Staining Intensity Of Tumor Cells Research Articles

Protein expression and localization of ABC transporters in pancreatic adenocarcinoma: Prognostic role of ABCC8

BackgroundATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients’ prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers – ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). MethodsProtein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. ResultsNo CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). ConclusionsThe study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.

Stress keratin 17 as a novel biomarker of response in immune checkpoint blockade–treated head and neck squamous cell carcinoma.

3117 Background: Low response rates in immune checkpoint blockade (ICB) treated head and neck squamous cell carcinoma (HNSCC)drive a critical need for robust clinically validated biomarkers that can predict response to ICB. Stress keratin 17 (K17) is a known prognostic marker in various types of cancer, including HNSCC; however, its predictive value for ICB response has not been investigated. Preclinical studies suggest K17 suppresses macrophage-mediated CXCL9/CXCL10 chemokine signaling involved in attracting activated CD8+ T cells into tumors. Furthermore, knocking out K17 results in restored response to ICB in a HNSCC mouse model. Here, we evaluated if K17 protein expression predicts response to ICB in human HNSCC patients. Methods: We conducted a retrospective analysis of 26 HNSCC patients that received at least one cycle of pembrolizumab at the University of Wisconsin-Madison Carbone Cancer Center. Pretreatment, archival, formalin-fixed, paraffin-embeded samples were stained by immunohistochemistry using a K17 monoclonal antibody. Clinical outcomes were investigator-assessed for all patients with at least one post-treatment scan or evidence of clinical progression after treatment initiation. Based on independent pathology review, cases were categorized into K17 high vs K17 low based on a cut-off of > 5% strong cytoplasmic staining intensity of tumor cells in the invasive carcinoma component. Correlation between K17 expression and clinical outcomes was assesed using Fischer's exact test and log rank test. Results: The 26 patients included in this study were 85% male, median age 60.5 years, 74% ECOG performance status < 2, with 80% having received prior chemotherapy. Primary site included oral cavity (54%), oropharynx (23%), larynx (4%), or other (19%). Seventeen tumors (65%) showed high K17 expression, and 9 tumors (35%) showed low K17 expression. Eleven patients (42%) had programmed death ligand 1 (PD-L1)+ tumors as determined by combined positive score. Six patients (23%, all K17 low) achieved clinical benefit, while 20 patients (77%, 17 K17 high and 2 K17 low) had progressive disease. High K17 expression was associated with lack of clinical benefit (p < 0.001), shorter time to treatment failure (p < 0.001), progression-free (p = 0.004) and overall survival (p = 0.02). PD-L1 expression by immunohistochemistry (clone 22C3) did not correlate with K17 expression or clinical outcome. Conclusions: Our findings suggest that K17 expression may predict clinical benefit from ICB in HNSCC patients, thus supporting further validation studies. [Table: see text]

Increased phosphorylated CREB1 protein correlates with poor prognosis in clear cell renal cell carcinoma.

BackgroundThis study aims to investigate the level of cAMP response element-binding protein 1 (phospho S133) (p-CREB1) protein in clear cell renal cell carcinoma (ccRCC) and evaluates its prognosis significance.MethodsImmunohistochemistry (IHC) method was performed to detect p-CREB1 staining in 233 ccRCC patients. Three or more high-power fields per tissue section were equally captured by a Leica DMRXA microphotographic system, and average staining intensity (optical density, OD) was analyzed by Leica Qwin Standard V2.6 system. Univariate and multivariate Cox proportional regression model was performed to assess the correlation of p-CREB1 staining and clinical outcomes.ResultsIHC proved that the level of p-CREB1 protein was significantly higher in tumor tissues than in adjacent normal tissues, and gradually increased from normal to tumor sections. On the basis of the receiver operating characteristic curve, patients were divided into low p-CREB1 staining (OD ≤0.28) and high p-CREB1 staining subgroup (OD >0.28) according to p-CREB1 protein staining intensity of tumor cells. Multivariate analyses showed that high p-CREB1staining was an independent risk factor for cancer-specific free survival, overall survival and progression-free survival.Conclusionsp-CREB1 protein is an independent prognostic biomarker for ccRCC patients.

Immunohistochemical study of the MSI phenotype of urothelial bladder cancer

To investigate the MSI phenotype of urothelial bladder cancer (BC) by immunohistochemistry (IHC) and to assess its relationship to the prognostic factors of the disease and PD-L1 status of BC. Using the surgical and biopsy materials obtained from 50 patients with BC during examination and treatment, the investigators studied the MSI phenotype by IHC. A system was proposed to assess the IHC expression of MSI proteins, by taking into account the intensity of nuclear staining and the area occupied by tumor cells with stained nuclei. An analysis of the results of an IHG study of MSI protein expression revealed a high direct correlation between the nuclear staining intensity of tumor cells and the percentage of tumor area occupied by the latter. The lack/decrease of the expression of the studied proteins was associated with the stage (T) and the presence of a high-grade tumor. The heterogeneous expression of the studied proteins (PMS2, MLH1, and MSH6) was noted to be 10, 30, and 40%, respectively. A high direct correlation was observed between the nuclear staining intensity of tumor cells and the percentage of the area occupied by the latter. There was a relationship between the lack and/or decrease of the expression of MSI proteins (mainly PMS2 and MLH1 and to a lesser extent MSH6) and the grade of BC, as well as stage T; there was a tendency to decrease the expression of the studied proteins in the area of invasive tumor growth, which confirms the prognostic role of MSI proteins. The most pronounced heterogeneity of IHC expression was noted with MSH6; the least one was seen with PMS2. There was a predominance of high-grade surface carcinomas (T1) among the heterogeneously stained tumors. In the case of positive PD-L1 status, there was decreased PMS2 and MLH1 expression and pronounced MSH6 expression; no significant relationship was found due to the small number of these cases. The authors consider it necessary to conduct further studies of the relationship between the MSI phenotype and the PD-L1 status of BC.

Association Between Programmed Death Ligand 1 Expression in Patients With Basal Cell Carcinomas and the Number of Treatment Modalities

Response to programmed death 1 (PD-1) inhibitors has been associated with programmed death ligand 1 (PD-L1) expression levels in several cancers, but PD-L1 expression and its clinical significance in basal cell carcinoma (BCC) are unknown to date. To assess PD-L1 expression in treatment-naive and treated BCCs. This investigation was a cross-sectional study at a single academic tertiary referral center. Immunohistochemical staining on formalin-fixed BCCs from a dermatology clinic were examined in masked fashion by a dermatopathologist and a dermatologist. The study dates were March 31, 2014, to June 7, 2016. Treated BCCs (including those recurrent after surgery, radiotherapy, systemic chemotherapy, or topical chemotherapy) vs treatment-naive BCCs. Percentage of tumor cells and tumor-infiltrating lymphocytes (TILs) with PD-L1 expression, intensities of expression, and association with treatment modalities. Among 138 BCCs from 62 patients (43 males and 19 females; mean [SD] age at biopsy, 61.6 [13.7] years), 89.9% (124 of 138) were positive for PD-L1 expression in tumor cells, and 94.9% (131 of 138) were positive for PD-L1 expression in TILs, defined as greater than 5% positive immunohistochemical staining in the respective cell populations. The PD-L1 immunohistochemical staining intensity of 78 treated BCCs compared with 60 treatment-naive BCCs was significantly different in tumor cells (32% vs 7%, P = .003) and TILs (47% vs 18%, P = .008) after adjusting for the age at diagnosis. In a multivariable model adjusting for age, sex, and BCC location, PD-L1 staining intensity in tumor cells increased with the number of distinct prior treatment modalities (median, 0.12; interquartile range, 0.03-0.20; P = .007). Our data suggest that PD-1 immunotherapy may have activity against BCCs, including in those that have been previously treated. This hypothesis needs to be tested in future clinical trials.

SULF2 Expression Is a Potential Diagnostic and Prognostic Marker in Lung Cancer.

AimsLung cancer is one of the most deadly cancers; median survival from diagnosis is less than one year in those with advanced disease. Novel lung cancer biomarkers are desperately needed. In this study, we evaluated SULF2 expression by immunohistochemistry and its association with overall survival in a cohort of patients with non-small cell lung cancer (NSCLC). We also looked for the presence of SULF2 protein in plasma to evaluate its potential as an early detection biomarker for NSCLC.MethodsWe identified patients who underwent surgical resection for pulmonary adenocarcinoma or squamous cell carcinoma at our institution. A section from each paraffin-embedded specimen was stained with a SULF2 antibody. A pathologist determined the percentage and intensity of tumor cell staining. Survival analysis was performed using a multivariate Cox proportional hazards model. Using a novel SULF2 ELISA assay, we analyzed plasma levels of SULF2 in a small cohort of healthy donors and patients with early stage NSCLC.ResultsSULF2 staining was present in 82% of the lung cancer samples. Squamous cell carcinomas had a higher mean percentage of staining than adenocarcinomas (100% vs. 60%; p<0.0005). After adjusting for age, sex, race, histologic type, stage, and neoadjuvant therapy, there was a non-significant (31%; p = 0.65) increase in the risk of death for patients with adenocarcinoma with SULF2 staining in tumor cells. In contrast, there was a significant decrease in the risk of death (89%; p = 0.02) for patients with squamous cell carcinoma with SULF2 staining in tumor cells. SULF2 protein was present in plasma of patients with early stage NSCLC, and soluble SULF2 levels increased with age. Finally, plasma SULF2 levels were significantly elevated in early stage NSCLC patients, compared to healthy controls.ConclusionsTumor expression of SULF2 may affect prognosis in NSCLC, while blood SULF2 levels may have a significant role in the diagnosis of this fatal disease.

Abstract 5308: CD9, CD63 and CD82 tetraspanins expression profile in vulvar carcinomas.

Abstract Background: Vulvar carcinomas are approximately 95 % of all vulvar tumors. This cancer presents good prognosis since early diagnosed, and survival depends of inguinal lymphonode commitment, with about 30% of patients presents lymphonode metastasis. The mechanisms involved in biological behavior of vulvar cancer remain unclear and its treatment involves physical mutilation and psychosocial factors of patients. Therefore, identification of features related with this tumors development or genesis can contribute to its knowledge and treatment. The tetraspanins are proteins that are involved in several biological processes including cell proliferation, adhesion, invasion and migration. Their reduction or lack of expression is frequently reported in metastatic and poor prognosis tumors. Our aim was to analyze CD9, CD63 and CD82 tetraspanins expression profile in vulvar carcinoma compared to normal tissue. Methods: All samples were obtained from Pathology Department of Hospital A. C. Camargo, Sao Paulo, Brazil. TMA block was constructed with 150 paraffin-embedded samples and was used in immunohistochemical assay. The tissue sections were incubated with specific antibodies against CD9, CD63 and CD82, following manufacturer's instruction. The slides were evaluated and scored semiquantitatively by assessing the staining intensity in tumor cells and the frequency of positive cells. Results: The Immunohistochemical analysis showed that all tetraspanins were expressed in tumor samples with different intensity and frequency. CD63 were expressed by a higher samples number (90%), with stronger staining both cytoplasm (79%) and nucleus (11%). Lack of CD63 expression was described in metastatic melanomas lesions, while no metastatic lesions shows higher levels of this protein. Seventy five percent of our samples expressed CD9, exclusively on cellular membrane. CD82 showed positive reaction in 45% of the samples with weakly or moderate staining, both nucleus and cytoplasm. The association of the downregulation of CD9 and/or CD82 with a worse prognosis has been shown in breast, cervical, endometrial, esophageal, head and neck, and others carcinomas. Moreover, CD82 is considered to be an important metastasis suppressor. Conclusion: Our preliminary results showed that tetraspanins may have an important role in vulvar carcinomas and CD82/CD63 nuclear expression must be better investigated. Further analyses are ongoing. Citation Format: Katia C. Carvalho, Rafael M. Rocha, Isabela W. Cunha, Fernando A. Soares. CD9, CD63 and CD82 tetraspanins expression profile in vulvar carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5308. doi:10.1158/1538-7445.AM2013-5308

Association of tissue factor expression in squamous cell head and neck carcinomas with well-differentiated tumors.

e16022 Background: Tissue factor (TF) is a glycoprotein that initiates coagulation when complexed with factor VIIa. High levels of TF expression have been found in many cancers and correlated with tumor aggressiveness. TF has been shown to be expressed in head and neck squamous cell carcinomas (HNSCC) but the role of TF on cell function and prognosis are not known. The epidermal growth factor receptor (EGFR) is overexpressed in many HNSCC and EGFR and TF expression are highly correlated in some tumor types. Our goal was to correlate the expression of TF and EGFR in HNSCC with tumor cell differentiation and prognosis. Methods: Twenty biopsy specimens of squamous cell head and neck carcinomas were studied. Immunohistochemistry for TF, EGFR, and p16 was performed. Cases were scored based upon the staining intensity of tumor cells using a semiquantitative scale as negative, weakly (+), moderately (++) or strongly (+++) positive. Results: The tumor stages ranged from T1N0 to T4N3. Of the 20 specimens, 5 were well differentiated, 12 were of moderate differentiation, and 3 were poorly differentiated. Median survival was 23 months, median follow-up 73 months. TF was expressed in 75% (15/20) of tumors at varying levels. Moderate TF expression was found in 6 specimens, and 9 showed weak expression. None of the poorly differentiated tumors expressed TF, and there was a definite negative correlation between TF expression and tumor differentiation (Pearson r=--0.747, p=0.0002). There was no association between TF expression and survival (Log-Rank 0.3115, p=0.8558). All of the tumors analyzed expressed EGFR and there was no correlation between TF expression and EGFR expression. Overall, 25% (5/20) of tumors expressed p16, suggesting human papilloma virus infection. Conclusions: Results from this retrospective study indicate that well differentiated HNSCC express higher levels of TF than poorly differentiated tumors and that TF and EGFR expression are not tightly linked. Moreover, unlike other cancer sites, HNSCC TF expression did not appear to worsen overall patient survival, and was actually associated with more well-differentiated tumors.

Expression level of pre-B-cell leukemia transcription factor 2 (PBX2) as a prognostic marker for gingival squamous cell carcinoma

In this study, we investigated the interrelationship between clinicopathologic findings and pre-B-cell leukemia transcription factor 2 (PBX2) expression in gingival squamous cell carcinoma (GSCC). Expression level of PBX2 was immunohistochemically examined in 66 GSCC subjects (30 men and 36 women) with ages ranging from 42 to 85 (median 64.5) years, in which staining intensity in tumor cells was categorized as either weaker (level 1) or equal to/stronger (level 2) than that in the endothelial cells. PBX2 expression is correlated with valosin-containing protein (VCP) expression. Univariate and multivariate analyses revealed a high level of PBX2 expression to be a poor prognosticator for disease-free survival (DFS) and overall survival (OS), and PBX2 expression was an independent prognostic factor for both DFS and OS in GSCC. PBX2 expression level in GSCC is prognostic. PBX2 may be a useful marker to identify the potential for progression in GSCC.

Abstract A50: Biomarker analysis of PTEN, targeted oncogenic mutations, and HPV in a phase II trial of the irreversible oral pan-human EGF receptor (HER) inhibitor dacomitinib (D) (PF-299804) as first-line treatment in recurrent and/or metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN).

Abstract Background: The pan-HER inhibitor D has demonstrated single-agent activity in a phase II trial of 69 patients (pts) with first-line RM SCCHN, reporting an objective response rate (ORR) of 12.7%, median progression-free survival (PFS) of 12.1 weeks (wks) and median overall survival (OS) of 35.9 wks (Siu et al. ASCO 2011). The identification of biomarkers predictive of sensitivity or resistance is of top priority to assist with pt selection in the development of D in SCCHN. Methods: Archival formalin-fixed paraffin-embedded (FFPE) tumor specimens were used for analysis. PTEN expression was measured by immunohistochemistry and predominant staining intensity of tumor cells was scored from 0 (absent) to 2+ (strong). Tumor DNA was subjected to mutation analysis using the OncoCarta panel 1.0 (238 mutations from 19 genes) on the Sequenom™ MassARRAY platform. Genotyping for HPV16 was performed using RT-PCR. Log-rank test was used to compare OS and PFS and Fisher's exact test was used to compare ORR between biomarker subgroups. Results: Results for PTEN expression, mutation analysis and HPV status were available for 40, 47 and 40 pts respectively. For PTEN, 7 pts (17.5%) scored 0, 30 pts (75%) scored 1+ and 3 pts (7.5%) scored 2+. For mutation analysis, no mutations were identified in 42 pts (89%); 5 pts (11%) had the following mutations: PIK3CA E545K (n=2), NRAS G12C plus PIK3CA E542K (n=1), NRAS G12C plus PIK3CA E545K (n=1), NRAS G12C (n=1). For HPV status, 14 pts (35%) were HPV16+. Summary of the results are displayed in the Table. Although statistical significance was not demonstrated for any of the subgroup analyzes, pts who harbored oncogenic mutations trended to have poorer OS compared to those without (17.6 wks vs 35.9 wks, p=0.20), with no responders among 4 response-evaluable pts. Pts with HPV16+ tumors had lower ORR to D (0% vs 17%, p=0.28) but better OS than those with HPV16-tumors (49.6 wks vs 32.7 wks, p=0.10). Conclusions: The data suggest a possible association between HPV16, PIK3CA and NRAS status and ORR or OS. Since these observations are based on a limited number of samples from a single arm study, a prospective trial designed for biomarker validation will be required to evaluate the strength of these associations and if these are prognostic vs predictive biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A50.

Different Serotonergic Expression in Nevomelanocytic Tumors

The neuromediator serotonin (5-hydroxytryptamine; 5-HT) has been proposed to play a role in tumor progression. Thus, the aim of the present investigation was to determine whether alterations in the serotonergic system occur in nevomelanocytic tumors. For this purpose, paraffin-embedded biopsies of superficial spreading malignant melanoma (SSM), dysplastic compound nevi (DN) and benign compound nevi (BCN) were characterized with regard to their expression of 5-HT, the 5-HT1A and 5-HT2A receptors, and the serotonin transporter protein (SERT), by immunohistochemical analysis. Melanocytes in the region surrounding the tumor were found to express both the 5-HT1A and 5-HT2A receptors. Tumor cells that immunostained positively for the different serotonergic markers were observed in the suprabasal epidermis of DN tissue and, to an even greater extent, in the case of SSM. Furthermore, some of these latter cells expressed both 5-HT1AR and 5-HT2AR. The level of expression of 5-HT1AR at the junctional area was lower for SSM than for DN or BCN. As the degree of atypia increased, the intensity of tumor cell staining in the dermis for 5-HT1AR and SERT declined. Vessel immunoreactivity for 5-HT2A was more intense in SSM than in BCN tissue. Round-to-dendritic cells that expressed both SERT and 5-HT1AR were seen to infiltrate into the dermal region of the tumor, this infiltration being more evident in the case of DN and SSM. These latter cells were also tryptase-positive, indicating that they are mast cells. Thus, alterations in serotonergic system may be involved in nevomelanocytic tumors and mast cells may play an important role in this connection.

Prognostic significance of CUB domain containing protein expression in endometrioid adenocarcinoma

CDCP1, a transmembrane protein with intracellular tyrosine residues which are phosphorylated upon activation, is supposed to be engaged in proliferative activities and resistance to apoptosis of cancer cells. High level of CDCP1 expression proved to be a poor prognosticator for lung adenocarcinoma. Here, expression level of CDCP1 was immunohistochemically examined in 110 cases (median age of 54.7 years) of endometrioid adenocarcinoma, and its clinical implications were evaluated. Tumor stage was stage I in 71 cases (64.5%), II in 5 (4.5%), III in 28 (25.5%), and IV in 6 (5.5%). Staining intensity of tumor cells was divided into two categories; tumor cells with no to weak and moderate to strong membrane staining. The intensity of CDCP1 expression in each case was defined by the staining of major population of cells as follows; cases with tumor cells showing no to weak and moderate to high membrane staining were categorized as CDCP1-low and CDCP1-high, respectively. Eighty-seven of 110 cases were categorized as CDCP1-high, and the remaining as CDCP1-low. Significant positive correlation was observed between low CDCP1 expression and stage (p=0.0091), relapse rate (p=0.0017), and poor prognosis (p=0.0009). Multivariate analysis revealed that low CDCP1 and advanced stage were independent poor prognostic factors for both OS and DFS. As compared to cancer cells, normal endometrium continuously expressed CDCP1. These suggested that the attitude of CDCP1 in cancers of lung and endometrium was different. Different role of CDCP1 by tissues and cancers is discussed.

Transcriptional and Post-Translational Upregulation of HER3 (ErbB3) Counteracts Antitumor Effect of HER2 Tyrosine Kinase Inhibitors.

Abstract We hypothesized that sustained inhibition of HER3 and its output to PI3K/Akt is required for the optimal antitumor effect of HER2 inhibitors. Therefore, we examined the temporal effect of the HER2 tyrosine kinase inhibitor (TKI) lapatinib (lap) on feedback upregulation of active HER3 in HER2-overexpressing breast cancer cells. A time course with lap-treated cells showed 3 to 5-fold upregulation of HER3 RNA and protein, beginning at 4 h and increasing through 48 h. P-Tyr immunoblot of HER3 immunoprecipitates revealed recovery of HER3 phosphorylation at and beyond 13 h of treatment. Site-specific antibodies revealed HER3 phosphorylation at Y1197 and Y1289, two of the six p85 binding sites in HER3. Recovery of P-HER3 correlated temporally with recovery of T308 P-Akt. The upregulation of HER3 RNA upon treatment with lap suggested that inhibition of active HER2 and PI3K/Akt derepresses the transcription factor FoxO3a. Putative FoxO3a binding sites were identified within the 5' flanking region upstream of the HER3 transcription start site. Transfection with FoxO3a siRNA reduced basal and lap-induced HER3 RNA levels 2 to 5-fold compared to control cells. Conversely, overexpression of FoxO3a increased HER3 RNA 2.5-fold, which could be further enhanced by lap treatment. In addition to these transcriptional mechanisms, the recovery of P-HER3 upon lap-induced inhibition of HER2 suggested engagement of another tyrosine kinase transactivating HER3 and/or that HER2 had been incompletely inhibited by the TKI. However, IGF-IR, Src, and MET TKIs did not inhibit the recovery of P-HER3. On the other hand, the addition of trastuzumab (tz) to lap-treated cells prevented recovery of P-HER3, suggesting that disruption of a ligand-independent HER2-HER3 interaction was involved in partial maintenance of HER3 phosphorylation.The upregulation of HER3 RNA and partial maintenance of P-HER3 and P-Akt suggested that combined inhibition of HER2 and HER3 will synergistically inhibit tumor cell viability. Transfection with HER3 siRNA sensitized HER2+ breast cancer cells to each lap and tz as assessed by Apo-BrdU (apoptosis) and 3D-Matrigel growth assays. Further, treatment with AMG-888, a HER3 monoclonal antibody (AMGEN-U3), sensitized cells to each lap and tz. Ongoing studies include the treatment of BT474 xenografts in athymic mice with lap ± AMG-888 using [18F]-FDG-PET as a non-invasive imaging biomarker to predict treatment outcome. Finally, we examined HER3 levels by immunohistochemistry in sections from tumor blocks of patients enrolled in a neoadjuvant trial where lap was given alone during the first 6 weeks of therapy. The percent and intensity of tumor cell staining was calculated as a histoscore (Human Pathol. 26:291, 1995). On week 2 of therapy, HER3 levels increased 135% above pre-therapy levels (n=8; p=0.03, Mann-Whitney). These data suggest that upon inhibition of the HER2 tyrosine kinase, HER2+ breast cancers 1) upregulate HER3 by transcriptional mechanisms and partially maintain HER3 function by post-translational mechanisms; 2) this compensatory phosphorylation of HER3 partially maintains PI3K/Akt; and 3) inhibition of HER3 sensitizes HER2-dependent breast cancer cells to HER2 inhibitors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 63.

Prognostic significance of HPV and p16 status in patients with oropharyngeal cancer treated on a large international phase III trial

6004 Background: Previous studies have reported that in patients with oropharyngeal cancer (OPC) the presence of human papilloma virus (HPV) is associated with an improved prognosis. We sought to determine the prognostic importance of HPV and p16 in patients with OPC treated with concurrent chemoradiation on a large international phase III trial. Methods: Patients with previously untreated Stage III or IV head and neck squamous cell cancer were randomized to receive definitive radiotherapy concurrently with either cisplatin or cisplatin plus tirapazamine. In this substudy, analyses were restricted to patients with OPC who received &gt; 60 Gy and did not have major radiation deviations predicted to impact on tumor control. HPV 16/18 were detected by in situ hybridization and scored as detected or undetected. p16 was detected by immunohistochemistry. Nuclear and cytoplasmic staining intensity of tumor cells was scored as grade 0–3, with grade 2 and 3 called positive. Log rank and Cox regression used for survival analyses. p values were 2-sided . Results: 384 out of 861 patients had OPC and met the eligibility criteria. Slides were available for HPV assay in 195 and for p16 in 186, and for both in 173. 54/195 (28%) were HPV positive, 107/186 (58%) were p16 positive. HPV pos tumors were associated with better 2-year overall survival (OS) (94 v 77%, p = 0.007) and better failure-free survival (FFS) (86 v 75%, p = 0.035) compared to HPV neg tumors. Similarly p16 pos tumors were associated with better 2-year OS (92 v 75%, p = 0.004) and FFS (87 v 72%, p = 0.003) compared to p16 neg . After adjustment for stage, Hb and ECOG PS, HPV pos had better OS than HPV neg (HR 0.29, p = 0.018), and p16 pos had better OS than p16 neg (HR 0.39, p = 0.013). When the HPV and p16 results were combined the relative HRs for OS were: HPVpos/p16pos 0.35 (45 patients, 26% of cases), HPVpos/p16neg 0 (3pts, 2%), HPVneg/p16pos 0.73 (58pts, 33%), HPVneg/p16neg 1.79 (67 pts, 39%). Conclusions: Our results confirm the prognostic significance of tumor HPV status in oropharyngeal cancer treated with chemoradiation, but also show that p16 identifies a larger group with an improved prognosis. The HPV neg/p16 pos population has a better prognosis compared to patients with HPV neg/p16 neg tumors. No significant financial relationships to disclose.

Expression of pre B cell leukemia transcription factor (PBX) is correlated with prognosis and survival of patients with non-small cell lung carcinoma

22222 Background: Pre B cell leukemia transcription factor 1 (PBX1) is a transcription factor for valosin-containing protein (VCP), which is involved in invasion and metastasis of cancers. To examine the prognostic significance of PBX1 and other PBX members, PBX2 and PBX3, in non-small cell lung cancer (NSCLC), the present study was designed. Methods: Expression of PBX1, PBX2, and PBX3 was immunohistochemically examined in 206 NSCLC patients (132 men and 74 women) with ages ranging from 35 to 78 (median, 62). Staining intensity of tumor cells was categorized as equal to or stronger expression than endothelial cells in less than 20% (score 1) or more than 20% (score 2). Results: PBX2 but not PBX1 and PBX3 nuclear expression correlated with high expression of VCP. Univariate analysis revealed score 1 PBX1 cytoplasmic expression, score 2 PBX2 nuclear expression, score 2 PBX2 cytoplasmic expression to be poor prognosticators for overall and disease-free survival. Multivariate analysis revealed that PBX1 cytoplasmic expression, visceral pleural invasion, and tumor size as independent prognostic factors for disease-free survival, and PBX1 cytoplasmic expression, PBX2 cytoplasmic expression, VCP expression, age, and visceral pleural invasion for overall survival. Conclusions: The present study showed that increased expression of PBX2 and decreased expression of PBX1 were poor prognosticators for NSCLC. Close correlation between PBX2 nuclear expression and VCP expression suggested the role of PBX2 as a transcription factor controlling VCP expression. No significant financial relationships to disclose.

What is your diagnosis? Lingual mass in a dog

A 3.5-year-old female spayed Rat Terrier was presented for evaluation of a submucosal lingual mass. Fine-needle aspiration of the mass revealed a population of neoplastic cells composed predominantly of small, round cells and large, round to spindle-shaped cells, which occasionally had blunt cytoplasmic borders and multiple nuclei. The neoplastic cells had moderate to marked anisocytosis and anisokaryosis. The cytologic interpretation was malignant neoplasia, most likely sarcoma. Histopathologic examination of a biopsy specimen revealed an unencapsulated, poorly demarcated, moderately cellular neoplasm composed of individualized, infiltrative spindle cells. Elongate skeletal muscle cells with prominent, rectangular borders (strap cells) were observed. A morphologic diagnosis of rhabdomyosarcoma was made. Staining with phosphotungstic acid-hematoxylin revealed haphazardly arranged skeletal muscle cross-striations. Immunohistochemical staining results for vimentin, Myo D1, desmin, and myoglobin were positive, though staining intensity of tumor cells varied with the degree of differentiation. Using transmission electron microscopy, irregular, disorganized Z-bands were identified. Rhabdomyosarcomas are uncommon tumors in the dog, and rarely may involve the tongue or oral cavity. Cytologic evaluation of a rhabdomyosarcoma may reveal a pleomorphic population of cells and definitive diagnosis may require histologic examination, immunohistochemical staining, and electron microscopy.

Occurrence of thymosin β4 in human breast cancer cells and in other cell types of the tumor microenvironment

Previous studies have shown that the G-actin sequestering polypeptide thymosin beta4 frequently is overexpressed in cancers and that such overexpression correlates to malignant progression. However, the localization of thymosin beta4 in human cancers has not been determined. We now demonstrate that there is a considerable heterogeneity in the cellular distribution of thymosin beta4 in breast cancer. In most tumors examined, cancer cells showed low or intermediate reactivity for thymosin beta4, whereas leukocytes and macrophages showed intense reactivity. In addition, endothelial cells showed variable reactivity to thymosin beta4, whereas myofibroblasts were negative. There was no correlation between the intensity of tumor cell staining and histological grade, whereas there was a tendency toward a correlation between endothelial cell staining and grade. These results demonstrate that multiple cell types within the tumor microenvironment produce thymosin beta4 and that such expression varies from tumor to tumor. Such heterogeneity of expression should be taken into account when the role of thymosin beta4 in tumor biology is assessed.

Bcl-2 and bax expression and prostate cancer outcome in men treated with radiotherapy in Radiation Therapy Oncology Group protocol 86-10

Bcl-2 and bax are proteins with opposing roles in apoptosis regulation; yet abnormal expression of either has been associated with failure after radiotherapy (RT). In this study we examined bcl-2 and bax expression as predictive markers in men treated with radiotherapy +/- androgen deprivation on Radiation Therapy Oncology Group (RTOG) protocol 86-10. Suitable archival diagnostic tissue was obtained from 119 (26%) patients for bcl-2 analysis and 104 (23%) patients for bax analysis. Cox proportional hazards multivariate analysis was used to determine the relationship of abnormal bcl-2 and bax expression to the end points of local failure, distant metastasis, cause-specific mortality, and overall mortality. Bcl-2 overexpression was classified as any tumor cell cytoplasmic staining and altered bax expression was classified as greater or lesser cytoplasmic staining intensity of tumor cells as compared with adjacent normal prostate epithelium. The study cohort exhibited bcl-2 overexpression in 26% (n = 30) of cases and abnormal bax expression in 47% (n = 49) of cases. A borderline significant relationship was observed between abnormal bax expression and higher Gleason score (p = 0.08). In univariate and multivariate analyses, there was no statistically significant relationship seen between abnormal bcl-2 or bax expression and outcome. Abnormal bcl-2 and bax expression were not related to any of the end points tested. The cohort examined was comprised of patients with locally advanced disease and it is possible that these markers may be of greater value in men with earlier-stage prostate cancer.

Increased Expression of Valosin-Containing Protein (p97) Is Correlated With Disease Recurrence in Follicular Thyroid Cancer

Valosin-containing protein (VCP) is involved in the ubiquitin/proteasome-degradation pathway, which works in proliferation and antiapoptosis in human cancer cells. Our previous study showed that VCP expression levels correlated with the recurrence and prognosis of several human cancers, such as hepatocellular carcinoma, gastric carcinoma, and colorectal carcinoma. In this study, the correlation of VCP expression with the prognosis of differentiated thyroid carcinoma was examined. VCP expression in 332 patients who underwent operation for differentiated thyroid carcinoma--257 with papillary thyroid carcinoma and 75 with follicular thyroid carcinoma (FTC)--was analyzed by immunohistochemistry. The staining intensity in tumor cells was categorized as weaker than (low expression), equal to (intermediate expression), or stronger than (high expression) that in endothelial cells in noncancerous tissue. One hundred ten (33.5%) cases showed low VCP expression, 117 (28.0%) showed intermediate expression, and 101 (30.8%) showed high expression. VCP expression significantly correlated with histological subtype (P < .05) and lymph node metastasis (P < .01). However, it correlated with neither any clinicopathologic factor nor prognosis in papillary thyroid carcinoma. VCP expression correlated with extrathyroidal extension (P < .05), pT classification (P < .05), and lymph node metastasis (P < .01) in FTC. Patients with low VCP expressing FTC showed better disease-free and overall survival rates than those with intermediate or high expression (P < .01 and P < .05, respectively). Multivariate analysis revealed VCP expression and extracapsular extension to be independent prognostic factors for disease-free survival in cases of FTC. The prognostic utility of VCP expression in FTC was demonstrated.

YKL-40 Is a Differential Diagnostic Marker for Histologic Subtypes of High-Grade Gliomas

In modern neuro-oncology, no variable affects therapeutic decisions and prognostic estimation more than tumor classification. We showed recently that class prediction models, based on gene expression profiles, classify diagnostically challenging malignant gliomas in a manner that better correlates with clinical outcome than standard pathology. In the present study, we used immunohistochemistry to investigate YKL-40 protein expression in independent sets of glioblastomas and anaplastic oligodendrogliomas to determine whether this single marker can aid classification of these high-grade gliomas. Glioblastomas show strikingly more YKL-40 expression than anaplastic oligodendrogliomas. Only 2 of 37 glioblastomas showed completely negative YKL-40 staining in both tumor cells and extracellular matrix, whereas 18 of 29 anaplastic oligodendrogliomas were completely negative in non-microgemistocytic tumor cells and extracellular matrix. Tumor cell staining intensity was also markedly different: 84% of glioblastomas showed strong staining intensities of 2+ or 3+ whereas 76% of anaplastic oligodendrogliomas either did not stain or stained at only 1+. YKL-40 staining provided a better class distinction of glioblastoma versus anaplastic oligodendroglioma than glial fibrillary acidic protein, the current standard immunohistochemical marker used to distinguish diagnostically challenging gliomas. Moreover, a combination of YKL-40 and glial fibrillary acidic protein immunohistochemistry afforded even greater diagnostic accuracy in anaplastic oligodendrogliomas.