyclin protein expression were assessed. Cyclin D3 immunohistochemical staining was measured in biopsy tissues obtained from patients before and after treatment with erlotinib. Erlotinib-sensitive lung cancer cells were transfected with cyclin D3 and changes in erlotinib sensitivity were examined. Results Individual transfection of cyclin D1, cyclin D3, and cyclin E expression plasmids each significantly reduced erlotinib sensitivity in NIH-3T3 cells. The erlotinib-resistant A549 cell line expressed high basal levels of cyclin D3 mRNA and protein. Comparison of tumor biopsies obtained from patients before and after treatment with erlotinib indicated an increase in the percentage of cancer cells expressing cyclin D3 following treatment with erlotinib ( P = .02). Transfection of cyclin D3 into an erlotinib-sensitive lung cancer cell line inhibited erlotinib-induced signaling changes and reduced the growth-suppressive effects of erlotinib. Conclusions High expression of cyclin D3 confers resistance to erlotinib in vitro and in vivo . Cyclin D3 immunohistochemical staining warrants investigation as a biomarker for predicting erlotinib resistance.

Associations between bronchioloalveolar carcinoma (BAC), mucinous differentiation, and epidermal growth factor receptor (EGFR) and KRAS mutations have been previously reported in studies of surgical specimens. We present the cytomorphology of lung adenocarcinomas, including metastases that were diagnosed by cytologic methods and the relationship to both EGFR and KRAS mutational status. We retrospectively reviewed the clinical and cytomorphologic features of 50 lung adenocarcinomas that were tested for both EGFR and KRAS mutations. Cytomorphologic features evaluated included cell size, architectural pattern, nucleoli, intranuclear cytoplasmic inclusions (INCI), mucin, necrosis, squamoid features, lymphocytic response, and histologic features of BAC differentiation. DNA was extracted from a paraffin-embedded cell block or frozen needle core fragments. Exon 19 deletions and the L858R mutation in exon 21 of EGFR were detected using PCR followed by capillary electrophoresis for fragment sizing. KRAS mutational analysis was performed by real-time PCR using a set of seven different Taqman(r) allelic discrimination assays to detect six mutations in codon 12 and one mutation in codon 13. Six cases (12%) showed EGFR mutations, 12 (24%) showed KRAS mutations, and 38 (62%) contained neither EGFR nor KRAS mutations. The majority of patients had stage IV disease (78%); 20 samples (40%) were from metastatic sites. The presence of prominent INCI (P = 0.036), papillary fragments (P = 0.041), and histologic features of BAC on paraffin block (P = 0.039) correlated with the presence of EGFR mutations. The presence of necrosis (P = 0.030), squamoid features (P = 0.048), and poorly differentiated tumors (P = 0.025) were more likely to be identified in the KRAS positive group.

Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA. Mol Cancer Ther; 10(3); 427–36. ©2011 AACR.

Objective: To describe a rare manifestation of parathyroid carcinoma in association with papillary thyroid carcinoma. Methods: We describe the clinical history, findings on physical examination, results of laboratory studies, imaging findings, and histopathologic features of a woman with 2 previously palpable thyroid nodules and mild hypercalcemia. Results: A 79-year-old woman presented to our endocrinology clinic for reevaluation of 2 thyroid nodules and long-standing hyperparathyroidism. She initially had undergone assessment 6 years previously, at which time an intact parathyroid hormone level was 89 pg/mL (reference range, 10 to 69) and the serum calcium concentration was 10.4 mg/dL (reference range, 8.2 to 10.2) in conjunction with normal alkaline phosphatase, creatinine, and 25-hydroxyvitamin D levels. Subsequently, she was found to have multifocal papillary thyroid cancer in the right thyroid lobe and a 5-cm parathyroid carcinoma in the left side. Conclusion: Synchronous parathyroid and thyroid carcinomas are extremely rare. To our knowledge, our current case is the first documented patient with parathyroid carci noma who had a clinical presentation consistent with longstanding mild adenoma in addition to synchronous papillary thyroid carcinoma. (Endocr Pract. 2010;16:664-668) Abbreviations: HPT = hyperparathyroidism; PTH = parathyroid hormone

Objective To describe a patient presenting with the rare findings of synchronous parathyroid adenoma and parathyroid sarcoidosis. Methods We describe the clinical history, physical examination findings, laboratory values, imaging findings, and pathologic data of a man who developed recurrent se- vere hypercalcemia after successful parathyroidectomy. Results A 67-year-old man had the following initial blood test results: calcium, 11.1 mg/dL (reference range, 8.5-10.6 mg/dL); albumin 4.0 g/dL (reference range, 3.2-5.2 g/dL); intact parathyroid hormone, 166 pg/mL (refer- ence range, 10-69 pg/mL); creatinine, 1.9 mg/dL; 25-hy- droxyvitamin D, 15 ng/mL (reference range, 30-80 ng/ mL); and 1, 25-dihydroxyvitamin D, 44 pg/mL (reference range, 16-72 pg/mL). Chest x-ray was normal, and delayed images from a technetium Tc 99m sestamibi scan showed increased activity in the right lower pole of the thyroid. Two months after successful parathyroidectomy, the pa- tient was admitted to the hospital with a serum calcium concentration of 17 mg/dL. Pathologic examination of the resected gland confirmed the diagnosis of parathyroid ad- enoma, and subsequent review disclosed the presence of noncaseating granulomas within the adenoma. Conclusions Sarcoidosis with parathyroid involve- ment causing severe hypercalcemia is unique to this case.Recurrent hypercalcemia after successful resection of a parathyroid adenoma may require consideration of poten- tial causes other than the initial diagnosis. (Endocr Pract. 2010;16:463-467)

Purpose: Cyclin-dependent kinases (Cdk) and their associated cyclins are targets for lung cancer therapy and chemoprevention given their frequent deregulation in lung carcinogenesis. This study uncovered previously unrecognized consequences of targeting the cyclin E–Cdk-2 complex in lung cancer. Experimental Design: Cyclin E, Cdk-1, and Cdk-2 were individually targeted for repression with siRNAs in lung cancer cell lines. Cdk-2 was also pharmacologically inhibited with the reversible kinase inhibitor seliciclib. Potential reversibility of seliciclib effects was assessed in washout experiments. Findings were extended to a large panel of cancer cell lines using a robotic-based platform. Consequences of cyclin E–Cdk-2 inhibition on chromosome stability and on in vivo tumorigenicity were explored as were effects of combining seliciclib with different taxanes in lung cancer cell lines. Results: Targeting the cyclin E–Cdk-2 complex, but not Cdk-1, resulted in marked growth inhibition through the induction of multipolar anaphases triggering apoptosis. Treatment with the Cdk-2 kinase inhibitor seliciclib reduced lung cancer formation in a murine syngeneic lung cancer model and decreased immunohistochemical detection of the proliferation markers Ki-67 and cyclin D1 in lung dysplasia spontaneously arising in a transgenic cyclin E–driven mouse model. Combining seliciclib with a taxane resulted in augmented growth inhibition and apoptosis in lung cancer cells. Pharmacogenomic analysis revealed that lung cancer cell lines with mutant ras were especially sensitive to seliciclib. Conclusions: Induction of multipolar anaphases leading to anaphase catastrophe is a previously unrecognized mechanism engaged by targeting the cyclin E–Cdk-2 complex. This exerts substantial antineoplastic effects in the lung. Clin Cancer Res; 16(1); 109–20

Purpose: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer. Experimental Design: miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal-malignant human lung tissue bank. Results: miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1 and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided a mechanism for observed growth suppression. Conclusions: miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.

Cyclin E is a critical G 1 -S cell cycle regulator aberrantly expressed in bronchial premalignancy and lung cancer. Cyclin E expression negatively affects lung cancer prognosis. Its role in lung carcinogenesis was explored. Retroviral cyclin E transduction promoted pulmonary epithelial cell growth, and small interfering RNA targeting of cyclin E repressed this growth. Murine transgenic lines were engineered to mimic aberrant cyclin E expression in the lung. Wild-type and proteasome degradation-resistant human cyclin E transgenic lines were independently driven by the human surfactant C (SP-C) promoter. Chromosome instability (CIN), pulmonary dysplasia, sonic hedgehog (Shh) pathway activation, adenocarcinomas, and metastases occurred. Notably, high expression of degradation-resistant cyclin E frequently caused dysplasia and multiple lung adenocarcinomas. Thus, recapitulation of aberrant cyclin E expression as seen in human premalignant and malignant lung lesions reproduces in the mouse frequent features of lung carcinogenesis, including CIN, Shh pathway activation, dysplasia, single or multiple lung cancers, or presence of metastases. This article reports unique mouse lung cancer models that replicate many carcinogenic changes found in patients. These models provide insights into the carcinogenesis process and implicate cyclin E as a therapeutic target in the lung.

Background USP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease. Methods Heterozygous USP18 +/− FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined. Results USP18 −/− FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6–12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441). Conclusions USP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1883-8) contains supplementary material, which is available to authorized users.

The lack of an effective detection method for lung circulating tumor cells (CTCs) presents a substantial challenge to elucidate the value of CTCs as a diagnostic or prognostic indicator in lung cancer, particularly in nonsmall cell lung cancer (NSCLC). In this study, we prepared a capture surface exploiting strong multivalent binding mediated by poly(amidoamine) (PAMAM) dendrimers to capture CTCs originating from lung cancers. Given that 85% of the tumor cells from NSCLC patients overexpress epidermal growth factor receptor (EGFR), anti-EGFR was chosen as a capture agent. Following in vitro confirmation using the murine lung cancer cell lines (ED-1 and ED1-SC), cyclin E-overexpressing (CEO) transgenic mice were employed as an in vivo lung tumor model to assess specificity and sensitivity of the capture surface. The numbers of CTCs in blood from the CEO transgenic mice were significantly higher than those from the healthy controls (on average 75.3 ± 14.9 vs 4.4 ± 1.2 CTCs/100 μL of blood, p < 0.005), indicating the high sensitivity and specificity of our surface. Furthermore, we found that the capture surface also offers a simple, effective method for monitoring treatment responses, as observed by the significant decrease in the CTC numbers from the CEO mice upon a treatment using a novel anti-miR-31 locked nucleic acid (LNA), compared to a vehicle treatment and a control-LNA treatment (p < 0.05). This in vivo evaluation study confirms that our capture surface is highly efficient in detecting in vivo CTCs and thus has translational potential as a diagnostic and prognostic tool for lung cancer.

The 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline updates lowered the threshold for HER2 positivity and altered the equivocal category. The goal of this study was to evaluate the impact of these changes on the distribution of HER2 fluorescence in situ hybridization (FISH) status. The utility of reflex HER2 immunohistochemistry (IHC) for FISH equivocal cases was also examined.We retrospectively reviewed all invasive breast cancers analyzed for HER2 via dual-probe FISH (PathVysion; Abbott Laboratories. Abbott Park, IL) 12 months before and after the HER2 guidelines updates were implemented. Reflex HER2 IHC results were recorded for HER2 FISH equivocal cases.There was a significant increase in the number of HER2 FISH equivocal results after the guideline updates (4.9% vs 1.4%, P = .0087) that was independent of specimen type (core vs surgical, P = .6). All 17 FISH equivocal cases after the updates had reflex HER2 IHC: two (12%) of 17 were positive, 12 (71%) of 17 remained equivocal, and three (18%) of 17 were negative.Implementation of the 2013 ASCO/CAP HER2 guideline updates resulted in an increase in HER2 FISH equivocal results, which can be attributed to HER2 copy number, regardless of the HER2/CEP17 ratio. Reflex IHC for FISH equivocal cases is of limited utility; however, IHC does assign HER2 positivity or negativity in a small percentage of cases.

A single monoclonal antibody (MAG-1) directed against the C-terminal 18-amino acid region (VAGc18) of provasopressin was examined as an agent for recognizing the tumor-specific NRSA marker common to small cell lung cancer (SCLC) in formalin-fixed tissues with ABC immunohistochemistry. SCLC tumors were obtained from several tissue locations and included primary, metastatic, and recurrent disease. Positive staining was found in 91% of cases (53/58). All five of the unreactive tumors were of the lungs or chest wall, and there did not appear to be an association of this negativity with disease stage, age, or sex. Alternatively, almost all primary lesions, almost all metastatic lesions, and all recurrent lesions examined gave a positive reaction with MAG-1. For this study, vasopressin-producing cells of the human anterior hypothalamus served as a positive control, while negative controls comprised normal lung tissue, tumor that received MAG-1 in the presence of an excess of antigen (VAGc18 peptide), or tumor reacted with a commercial IgG1 isotype as primary antibody. All of the results indicate that MAG-1 can be effectively used to selectively identify the NRSA marker on almost all SCLC tumors, at all disease stages, and at all locations. Since all four tumors tested showing no reactivity with MAG-1 gave a positive reaction for synaptophysin, it is proposed that a combined use of MAG-1 with synaptophysin antibodies could allow all SCLC tumors to be detected by ABC immunohistochemistry.

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2BNNK), and RA-chemoprevented (BEAS-2BNNK/RA) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.

Erdheim-Chester disease (lipoid granulomatosis) is a rare type II (non-Langerhans cell) histiocytosis with systemic manifestations. The disease causes nonspecific disturbances in the function of multiple extraosseous organs, most commonly the eyes, lungs, pituitary gland, and kidneys. Diagnosis is usually made on the basis of radiologic evidence of cortical expansion of long bones. While the osseous and systemic changes have been well documented in the current literature, pathologic changes in the myocardium have not been well characterized since Erdheim and Chester's first description of this disease in 1930. In the 2 autopsy cases from Dartmouth-Hitchcock Medical Center (Lebanon, NH) reported in the present study, myocardial involvement was severe and had contributed significantly to the patients' morbidity and death. We describe the autopsy results and correlate them with Erdheim's original descriptions of this disease. In neither of our cases was bony involvement characteristic of the disease, and the diagnosis was made postmortem on the basis of soft tissue findings at autopsy.

We report the identification of a virulent Streptococcus organism associated with fulminant endocarditis, using 16S rRNA gene amplification, sequencing and assembly from formalin-fixed, paraffin-embedded archival heart valve tissue, years after the autopsy of a patient.

We previously found that expression of the vasopressin gene is a common feature of human breast cancer. In the present study we first examined 21 different cases of benign fibrocystic breast disease for vasopressin expression using immunohistochemistry and antibodies directed against vasopressin (anti-VP) and against vasopressin-associated glycopeptide (anti-VAG). All cases examined were negative for vasopressin gene expression using these antibodies. Alternatively, we examined 16 cases of breast ductal carcinoma in situ (DCIS) using the second of these antibodies (anti-VAG), and all of these cases were positive for vasopressin gene expression. Our results suggest that products of vasopressin gene expression are not markers of cellular proliferation in the breast, and might rather represent an early part of the carcinogenic process in this tissue.

To characterize the impact of increased production of TGF-beta in a xenograft model of human breast cancer, TGF-beta-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-beta1. Compared with control cells, cells that produced increased amounts of TGF-beta proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-beta signaling, cells were also transfected with a dominant-negative truncated type II TGF-beta receptor (TbetaRII). Disruption of autocrine TGF-beta signaling in the TGF-beta-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-beta-overexpressing cells expressing the truncated TbetaRII compensated for their diminished in vivo growth capacity, compared with the TGF-beta-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-beta-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-beta-overexpressing cells, irrespective of the status of the autocrine TGF-beta-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-beta's enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-beta that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.

The purpose of this article is to characterize the histologic vascular features and distinguishing MRI features of cystic apocrine metaplasia to better understand imaging-pathology concordance.Retrospective review of 261 consecutive MRI-guided biopsy cases was performed. Pathology results were reviewed for all biopsies; cystic apocrine metaplasia was identified as the predominant finding in 19 cases (7%). CD31 immunohistochemistry was subsequently performed on the most representative block of cystic apocrine metaplasia, and microvasculature was evaluated using computer-assisted image analysis. The contrast-enhanced MRI examinations correlating with the cystic apocrine metaplasia cases were independently reviewed by two radiologists specializing in breast imaging; lesions were analyzed for morphologic, kinetic, and T2 characteristics.On MRI review, 17 of 19 (89%) lesions were 10 mm or smaller. Washout kinetics were present in 11 of 19 (58%) lesions, and 14 of 19 (74%) lesions were at least partially hyperintense on T2-weighted sequences relative to adjacent glandular tissue. Cystic apocrine metaplasia had a higher percentage area (mean, 4.1%) of CD31-immunostained microvessels compared with background fibroglandular tissue (mean, 1.2%).Cystic apocrine metaplasia should be considered in the differential diagnosis of a T2-hyperintense enhancing focus or subcentimeter smoothly marginated mass, even if associated with washout kinetics. Cystic apocrine metaplasia contains a statistically significant increase in microvessel area compared with background fibroglandular tissue and fat and, therefore, may be considered a concordant result for this set of imaging findings.

Purpose Defects in the apoptotic pathway confer insensitiviry to the cytotoxic effects of chemotherapy and hence represent an important distal mechanism for the development of chemoresistance. In this study, we sought to obtain results on which to base clinical hypotheses about this mechanism. The aim of this study was to analyze the correlation of the expression of three of these apoptotic molecules, Bc1-2, Bax and Bc1-X, with chemoradiotherapy response and clinical outcome in patients with esophageal tumors.

Mutation of the p53 tumor suppressor gene is found in a large number of exocrine pancreatic tumors. The majority of these tumors is of the ductal cell phenotype. We examined 12 human acinar cell carcinomas and 42 transgenic mouse carcinomas (including 36 acinar cell tumors, four islet cell tumors, and two liver metastases of primary acinar cell tumors) for evidence of p53 mutation. Immunohistochemistry was used to identify p53 protein in tumor sections. To evaluate p53 exons 5-8, heteroduplex analysis was used on formalin-fixed, paraffin-embedded human tumor DNA, and single-strand conformation polymorphism analysis was used on frozen mouse tumor DNA. No molecular evidence of p53 mutation was found in any of the tumor DNAs and immunohistochemical data were regarded as negative. This study provides evidence that acinar cell carcinogenesis in both humans and transgenic mice is independent of p53 mutation.

Molecular testing is the current standard of care for nonesmall-cell lung cancer particularly adenocarcinoma (ADC). Novel tyrosine kinase inhibitors are currently available to target aberrations in epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) in these tumors. Often, this testing is performed using diagnostic fine needle aspiration (FNA) or core biopsy. Pathologists use immunohistochemistry to identify biomarkers to distinguish histologic types and to triage tumors for molecular studies. Here, we report a case of an adenosquamous carcinoma initially diagnosed as a squamous cell carcinoma in a FNA biopsy that was found on resection to have an ADC component and harbor an ALK rearrangement. Using current guidelines, this ALK rearrangement might have not been identified and the patient would not have been considered for crizotinib therapy. A small biopsy might not be representative of a lung cancer with mixed histologies; in instances when an ADC component cannot be excluded, testing for EGFR or ALK might be warranted and clinical criteria could be used to select patients for testing.

BACKGROUND The functional significance of cytokines expressed in situ by tumor cells and tumor infiltrating lymphocytes (TIL) in human colon carcinomas is largely unknown. METHODS We assessed TIL expression of interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10, and transforming growth factor-beta (TGF-beta), and tumor cell expression of IL-10 and TGF-beta in situ in 49 primary colon carcinomas and 20 metastases using immunohistochemistry. RESULTS The percentage of primary colon carcinoma samples in which > 20% of TIL expressed each cytokine was as follows: IL-4: 47%; TNF-alpha: 22%; TGF-beta: 10%; IFN-gamma: 6%; IL-2:2%; IL-10: 0%; and GM-CSF: 0%. Lymphocytes more commonly infiltrated colon carcinoma primaries than metastases, and TIL expression of IL-4 and TNF-alpha was more common in primary than metastastatic carcinomas. Expression of TNF-alpha by even a small proportion (> or = 3%) of the TIL in a colon carcinoma specimen was associated with better overall survival (P = 0.01) when compared with patients with little or no TIL TNF-alpha expression (5-year survival 82% vs. 47%). Expression of IL-4 by > or = 20% of colon carcinoma TIL was also associated with improved survival (P = 0.01; 5-year survival 87% vs. 50%). The expression of IL-10 or TGF-beta by colon carcinoma TIL or colon tumor cells themselves was not associated with impaired survival. Benign epithelial cells stained positively for IL-10 and TGF-beta more frequently than tumor cells (P < 0.001). CONCLUSIONS There are differences between the immune microenvironment of primary tumors and metastases. Although IL-10 is expressed by colon carcinoma cells and TIL, it is unlikely that it plays an important immunosuppressive role. TNF-alpha and IL-4 are commonly expressed by colon carcinoma TIL and both are associated with improved survival.

Human breast cancers progressively grow despite the presence of extensive lymphocytic infiltration and specific antitumor immune recognition, thereby calling into question the competency of breast tumor-infiltrating lymphocytes (TIL). The function of breast TILs in vivo and their possible role in the suppression of an antitumor immune response are largely unknown.The cytokines produced in situ by lymphocytes in 89 breast carcinomas and 14 benign breast lesions were assessed using immunohistochemistry.The majority of tumor and benign breast samples contained T-cell infiltrates, which were disclosed using an anti-CD3 antibody stain. The percentage of tumor samples in whichor =3% of the lymphocytes were producing cytokines was as follows: interleukin (IL)-2 45%, IL-4 36%, tumor necrosis factor-alpha (TNF-alpha) 28%, transforming growth factor-beta 1 (TGF-beta 1) 20%, IL-10 11%, interferon-gamma (IFN-gamma) 4%, and granulocyte-macrophage colony-stimulating factor (GM-CSF) 3%. Production of IL-2, IL-4, and TGF-beta 1 by TILs in breast cancers exceeded that detected in benign breast lesions (p0.005). Significantly more tumor samples contained lymphocytes producing IL-2, IL-4, TGF-beta 1, and TNF-alpha than IFN-gamma and GM-CSF (p0.002 for each comparison). One or more of the potentially immunoinhibitory cytokines-IL-4, IL-10, or TGF-beta 1-were produced by lymphocytes in 44% of the specimens. No significant associations were seen between lymphocyte production of a particular cytokine and disease-free survival (median follow-up 43 months).Immunohistochemical techniques can be used to detect cytokine secretion by TILs in preserved tissue. The relative lack of secretion of IFN-gamma and GM-CSF, rather than a deficiency of IL-2, may explain why the antitumor immune response to breast cancer is impaired.

PURPOSE MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2.

Immunohistochemical analysis for products of vasopressin and oxytocin gene expression was performed on acetone-fixed tissues from 19 breast cancers representing a variety of tumor sub-types. Studies employed the avidin-biotin complex (ABC) immunohistochemical procedure and utilized rabbit polyclonal antibodies to arginine vasopressin (VP), provasopressin (ProVP), vasopressin-associated human glycopeptide (VAG), oxytocin (OT), oxytocin-associated human neurophysin (OT-HNP), and a mouse monoclonal antibody to vasopressin-associated human neurophysin (VP-HNP). Western Blot analysis was performed on protein extracts of fresh-frozen tissues from 12 additional breast tumors. While VP gene related proteins were not detected in normal breast tissue, immunohistochemistry revealed the presence of VP, Pro VP, and VAG in all neoplastic cells for all of the tumor tissues examined. Vasopressin-associated human neurophysin was evident in only one of 19 acetone-fixed tumor preparations. However, Western blot analysis for all 12 fresh-frozen tumor samples showed the presence of two proteins, 42,000 and 20,000 daltons, that were immunoreactive with antibodies to VP, VP-HNP, and VAG. Oxytocin and OT-HNP, by immunohistochemistry, were found to be common to cells of normal breast tissues. For tumors, positive staining for OT was observed in 8 of 18 tumors, while OT-HNP was not detected in any of the tumors examined. These findings indicate that VP gene expression is a selective feature of all breast cancers, and that products of this expression might therefore be useful as markers for early detection of this disease and as possible targets for immunotherapy.

KRAS is frequently mutated in lung cancers. Innovative strategies are needed to combat KRAS mutant lung cancers because these tumors are often resistant to therapy. This study reports that loss of the deubiquitinase USP18 leads to destabilization of the KRAS oncogenic protein in panels of murine and human lung cancer cells. In marked contrast, engineered gain of USP18 expression using retroviral vectors introduced into the same panel of murine and human lung cancer cells stabilized KRAS protein. Loss of USP18 expression in KRAS mutant-expressing lung cancer cells inhibited their growth while gain of USP18 expression opposed this effect. We sought to identify mechanisms engaged in this KRAS protein destabilization. Intriguingly, immunoprecipitation assays established that KRAS conjugates with the ubiquitin-like protein ISG15. This leads to KRAS protein destabilization. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown was shown to destabilize mutant KRAS more prominently than wild-type KRAS protein. To independently confirm that KRAS directly complexes with ISG15, site-directed mutagenesis was performed to render the C-terminal domain of KRAS lysine-less. This led to stabilization of KRAS, despite knockdown of USP18. These studies were confirmed and extended in the in vivo setting of KRAS-driven lung cancers in KRASLA2/+ mice within the FVB strain background. We crossed these mice with USP18 null (USP18-/-) mice to obtain KRASLA2/+;USP18-/- compound mice. Strikingly, these compound mice had statistically significantly (P < 0.05) reduced lung cancers as compared to parental KRASLA2/+ mice. To establish the biological importance of this finding, USP18 immunohistochemical expression was compared in these engineered mice versus a second engineered mouse lung cancer model that was not driven by KRAS, but by aberrant cyclin E expression. USP18 immunohistochemical expression was substantially higher in the KRAS-driven than cyclin E-dependent lung cancers. To explore the translational relevance of this work, lung cancer tissue arrays were examined in 551 lung cancers where a clinical database was available. USP18 immunohistochemical expression was significantly (P < 0.001) higher in KRAS mutant adenocarcinomas as compared to wild-type cases. There was also a significant increase in USP18 expression in adenocarcinoma versus squamous cell carcinoma cases. Taken together, these studies broaden the role of USP18 as an antineoplastic target to combat lung cancers that harbor KRAS mutations. Citation Format: Lisa Maria Mustachio, Yun Lu, Laura J. Tafe, Angeline S. Andrew, Vincent Memoli, Jaime Rodriguez-Canales, Pamela A. Villalobos, Ignacio Wistuba, Jun Yu, Jack J. Lee, Fadzai Chinyengetere, David J. Sekula, Xi Liu, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1255.

e23168Background: Genetic profiling of tumors is quickly increasing as are trials targeting genetic alterations. Furthermore, genetic alterations are a relatively permanent part of a tumor profile,...

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

New pharmacologic targets are needed for lung cancer. One candidate pathway to target is composed of the E1-like ubiquitin-activating enzyme (UBE1L) that associates with interferon-stimulated gene 15 (ISG15), which complexes with and destabilizes cyclin D1. Ubiquitin protease 43 (UBP43/USP18) removes ISG15 from conjugated proteins. This study reports that gain of UBP43 stabilized cyclin D1, but not other D-type cyclins or cyclin E. This depended on UBP43 enzymatic activity; an enzymatically inactive UBP43 did not affect cyclin D1 stability. As expected, small interfering RNAs that reduced UBP43 expression also decreased cyclin D1 levels and increased apoptosis in a panel of lung cancer cell lines. Forced cyclin D1 expression rescued UBP43 apoptotic effects, which highlighted the importance of cyclin D1 in conferring this. Short hairpin RNA-mediated reduction of UBP43 significantly increased apoptosis and reduced murine lung cancer growth in vitro and in vivo after transplantation of these cells into syngeneic mice. These cells also exhibited increased response to all-trans-retinoic acid, interferon, or cisplatin treatments. Notably, gain of UBP43 expression antagonized these effects. Normal-malignant human lung tissue arrays were examined independently for UBP43, cyclin D1, and cyclin E immunohistochemical expression. UBP43 was significantly (P < 0.01) increased in the malignant versus normal lung. A direct relationship was found between UBP43 and cyclin D1 (but not cyclin E) expression. Differential UBP43 expression was independently detected in a normal-malignant tissue array with diverse human cancers. Taken together, these findings uncovered UBP43 as a previously unrecognized antineoplastic target. Mol Cancer Ther; 11(9); 1968–77. ©2012 AACR.