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The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted. Cancer Immunol Res; 2(2); 154–66. ©2013 AACR.

PDF file - 1196K, Supplementary Figure 1. Phenotypic analysis of FAP+ stromal cells in untreated TC1 flank tumors in C57BL/6 mice. Supplementary Figure 2. Depletion of FAP+ cells. Supplementary Figure 3. Persistence and antitumor activities of FAP-CAR T cells employing either human 4-1BB (73.3-hBBz) or mouse CD28 (73.3-m28z) co-stimulatory domain in mice and their in vitro activity. Supplementary Figure 4. Deletion of DGKzeta enhanced effector functions and in vivo persistence of FAP-CAR T cells. Supplementary Figure 5. Body weight of FAP-CAR T cells-treated mice remained constant or increased. Supplementary Figure 6. Histology of bone marrows and pancreas following treatment with FAP-CAR T cells. Supplementary Figure 7. Activation-induced cell death of FAP-CAR T cells. Supplementary Figure 8. Differential FAP expression on tumor and pancreatic FAP+ stromal cells. Supplementary Figure 9. mAb 73.3 and mAb FAP5 are specific for distinct epitopes of murine FAP expressed by fibroblasts.

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Abdominal aortic aneurism (AAA) is a complex immunological disease with a strong genetic component, and one of the ten leading causes of death of individuals 55-74 years old worldwide. Strong evidence has been accumulated suggesting that AAA is an autoimmune specific antigen-driven disease. Mononuclear cells infiltrating AAA lesions comprised of T and B lymphocytes and other cells expressing early-, intermediate- and late-activation antigens, and the presence of antigen-presenting cells have been documented, demonstrating an ongoing immune response. The three components of the trimolecular complex, T-cell receptor (TCR)/peptide (antigen)/HLA have been identified in AAA, and specifically: (i) clonal expansions of T-cell clones in AAA lesions; (ii) the association of AAA with particular HLA Class I and Class II; and (iii) self or nonself putative AAA-associated antigens. IgG autoantibodies recognizing proteins present in normal aortic tissue have been reported in patients with AAA. Molecular mimicry, defined as the sharing of antigenic epitopes between microorganisms (bacteria, viruses) and self antigens, maybe is responsible for T-cell responses and antibody production in AAA. Also, the frequency and the suppressor activity of CD4+ CD25+ FOXP3+ Tregs and the expression of FOXP3 transcripts and protein have been reported to be significantly impaired in AAA patients vs normal donors.

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the preferred method for diagnosing pancreatic masses. While the diagnostic success of EUS-FNA is widely accepted, the actual performance of EUS-FNA is not known. This study sought to define the EUS-FNA accuracy compared with the gold standard, surgically resected specimens. The study was a single institution, retrospective, and chart review of patients with surgically resected pancreatic specimens from 2005 to 2015 with a preoperative EUS-FNA or biliary brushing. Cytological reports were organized from least concerning (i.e., low chance of malignancy) to most concerning (high chance of malignancy) into eight cytologic categories. We identified 741 cytologic cases: 530 EUS-FNA and 211 endoscopic brushings. For EUS-FNA samples, 62.5% of "benign" samples proved to be "benign" on surgical pathology. A cytologic diagnosis of "suspicious for malignancy" or "positive for malignancy" were concordant with a cancer diagnosis on surgical pathology 93.3% and 98.0% of cases, respectively. EUS-FNA proved to be highly reliable at diagnosing malignancy for cytologic samples that were "suspicious" or "positive" for malignancy. Paired with supportive clinical data, these interpretations may be used to justify cancer treatment.

Despite optimal surgical resection, meningiomas may recur, with increasing grade and the degree of resection being predictive of risk. We hypothesize that an increasing Ki67 correlates with a higher risk of recurrence of resected WHO grade I meningiomas. The study population consisted of patients with resected WHO grade 1 meningiomas in locations outside of the base of skull. Digitally scanned slides stained for Ki67 were analyzed using automatic image analysis software in a standardized fashion. Recurrence was observed in 53 (17.7%) of cases with a median follow up time of 25.8 months. Ki67 ranged from 0 to 30%. Median Ki67 was 5.1% for patients with recurrence and 3.5% for patients without recurrence. In unadjusted analyses, high Ki-67 (≥ 5 vs. < 5) vs. ≥ 5) was associated with over a twofold increased risk of recurrence (13.1% vs. 27% respectively; HR 2.1731; 95% CI [1.2534, 3.764]; p = 0.006). After Adjusting for patient or tumor characteristics, elevated Ki-67 remained significantly correlated with recurrence. Grade 4 Simpson resection was noted in 71 (23.7%) of patients and it was associated with a significantly increased risk of recurrence (HR 2.56; 95% CI [1.41, 4.6364]; p = 0.002). WHO grade 1 meningiomas exhibit a significant rate of recurrence following resection. While Ki-67 is not part of the WHO grading criteria of meningiomas, a value greater than 5% is an independent predictor for increased risk of local recurrence following surgical resection.

Background Basaloid salivary gland neoplasms (BSNs), which include benign primary tumors and primary or metastatic malignancies, show overlapping morphology in fine-needle aspiration (FNA). The Milan system recommends assigning a grade (low or high) to malignant salivary neoplasms because of the impact on surgical planning. This study investigated cytomorphologic features of BSNs on FNA that would help to favor a high-grade malignancy over a low-grade malignancy or a benign tumor. Methods Two pathologists performed a double-blinded cytologic evaluation of FNA cases diagnosed as BSNs that had corresponding surgical resections. The diagnosis made with the Milan system was correlated with the final surgical diagnosis and grade. Cytologic sensitivity, specificity, and predictive values were calculated. Results There were 132 BSN FNA cases; cytology slides were available for 77 of 87 patients who had undergone resection. The risk of malignancy for the benign neoplasm (BN), salivary gland neoplasm of uncertain malignant potential (SUMP), suspicious for malignancy (SFM), and malignant categories were 13.6%, 22%, 100%, and 100%, respectively. The sensitivity of the malignant/SFM category was 51.7%; another 37.9% of confirmed malignancies were diagnosed as SUMP. The specificity of the BN category was 86%. Favoring a high-grade malignancy on FNA had 100% accuracy (5 of 5). Favoring a low-grade malignancy on FNA had 75% accuracy (6 of 8). The most specific cytomorphologic clues for a high-grade malignancy were necrotic/apoptotic debris, mitoses, discohesion, and anisonucleosis. Conclusions BSNs encompass a broad spectrum of primary and metastatic tumors. Necrotic/apoptotic debris, mitotic activity, discohesion, and significant anisonucleosis, alone or especially in combination, should make a cytopathologist suspect a high-grade malignancy.

Introduction Fine-needle aspiration (FNA) of nodal metastases plays a key role in the diagnosis of oropharyngeal squamous cell carcinoma (OPSCC). Because of significant clinical implications of human papillomavirus (HPV)-related OPSCC, immunohistochemistry for p16 as a surrogate marker for high-risk HPV is an important ancillary test. After our laboratory switched from CytoLyt to formalin fixative for FNA needle rinses generating cell block (CB) material, we investigated the impact of this protocol change on the accuracy of p16 results. Materials and methods FNA specimens of head and neck lesions with p16 staining performed on CB, from 1 year before and after the implementation of formalin-fixed CB (FCB) were identified. Nuclear and cytoplasmic p16 expression was scored and compared to p16 status on corresponding surgical specimens. Results There were no false-positive results with either fixative. CytoLyt-fixed CB (CCB) had 47% (7 of 15) false-negative cases, whereas FCB had none, with 100% diagnostic accuracy for p16-negative (n = 6) and p16-positive (n = 15) results. False-negative CCB showed 0% to 10% nuclear and 0% to 65% weak cytoplasmic staining, whereas true-positive CCB showed 10% to 85% nuclear and 35% to 90% cytoplasmic staining. p16-negative FCB showed 0% nuclear and cytoplasmic staining, and p16-positive FCB showed 30% to 100% moderate-strong nuclear and cytoplasmic staining. Interobserver variability was greater with CCB. Conclusions In our laboratory, formalin fixation of CB material improved the accuracy of p16 interpretation. Staining in FCB was also more robust than CCB, which showed weaker cytoplasmic and more focal nuclear staining. Therefore, we advocate formalin fixation for head and neck cytology specimens that may require p16 testing.

PURPOSE: The purpose of this study was to assess the performance of molecular ultrasound with dual-targeted microbubbles to detect metastatic disease in the sentinel lymph nodes (SLNs) in swine model of naturally occurring melanoma. The SLN is the first lymph node in the lymphatic chain draining primary tumor, and early detection of metastatic SLN involvement is critical in the appropriate management of melanoma. PROCEDURE: Nine Sinclair swine (weight 3–7 kg; Sinclair BioResources, Columbia, MO, USA) with naturally occurring melanoma were examined. Siemens S3000 scanner with a 9L4 probe was used for imaging (Siemens Healthineers, Mountain View, CA). Dual-targeted contrast agent was created using Targestar SA microbubbles (Targeson, San Diego, CA, USA) labeled with α(v)β(3)-integrin and P-selectin antibodies. Targestar SA microbubbles labeled with IgG-labeled were used as control. First, peritumoral injection of Sonazoid contrast agent (GE Healthcare, Oslo, Norway) was performed to detect SLNs. After that, dual-targeted and IGG control Targestar SA microbubbles were injected intravenously with a 30-min interval between injections. Labeled Targestar SA microbubbles were allowed to circulate for 4 min to enable binding. After that, two sets of image clips were acquired several seconds before and after a high-power destruction sequence. The mean intensity difference pre- to post-bubble destruction within the region of interest placed over SLN was calculated as a relative measure of targeted microbubble contrast agent retention. This process was repeated for non-SLNs as controls. All lymph nodes evaluated on imaging were surgically removed and histologically examined for presence of metastatic involvement. RESULTS: A total of 43 lymph nodes (25 SLNs and 18 non-SLNs) were included in the analysis with 18 SLNs demonstrating metastatic involvement greater than 5 % on histology. All non-SLNs were benign. The mean intensity (± SD) of the dual-targeted microbubbles for metastatic SLNs was significantly higher than that of benign LNs (18.05 ± 19.11 vs. 3.30 ± 6.65 AU; p = 0.0008), while IgG-labeled control microbubbles demonstrated no difference in retained contrast intensity between metastatic and benign lymph nodes (0.39 ± 1.14 vs. 0.03 ± 0.24 AU; p = 0.14). CONCLUSIONS: The results indicate that dual-targeted microbubbles labeled with P-selectin and α(v)β(3)-integrin antibodies may aid in detecting metastatic involvement in SLNs of melanoma.

Insulin-like growth factor binding protein 3 (IGFBP3) modulates cell growth through IGF-dependent and -independent mechanisms. Reports suggest that the serum levels of IGFBP3 are associated with various cancers and that IGFBP3 expression is significantly decreased in cisplatin (CDDP)-resistant lung cancer cells. Based on these findings, we investigated whether Igfbp3 deficiency accelerates mouse lung tumorigenesis and if expression of IGFBP3 enhances CDDP response by focusing on the IGF1 signaling cascade. To this end, an Igfbp3-null mouse model was generated in combination with KrasG12D to compare the tumor burden. Then, IGF-dependent signaling was assessed after expressing wild-type or a mutant IGFBP3 without IGF binding capacity in non–small cell lung cancer (NSCLC) cells. Finally, the treatment response to CDDP chemotherapy was evaluated under conditions of IGFBP3 overexpression. Igfbp3-null mice had increased lung tumor burden (>2-fold) and only half of human lung cancer cells survived after expression of IGFBP3, which corresponded to increased cleaved caspase-3 (10-fold), inactivation of IGF1 and MAPK signaling. In addition, overexpression of IGFBP3 increased susceptibility to CDDP treatment in lung cancer cells. These results, for the first time, demonstrate that IGFBP3 mediates lung cancer progression in a KrasG12D mouse model. Furthermore, overexpression of IGFBP3 induced apoptosis and enhanced cisplatin response in vitro and confirmed that the suppression is in part by blocking IGF1 signaling. Implications: These findings reveal that IGFBP3 is effective in lung cancer cells with high IGF1 signaling activity and imply that relevant biomarkers are essential in selecting lung cancer patients for IGF1-targeted therapy. Mol Cancer Res; 15(7); 896–904. ©2017 AACR.

Objective To validate a hypothesis that prostate cancer can be detected non-invasively by a simple and reliable assay by targeting genomic VPAC receptors expressed on malignant prostate cancer cells shed in voided urine. Patients/Subjects and Methods VPAC receptors were targeted with a specific biomolecule, TP4303, developed in our laboratory. With an Institutional Review Board exempt approval of use of de-identified discarded samples, an aliquot of urine collected as a standard of care, from patients presenting to the urology clinic (207 patients, 176 men and 31 women, aged ≥21 years) was cytospun. The cells were fixed and treated with TP4303 and 4,6-diamidino-2-phenylindole (DAPI). The cells were then observed under a microscope and cells with TP4303 orange fluorescence around the blue (DAPI) nucleus were considered ‘malignant’ and those only with a blue nucleus were regarded as ‘normal’. VPAC presence was validated using receptor blocking assay and cell malignancy was confirmed by prostate cancer gene profile examination. Results The urine specimens were labelled only with gender and presenting diagnosis, with no personal health identifiers or other clinical data. The assay detected VPAC positive cells in 98.6% of the men with a prostate cancer diagnosis (141), and none of the 10 men with benign prostatic hyperplasia. Of the 56 ‘normal’ patients, 62.5% (35 patients, 10 men and 25 women) were negative for VPAC cells; 19.6% (11, 11 men and no women) had VPAC positive cells; and 17.8% (10, four men and six women) were uninterpretable due to excessive crystals in the urine. Although data are limited, the sensitivity of the assay was 99.3% with a confidence interval (CI) of 96.1–100% and the specificity was 100% with a CI of 69.2–100%. Receptor blocking assay and fluorescence-activated cell sorting (FACS) analyses demonstrated the presence of VPAC receptors and gene profiling examinations confirmed that the cells expressing VPAC receptors were malignant prostate cancer cells. Conclusion These preliminary data are highly encouraging and warrant further evaluation of the assay to serve as a simple and reliable tool to detect prostate cancer non-invasively.

OBJECTIVES Alterations of cellular metabolism have been implicated in immune dysfunction in the tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC). Metformin has recently emerged as a candidate of interest for combination with immunotherapy in HNSCC. This study investigated the effect of metformin on immune cell infiltrates of HNSCC. METHODS Retrospective analysis of T cell infiltrates in primary tumor specimens from patients enrolled in a clinical window of opportunity trial of presurgical metformin. Metformin was titrated to a standard diabetic dose (2000 mg/day) for a minimum of 9 days (mean 13.6 days) prior to surgical resection. Pre and posttreatment surgical specimens from 36 patients (16 HPV+ , 20 HPV- ) were comparatively analyzed. FOXP3+ and CD8+ immune cell infiltrates in the tumor and peritumoral stroma of pre and posttreatment HNSCC specimens were quantified by digital image analysis using Visiopharm software. RESULTS Metformin treatment was associated with a 41.4% decrease in FOXP3+ T cells in intratumor regions of interest (P = .004) and a 66.5% increase in stromal CD8+ T cells at the leading edge of the tumor (P = .021) when compared to pretreatment biopsies. This was reflected in increased CD8+ /FOXP3+ cell ratios within the tumor (P

Objective Multifocal non–small cell lung cancer has historically been separated into synchronous primary lung cancers or intrapulmonary metastases with the use of histopathology. We hypothesize that using targeted next-generation sequencing of key driver mutations in multifocal non–small cell lung cancer will improve our ability to differentiate intrapulmonary metastases from synchronous primary lung cancers. Methods We identified patients who underwent surgery for non–small cell lung cancer between 2013 and 2018 with multifocal tumors. Archived specimens were reviewed with a 4-gene next-generation sequencing panel identifying mutations of EGFR, KRAS, BRAF, and NRAS. Synchronous primary lung cancers were classified as lesions with different histopathologic subtypes or driver mutations. Tests of hypotheses were performed with the Fisher exact test. Calculations were performed in Stata (v13.0; StataCorp LLC, College Station, Tex). Results A total of 18 patients had non–small cell lung cancer tumor specimens (n = 41) available from 2 or more sites. The pathologic diagnosis was predominantly adenocarcinoma (39/41 specimens). We detected a driver mutation in 68.3% (28/41) of all tumors. The most common mutations observed were in KRAS (n = 17/41) and EGFR (n = 7/41). Eleven patients had synchronous primary lung cancers, and 4 patients had intrapulmonary metastases based on combined histopathologic and molecular profiling results. Three lacked driver mutations in either lesion. Eight synchronous primary lung cancers (8/18, 44%) were downstaged when compared with their original diagnosis (P = .08). Of these, 4 patients received adjuvant chemotherapy unnecessarily in hindsight. Conclusions Molecular non–small cell lung cancer profiling using a 4-gene next-generation sequencing panel allows for better distinction between synchronous primary lung cancers and intrapulmonary metastases than histopathology alone. Routine use of next-generation sequencing for multifocal lesions prevents unnecessary adjuvant treatment for patients with histologically similar synchronous primary lung cancers.

AAA is a life-threatening vascular disease and one of the ten leading causes of death of 55–74 years old men and women. Although autoimmunity may be responsible for the initiation and/or progression of the disease, limited information is available about the pathogenesis of AAA. Mononuclear infiltrates comprised mostly of T and B cells and monocytes are present in AAA lesions. We have reported (J. Immunol. 192:4897, 2014) that clonally expanded beta-chain TCR transcripts are present in AAA lesions, providing evidence suggesting that AAA is a specific antigen-driven T-cell disease. In addition to the beta-chain TCR, there are several reasons to study the clonality and the repertoire of the alpha-chain TCR in AAA. Alpha-chain TCR transcripts from AAA lesions from these patients were amplified by the non-palindromic adaptor-PCR (NPA-PCR)/Vα-specific PCR and/or the Vα-specific PCR and the amplified transcripts were cloned and sequenced. Sequence analysis revealed substantial proportions of identical alpha-chain TCR transcripts in 4 of 5 AAA patients, demonstrating that clonally expanded T cells are present in these AAA lesions. These results are statistically significant. These T-cell clones very likely have undergone proliferation and clonal expansion in vivo in response to specific, although, as yet unidentified, antigen(s) (self or non-self) recognized by the T-cell clones. Three of 5 patients with AAA were typed by DNA-based HLA-typing and all expressed DRB1 alleles positive for the DRbGln70 amino acid residue, reported to be associated with AAA. Clonally expanded T cells in AAA lesions were found in all these 3 patients. These findings provide additional evidence suggesting that AAA is a specific antigen-driven T-cell disease.

Objective: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has recently been used for the evaluation of various lesions arising in the porta hepatis. The purpose of this study is to evaluate the diagnostic yield of this increasingly utilized approach to porta hepatis lesions. Study Design: A retrospective study of 72 consecutive samples of porta hepatis lesions obtained via EUS-FNA between 2004 and 2015 was conducted. Clinical histories and endoscopic findings were available prior to the diagnostic interpretation. The diagnosis of each lesion was based on its cytologic features on smears, its histologic features on cell block, a comparison with any relevant prior specimens, immunohistochemistry and flow cytometric studies when applicable. Results: A total of 72 lesions (59 lymph nodes, 2 cysts and 11 masses) were biopsied in 70 patients. Adequate specimens were obtained in 65/72 cases (90%). Most of the lymph nodes were benign (n = 40, 67%) and most of the masses were malignant or suspicious (n = 8, 73%). A variety of diagnoses, primary and metastatic, were made, including hepatocellular carcinoma, cholangiocarcinoma and lymphoma. In addition, we have noted a significant increase in the number of EUS-FNAs in recent years. Conclusion: EUS-FNA is an effective and increasingly utilized diagnostic approach for the evaluation of multiple types of lesions in the porta hepatis.

9051 Background: Patients (pts) with resectable stage IB (≥4cm)-IIIA non-small-cell lung cancer (NSCLC) derive modest overall survival benefit with neoadjuvant or postoperative adjuvant chemotherapy. Neoadjuvant therapy can speed the discovery of promising regimens by using pathologic response as a surrogate for OS. Major pathologic response (MPR), defined as < 10% viable tumor, was strongly associated with improved OS. PD-(L)1 checkpoint inhibitors in combination with chemotherapy are standard of care in advanced NSCLC. We hypothesize that the addition of N to neoadjuvant CP or CG will increase the MPR rate compared to historical controls. Methods: This is an investigator-initiated trial for pts with newly diagnosed AJCC 8th stage IB (≥4cm)-IIIA squamous or non-squamous EGFR/ALK WT NSCLC with a plan to have surgery. Pts receive 3 courses of N 360mg IV q 3w added to C 75mg/m2 IV q 3w plus P 500 mg/m2 IV q 3wks or G 1250mg/m2 IV d1, d8 with surgery 3 wks after the last dose. The primary objective is MPR. To estimate pathologic response, the resected pathology specimens are cut >1 section/cm. Using the Aperio Digital scanning system©, slides were imaged, and then annotated by at least 2 pathologists for viable tumor vs. treatment effect with respective areas then automatically calculated and percentage of viable tumor calculated. Our primary endpoint will be reached if 10/34 (29%) planned pts have at least an MPR. Results: From 6/2018-8/2019, 13 pts were enrolled all of whom had surgery. Median age was 69 (49-80), 38% women, 31% nonsquamous, 54% stage IIIA, and 77% PD-L1 positive (≥1%, SP263). Pre-surgical grade 3 toxicity occurred in 2/13 pts, one of whom was changed to carboplatin for courses 2 and 3. Grade 3 toxicities were neutropenia (2/13), anemia (1/13), and renal (1/13). One pt. developed hypothyroidism 4 mos after surgery. One pt died 6 weeks after surgery from complications unrelated to study drugs. Our primary endpoint was met; 11/13 (85%), had at least an MPR with 6/13 (46%) and 5/13 (38%) having an MPR and pCR respectively. Radiologic response rate was 46% (PR 5, CR 1). Pts with either PD-L1+ or PD-L1- had MPRs. With a median follow-up of 10 months there are no recurrences. Conclusions: The combination of nivolumab added to platinum doublets was well tolerated. The primary endpoint of MPR in at least 10/34 pts was surpassed with MPR or pCR in 11/13 pts post-surgery. MPR was seen independent of PD-L1 score. Exploratory outcomes assessing markers of immune bias in tumor tissue and plasma are in process. Clinical trial information: NCT03366766.

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

OBJECTIVES To assess the performance of molecular lymphosonography with dual-targeted microbubbles in detecting and quantifying the metastatic involvement in sentinel lymph nodes (SLNs) using a swine melanoma model. METHODS Targeted microbubbles were labeled with P-selectin and αV β3 -integrin antibodies. Control microbubbles were labeled with immunoglobulin G antibodies. First lymphosonography with Sonazoid (GE Healthcare, Oslo, Norway) was used to identify SLNs. Then dual-targeted and control microbubbles were injected intravenously to detect and quantify metastatic disease in the SLNs. Distant non-SLNs were imaged as benign controls. All evaluated lymph nodes (LNs) were surgically removed, and metastatic involvement was characterized by a histopathologic analysis. Two radiologists blinded to histopathologic results assessed the baseline B-mode images of LNs, and the results were compared to the histologic reference standard. The mean intensities of targeted and control microbubbles within the examined LNs were measured and compared to the LN histologic results. RESULTS Thirty-five SLNs and 34 non-SLNs from 13 Sinclair swine were included in this study. Twenty-one SLNs (62%) were malignant, whereas 100% of non-SLNs were benign. The sensitivity of B-mode imaging for metastatic LN diagnosis for both readers was relatively high (90% and 71%), but the specificity was very poor (50% and 58%). The sensitivity and specificity of molecular lymphosonography for metastatic LN detection were 91% and 67%, respectively. The mean intensities from dual-targeted microbubbles correlated well with the degree of metastatic LN involvement (r = 0.6; P

Background and Aims: The benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) patients remains unclear, emphasizing the need for improved prognostic biomarkers to identify patients at risk of metastatic recurrence. To address this unmet clinical need, we examined the expression and phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) in CRC tumor progression. VASP, a processive actin polymerase, promotes the formation of invasive membrane structures leading to extracellular matrix remodeling and tumor invasion. Phosphorylation of VASP serine (Ser) residues 157 and 239 regulate VASP function, directing subcellular localization and inhibiting actin polymerization, respectively. Methods: The expression levels of VASP protein, pSer157-VASP, and pSer239-VASP were determined by immunohistochemistry in tumors and matched normal adjacent tissue from 141 CRC patients, divided into 2 cohorts, and the association of VASP biomarker expression with clinicopathologic features and disease recurrence was examined. Results: We report that changes in VASP expression and phosphorylation were significantly associated with tumor invasion and disease recurrence. Furthermore, we disclose a novel 2-tiered methodology to maximize VASP positive and negative predictive value performance for prognostication. Conclusion: VASP biomarkers may serve as prognostic biomarkers in CRC and should be evaluated in a larger clinical study.

Introduction Arginase-1 is a novel immunohistochemical (IHC) marker for hepatocellular differentiation. The purpose of this study was to evaluate the expression of Arginase-1 and HepPar-1 in lung adenocarcinoma to assess the potential value of these markers for diagnosing metastatic lung tumors, especially to the liver in fine-needle aspiration specimens. Materials and methods Forty-four cytology specimens of lung adenocarcinoma, obtained by endobronchial ultrasound-guided fine-needle aspiration were retrospectively reviewed. IHC stains for Arginase-1 and HepPar-1 were performed on formalin-fixed paraffin-embedded cell blocks. Tissue from confirmed hepatocellular carcinoma was used as the positive control. TTF-1 IHC stain was performed in all cases. Results All 44 lung adenocarcinoma cases (100%) were negative for Arginase-1, whereas HepPar-1 expression was detected in 3 (7%) of lung adenocarcinomas and negative in 41 (93%). The 3 HepPar-1–positive lung adenocarcinoma cases demonstrated positive TTF-1 IHC stain performed on the same cell block. Although both Arginase-1 and HepPar-1 are useful diagnostic IHC markers to differentiate metastatic lung adenocarcinoma from hepatocellular carcinoma, Arginase-1 IHC stain shows better specificity than HepPar-1 does (Arginase-1 specificity 100% and HepPar-1 specificity 93%). Conclusions Arginase-1 IHC can be used in combination with other markers in the workup of metastatic lung adenocarcinoma, especially to the liver.

Background Syringocystadenoma papilliferum (SCAP) is an uncommon cutaneous adnexal proliferation. There have been several reports describing collision lesions of SCAP and verruca, although little is known about the frequency of this association. Molecular testing has revealed the BRAFV600E mutation in a large proportion of SCAP cases, although its expression pattern has not been previously evaluated. Methods In this retrospective analysis, we explored the potential histopathological association between verruca and SCAP. We also evaluated mutation-specific BRAFV600E expression in these lesions by immunohistochemistry. Cases of SCAP diagnosed over a 7-year period were closely reviewed for the presence of contiguous verrucous proliferations. Additional sections were cut and stained using the BRAFV600E-specific clone VE1 antibody. Results Contiguous verrucous proliferations were identified in 9 out of 12 identified cases. Furthermore, expression of the BRAFV600E mutation was identified in 7 out of 12 cases. Interestingly, in SCAP associated with endophytic verrucous proliferations (n = 4), expression of BRAFV600E was found in both the glandular and the contiguous hyperplastic squamous epithelium. Conclusion Overall, these findings suggest that contiguous verrucous proliferations in SCAP are common. Both components of the neoplasm may express the BRAFV600E mutation, which is suggestive of a common origin.

Background Atypical lipomatous tumor/well-differentiated liposarcoma (ALT-WDLPS) and dedifferentiated liposarcoma (DDLPS) are characterized cytogenetically by a 12q13–15 amplification involving the mouse double minute 2 (MDM2) oncogene. Fluorescence in situ hybridization (FISH) is used frequently to detect this amplification and aid with the diagnosis of these entities, which is difficult by morphology alone. Recently, bright-field in situ hybridization techniques such as chromogenic in situ hybridization (CISH) have been introduced for the determination of MDM2 amplification status. Methods The present study compared the results of FISH and CISH for detecting MDM2 amplification in 41 cases of adipocytic tumors. Amplification was defined in both techniques as a MDM2/CEN12 ratio of 2 or greater. Results Eleven cases showed amplification with both FISH and CISH, and 26 cases showed no amplification with both methods. Two cases had discordant results between CISH and FISH, and two cases were not interpretable by CISH. Conclusion CISH is advantageous for allowing pathologists to evaluate the histologic and molecular alterations occurring simultaneously in a specimen. Moreover, CISH is found to be more cost- and time-efficient when used with automation, and the signals do not quench over time. CISH technique is a reliable alternative to FISH in the evaluation of adipocytic tumors for MDM2 amplification.

The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted. Cancer Immunol Res; 2(2); 154–66. ©2013 AACR.

Objective: Liquid-based cytology of nongynecological specimens is commonly used in cytology laboratories throughout the world and various processing methods, such as ThinPrep and SurePath, have been reported. The cytological features and performance of liquid-based cytology for various cytology specimens, including body cavity fluids, urine, brushing specimens and fine-needle aspiration of various lesions, were reviewed and compared with the experience of our laboratory and the literature published in PubMed. Study Design: The parameters for the evaluation of liquid-based cytology and conventional smears were described in the various types of specimens. Criteria for the interpretation of nongynecological liquid-based cytology were highlighted to show differences in cell morphology, background and artifacts. Results: The interpretation requires familiarity with the appearance of liquid-based cytology in the various types of preparations to avoid misdiagnosis. Conclusions: Cell blocks can be prepared with specimens preserved in a liquid-based cytology medium and immunocytochemical stains and molecular testing can be successfully performed. These are important adjuncts in order to reach a definitive diagnosis.

Objective: Fine needle aspiration (FNA) cytology with thyroglobulin wash (TG-W) testing is recommended for follow-up of patients with differentiated thyroid carcinoma (DTC). The goal of this retrospective study was to determine if TG-W results contributed to the management of cases with positive FNA cytology. Study Design: We reviewed data on patients with positive and suspicious cytology results, undergoing lymph node or thyroid bed FNA with TG-W testing as part of the preoperative or follow-up investigation of histologically proven DTC in our institution and from the literature. Results: Of 30 positive/suspicious lymph node and thyroid bed FNAs in our institution, 22 (73%) had an elevated (>1 ng/ml) TG-W level. Seven of 8 TG-W-negative cases had DTC on follow-up. Of 577 cytology-positive/suspicious FNAs in the literature, 557 (97%) showed TG-W-positive results. Fourteen of 20 TG-W-negative cases had DTC on follow-up. All patients in retrospective and literature review groups with positive and suspicious FNA cytology and available follow-up were treated for recurrent or metastatic disease regardless of TG-W results. Conclusion: Observations of both our and other institutions support a recommendation of reflex FNA TG-W testing only for cases with negative or indeterminate cytology results.

Many studies recently focus on complicated and expensive genomic tests, but the prognostic values of biochemical markers which are easily obtained in clinics are largely overlooked and without further exploration. This study assesses the association of neutrophil–lymphocyte-ratio (NLR) with prognosis of lung cancer patients. In 1032 patients with histologically confirmed lung cancer, the association of pretreatment NLR values with overall survival (OS) was evaluated using a Cox proportional hazards model and the temporal relationship of longitudinal NLR was assessed using a mixed effects model. Compared to the patients with a low pretreatment NLR value, those with elevated NLR exhibited a statistically significant worse OS with a hazard ratio (HR) of 1.50 (P

The purpose of this ongoing study is to evaluate the ability of molecular ultrasound imaging to detect the metastatic involvement in the sentinel lymph node (SLN) of melanoma. The SLN is the first node along the lymphatic channel from the primary tumor and accurate characterization of the SLN helps in the clinical management of melanoma. To date, eight swine (3–7 kg; Sinclair BioResources, Columbia, MO, USA) with naturally occurring melanoma have been studied. Contrast enhanced ultrasound on a S3000 scanner with a 9L4 probe (Siemens Healthineers, Mountain View, CA, USA) was used for imaging. Dual-targeted microbubbles were created with Targestar SA (Targeson, San Diego, CA, USA) labeled with P-selectin and α V β 3 -integrin. IgG labeled Targestar SA was used as control. A two stage imaging approach was used to first identify and then characterize SLNs. First, 0.25 ml of Sonazoid (GE Healthcare, Oslo, Norway) was injected around the tumor and SLNs were identified. Next, dual-targeted and control microbubbles were injected IV with a 30 min interval between injections. Agents were allowed to circulate for 4 min to enable binding. Then 2 sets of image clips were collected before and after a high-power destruction sequence. The mean intensity difference in pre- and post- destruction images was calculated as a relative bubble retention measure. Some non-SLNs were also imaged in this fashion as control. All examined nodes were dissected and histologically examined for the presence of metastatic involvement. A total of 30 lymph nodes (16 SLNs and 14 non-SLNs) were analyzed with 10 SLNs showing metastatic involvement greater than 5%. All non-SLNs were benign. The mean intensity of the dual-targeted bubbles was significantly higher than that of IgG control bubbles in the metastatic SLNs (19.23 ± 23.95 AU vs. 0.20 ± 0.26 AU; p=0.03). Benign nodes did not show significant difference in the mean intensity of the dual-targeted and control bubbles (0.85 ± 1.81 AU vs. 0.03 ± 0.26 AU; p=0.07). Additionally, the mean intensity of dual-targeted bubbles for metastatic nodes was significantly different from that of benign nodes (p=0.002), while control bubbles did not differentiate between metastatic and benign nodes (p=0.09). The results indicate that dual-targeted microbubbles labeled with P-selectin and α V β 3 -integrin can help to characterize SLNs.

Nrf2 and its role in controlling levels of the AKR family of aldo–keto reductases which have been implicated in resistance to platinum-based chemotherapy was studied in ovarian, cervical and lung cell lines. Nrf2 shRNA knockdowns of cells from different tumor origins were prepared to determine the role of this factor in producing resistance to platinum chemotherapy. Nrf2 knockdowns resulted in marked decreases in AKR1C1, AKR1C2 and to a lesser extent AKR1C3. Additionally, all other candidate enzymes GSTπ and TRX1 were decreased, but their role was difficult to correlate to cytotoxicity. Nrf2 knockdowns exhibited marked increases in mitochondrial membrane depolarization and ROS production following cisplatin treatment, with the cervical ME180R knockdowns exhibiting the greatest effect (AKR1C1 and AKR1C2 levels were decreased in the ME180R and SKOV3 cells to near zero). Oxaliplatin tended to parallel cisplatin, except it markedly stimulated O2 − production not $$ {\text{H}}_{2} {\text{O}}_{2}$$ by oxaliplatin treatment of the ME180R cells. The pJNK/p38 pathway has been implicated in cisplatin cytotoxicity, and significant phosphorylation of pJNK was observed in the SKOV3 and ME180R and p38 in the SKOV3 knockdowns. Phosphorylation of ATF2 was decreased in the Nrf2 knockdowns (Crf38, Srf6, Arf5) which could affect its interaction with JNK and p38. Oxaliplatin treatment showed minimal effects on the JNK/p38 pathway, showing that its mode of action is different although ROS generation appeared an initial step with both drugs. Nrf2 controls a multitude of different candidate genes; however, it did markedly modulate cisplatin resistance through the AKR family. This involved ROS production and activation of the pJNK/p38 pathway with involvement of ATF2.

Objective: We performed a retrospective review of Papanicolaou (Pap) testing to assess whether the cytology practice in our institution was affected by the introduction of high-risk (HR) human papillomavirus (HPV) assays over time. Study Design: Cytology, HPV and histopathology records were retrieved from our laboratory information system from 2003 to 2015. Records for Digene Hybrid Capture 2®, Hologic Cervista® and Roche Cobas® HPV assays were obtained. A 3-month follow-up for HPV detected cases was performed, and results were correlated with cytology and biopsies. A 1-year follow-up of HPV 16/18 and other HR HPV detected cases was also performed. Results: From 2008 to 2015, a noticeable decrease in Pap testing volume occurred, from 11,792 to 4,664, while the percentage of HPV testing increased from 19 to 59%. Similar HPV detection rates and follow-up results for both reflex and cotesting were observed in the 3 HPV assays. Conclusions: The decrease in Pap testing was due to the lengthening of the test interval when cotesting results were negative. Practitioners adhering to guidelines accounts for increased molecular testing volume. A trend towards higher-grade cervical intraepithelial neoplasia in the follow-up of detected HPV 16/18 was noted. So far there has been no demand for HPV as a stand-alone test.

Recent studies have shown that intratumoral heterogeneity (ITH) is prevalent in clear cell renal cell carcinoma (ccRCC), based on DNA sequencing and chromosome aberration analysis of multiple regions from the same tumor. VHL mutations were found to be universal throughout individual tumors when it occurred (ubiquitous), while the mutations in other tumor suppressor genes tended to be detected only in parts of the tumors (subclonal). ITH has been studied mostly by DNA sequencing in limited numbers of samples, either by whole genome sequencing or by targeted sequencing. It is not known whether immunohistochemistry (IHC) can be used as a tool to study ITH. To address this question, we examined the protein expression of PBRM1, and PBRM1-related proteins such as ARID1A, SETD2, BRG1, and BRM. Altogether, 160 ccRCC (40 per stage) were used to generate a tissue microarray (TMA), with four foci from each tumor included. Loss of expression was defined as 0-5% of tumor cells with positive nuclear staining in an individual focus. We found that 49/160 (31%), 81/160 (51%), 23/160 (14%), 24/160 (15%), and 61/160 (38%) of ccRCC showed loss of expression of PBRM1, ARID1A, SETD2, BRG1, and BRM, respectively, and that IHC could successfully detect a high prevalence of ITH. Phylogenetic trees were constructed that reflected the ITH. Striking co-losses among proteins were also observed. For instance, ARID1A loss almost always accompanied PBRM1 loss, whereas BRM loss accompanied loss of BRG1, PBRM1 or ARID1A. SETD2 loss frequently occurred with loss of one or more of the other four proteins. Finally, in order to learn the impact of combined losses, we compared the tumor growth after cells acquired losses of ARID1A, PBRM1, or both in a xenograft model. The results suggest that ARID1A loss has a greater tumor-promoting effect than PBRM1 loss, indicating that xenograft analysis is a useful tool to investigate how these losses impact on tumor behavior, either alone or in combination.

Objective: We aimed to supplement microscopic examination of biliary cytobrush specimens to improve sensitivity by mutational profiling of: (1) selected cells microdissected from cytology slides and (2) corresponding cell-free DNA in residual supernatant fluid. Study Design: From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from microdissected cells and 1–2 ml of cytocentrifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and loss of heterozygosity (LOH) analysis of microsatellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. Results: Mutations were found in 25/28 patients with malignancy, and no mutations were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer. Mutational profiling of supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. Conclusion: Brush cytology specimens yielded supernatant fluid enriched with DNA, probably from actively proliferating cells. Mutational profiling can enhance the cytologic evaluation and characterization of specimens suspected to contain pancreatic or bile duct cancer.

BACKGROUND: The aim of this study was to evaluate the expression of epidermal growth factor receptor (EGFR) and insulinlike growth factor 1 receptor (IGF-1R) proteins and IGF-1R gene copy numbers in pancreatic ductal adenocarcinoma in relation to patients' characteristics and prognosis. METHODS: Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue derived from tumor specimens recovered during surgery. Slides were evaluated for membranous EGFR and IGF-1R staining using both the HercepTest and the semiquantitative H-score systems. Chromogenic in situ hybridization was performed to quantify IGF-1R gene copy number. The primary outcome was the association between EGFR expression, IGF-1R expression—in both neoplastic epithelial and stromal cells—or IGF-1R gene copy number and overall survival. Secondary outcomes included associations between EFGR and IGF-1R expression and pathologic variables. RESULTS: A total of 105 patients were included. EGFR expression was present in 30.4% of cases and was associated with lymph node metastasis (P = .038). IGF-1R was overexpressed in 53% of tumors and correlated with higher tumor grade (P = .033). High membranous expression of EGFR (P < .001) and/or IGF-1R (P = .004), the cytoplasmic detection of EGFR (P = .027), and high expression levels of IGF-1R in the tumoral stroma (P < .001) were all associated with shorter overall survival, being significantly better in patients who simultaneously do not express membranous EGFR or stromal IGF-1R. CONCLUSIONS: EGFR and IGF-1R expression, in neoplastic and stromal cells, seems to be an important prognostic factor. Cancer 2012;3484–3493. © 2011 American Cancer Society.

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is central to discerning the diagnosis of solid pancreatic tumors through tissue acquisition. Test performance is affected by a number of factors including location of mass within the pancreas, presence of onsite cytology technologist, and number of passes with the needle. The influence of tumor size has not been well studied.The objective of the current study was to determine whether the size of mass affects the diagnostic accuracy for solid pancreatic lesions aspirated under EUS guidance.Data were collected retrospectively on all patients with solid pancreatic masses undergoing EUS-FNA from June 2003 to August 2010. The cytology samples were reported as positive, suspicious for malignancy, atypical, negative, or nondiagnostic. The gold standard for a cytological diagnosis was histological confirmation or clinical follow-up of more than 6 months with repeat imaging. Patients were divided into five groups based upon lesion size as follows: (a) less than 1 cm, (b) 1-2 cm, (c) 2-3 cm, (d) 3-4 cm, and (e) greater than 4 cm. Performance characteristics of EUS-FNA including sensitivity, specificity, and accuracy were compared for each group. Accuracy was defined as the ratio of the sum of true-positive and true-negative values divided by the number of lesions.We identified 583 patients with solid pancreatic lesions in which EUS-FNA was performed and adequate cellularity was obtained (47% men, mean age 65 ± 1.4 (SE) years). Overall, 486 (83%) of lesions were pancreatic adenocarcinoma, 18 (3%) were neuroendocrine tumors, 12 (2%) were lymphomas, and 67 (12%) were benign lesions. The median size of the mass was 3 cm (range, 0.5-7 cm). A mean of 4.9 passes (range, 1-9 passes) was needed to obtain adequate samples from lesions. The overall yield of obtaining adequate samples for diagnosis was 85%. When stratified by size, the EUS-FNA sensitivity for lesions with size1, 1-2, 2-3, 3-4, and4 cm was 40, 75.9, 86.9, 93.2, and 91.6%, respectively; EUS-FNA sensitivity strongly correlate with tumor size (p 0.001). Similarly, the accuracy of EUS-FNA increased as lesion size increased, ranging from 47% for tumors less than 1 cm to 88% for tumors greater than 4 cm (p 0.05). Location of tumor and number of needle passes did not significantly influence EUS-FNA performance characteristics.The sensitivity and diagnostic accuracy of EUS-FNA for solid pancreatic lesions is strongly correlated with tumor size. Sensitivity and accuracy decrease significantly for tumors that are smaller than 1 cm.