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Purpose: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002.Experimental Design: Using Automated Quantitative Analysis, we quantified expression of p85 and p110α subunits in 540 nevi and 523 melanomas. We determined the IC50 for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002.Results: p85 and p110α tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110α, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser235 and Ser240 were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively).Conclusions: Expression of p85 and p110α subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.

Purpose: The cell cycle mediators Aurora A and B are targets of drugs currently in clinical development. As with other targeted therapies in breast cancer, response to therapy might be associated with target expression in tumors. We therefore assessed expression of Aurora A and B in breast tumors and studied associations with clinical/pathologic variables.Experimental Design: Tissue microarrays containing primary specimens from 638 patients with 15-year follow-up were employed to assess expression of Aurora A and B using our automated quantitative analysis method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured Aurora A and B expression within the mask using Cy5-conjugated antibodies.Results: Aurora A and B expression was variable in primary breast tumors. High Aurora A expression was strongly associated with decreased survival (P = 0.0005). On multivariable analysis, it remained an independent prognostic marker. High Aurora A expression was associated with high nuclear grade and high HER-2/neu and progesterone receptor expression. Aurora B expression was not associated with survival.Conclusions: Aurora A expression defines a population of patients with decreased survival, whereas Aurora B expression does not, suggesting that Aurora A might be the preferred drug target in breast cancer. Aurora A expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of Aurora A as well as the predictive role of Aurora A expression in patients treated with Aurora A inhibitors.

Background: Sorafenib, a multitarget kinase inhibitor, inhibits members of the mitogen-activated protein kinase (MAPK) pathway and receptor tyrosine kinases, including vascular endothelial growth factor receptor 2 (VEGF-R2). Sorafenib, carboplatin, and paclitaxel (SCP) has antitumor activity in melanoma patients, but no association was found between response and activating B-Raf V600E mutations. We assessed the expression of sorafenib targets in SCP-treated patient specimens and evaluated the association with response and progression-free survival.Experimental Design: Using automated quantitative analysis, we quantified the expression of VEGF-R1, VEGF-R2, VEGF-R3, fibroblast growth factor receptor 1, platelet-derived growth factor receptor β, c-Kit, B-Raf, C-Raf, meiosis-specific serine/threonine protein kinase 1, and extracellular regulated kinase 1/2 (ERK1/2) in pretreatment specimens from 46 patients. Furthermore, we assessed ERK1/2 expression in 429 archival melanomas.Results: VEGF-R2 expression was significantly higher in patients with a complete or partial response (P = 0.0435), whereas ERK1/2 was higher in patients who did not respond (P = 0.0417). High ERK1/2 was an independent predictor of poor survival. High ERK1/2 was associated with decreased survival in the archival melanoma cohort, suggesting that high ERK1/2-expressing tumors are biologically more aggressive. All of the six patients with both high VEGF-R2 and low ERK1/2 responded to SCP.Conclusions: High VEGF-R2 expression is associated with response to SCP in melanoma, whereas high ERK1/2 is associated with resistance. Collection of specimens from SCP-treated melanoma patients in a cooperative group phase III trial comparing this regimen with the chemotherapy alone is ongoing, and confirmation of these findings is necessary. These markers might be useful for predicting response to sorafenib when given with other chemotherapies and in other diseases, resulting in the possible elimination of unnecessary treatment of patients unlikely to respond.

Purpose: Raf-kinases include three major isoforms. Although the role of B-Raf in melanoma is well established, little is known about C-Raf. We studied effects of C-Raf knockdown in vitro and assessed expression of C-Raf in a large cohort of melanomas and nevi.Experimental Design: Using specific siRNAs, we knocked down C-Raf expression, and determined the effect on viability, MAP extracellular signal-regulated kinase (ERK)/ERK kinase signaling, and apoptosis in seven melanoma cell lines. We determined the IC50 of the C-Raf inhibitors sorafenib and GW5074, and studied the effects of GW5074 on cell signaling. Using an automated method to measure in situ protein expression, we quantified C-Raf expression in 263 nevi and 523 melanomas.Results: C-Raf was knocked down in three cell lines with detectable phospho-C-Raf, resulting in decreased viability in two of the three (YULAC and YUROB). This resulted in decreased Bcl-2 expression and phospho-Bad cleavage, without affecting phospho-MEK and phospho-ERK. Sensitivity to sorafenib and GW5074 varied. GW5074 inhibited mitogen-activated protein kinase signaling without Bcl-2 and phospho-Bad down-regulation. C-Raf was highly expressed in melanomas compared with nevi (P < 0.0001), and no nevi had high C-Raf expression. C-Raf expression was higher in metastatic than primary specimens (P = 0.0225).Conclusions: C-Raf siRNA knock-down results in decreased viability of YULAC (B-RafV600K) and YUROB (B-RafWT) melanoma cells, likely mediated by Bcl-2 inhibition rather than mitogen-activated protein kinase inhibition. Cotargeting C-Raf and parallel pathways might be an effective therapeutic approach for melanoma. C-Raf expression is up-regulated in a subset of melanomas but not in nevi, suggesting that it might be a valuable diagnostic marker and therapeutic target. (Clin Cancer Res 2009;15(18):5704–13)

Supplementary Data from C-Raf Is Associated with Disease Progression and Cell Proliferation in a Subset of Melanomas

Supplementary Data from Phosphatidylinositol-3-Kinase as a Therapeutic Target in Melanoma

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Multivariable analysis of clinical and pathologic variables and their association with overall survival in RCC

Purpose: A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid–based analysis to predict patient outcome.Experimental Design: We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section.Results: The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n = 223, 5-year survival = 95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n = 149).Conclusions: With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.

Purpose: Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K.Methods: Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110α. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244.Results: Strong coexpression of mTOR and p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC50 values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage.Conclusions: mTOR and p110α are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors.

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Supplementary Table 1, Figure 1 from Vertical Targeting of the Phosphatidylinositol-3 Kinase Pathway as a Strategy for Treating Melanoma

Purpose: Measurement of autophagy in cancer and correlation with histopathologic grading or clinical outcomes has been limited. Accordingly, we investigated LC3B as an autophagosome marker by analyzing nearly 1,400 tumors from 20 types of cancer, focusing on correlations with clinical outcomes in melanoma and breast cancer.Experimental Design: Staining protocols were developed for automated quantitative analysis (AQUA) using antibodies versus LC3 isoform B (LC3B) and Ki-67. Clinically annotated breast and melanoma tissue microarrays (TMA) and a multitumor array were used. An AQUA program was developed to quantitate LC3B distribution in punctate and diffuse compartments of the cell.Results: LC3B staining was moderate to high in the large majority of tumors. The percentage of area occupied by punctate LC3B was elevated by 3- to 5-fold at high LC3B intensities. In breast cancer and melanoma TMAs, LC3B and Ki-67 showed strong correlations (P < 0.0001), and in multitumor TMAs, mitotic figures were most often seen in tumors with the highest LC3B expression (P < 0.002). In breast cancer, LC3B expression was elevated in node-positive versus node-negative primaries and associated with increased nuclear grade and shortened survival. In a melanoma TMA with no survival data, LC3B levels were highest in nodal, visceral, and cutaneous metastases.Conclusions: The results reveal a common expression of LC3B in malignancy and support emerging evidence that autophagy plays a significant role in cancer progression. High LC3B was associated proliferation, invasion and metastasis, high nuclear grade, and worse outcome. Thus, autophagy presents a key target of therapeutic vulnerability in solid tumors. Clin Cancer Res; 18(2); 370–9. ©2011 AACR.

Supplementary Figure 1 and Table 1 from Expression of Aurora A (but Not Aurora B) Is Predictive of Survival in Breast Cancer

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Supplementary Data from Expression of Sorafenib Targets in Melanoma Patients Treated with Carboplatin, Paclitaxel and Sorafenib

Purpose: Increased vascularity is a hallmark of renal cell carcinoma (RCC). Microvessel density (MVD) is one measurement of tumor angiogenesis; however, its utility as a biomarker of outcome is unknown. ECOG-ACRIN 2805 (E2805) enrolled 1,943 resected high-risk RCC patients randomized to adjuvant sunitinib, sorafenib, or placebo. We aimed to determine the prognostic and predictive role of MVD in RCC.Experimental Design: We obtained pretreatment primary RCC nephrectomy tissues from 822 patients on E2805 and constructed tissue microarrays. Using quantitative immunofluorescence, we measured tumor MVD as the area of CD34-expressing cells. We determined the association with disease-free survival (DFS), overall survival (OS), treatment arm, and clinicopathologic variables.Results: High MVD (above the median) was associated with prolonged OS for the entire cohort (P = 0.021) and for patients treated with placebo (P = 0.028). The association between high MVD and OS was weaker in patients treated with sunitinib or sorafenib (P = 0.060). MVD was not associated with DFS (P = 1.00). On multivariable analysis, MVD remained independently associated with improved OS (P = 0.013). High MVD correlated with Fuhrman grade 1–2 (P < 0.001), clear cell histology (P < 0.001), and absence of necrosis (P < 0.001) but not with gender, age, sarcomatoid features, lymphovascular invasion, or tumor size.Conclusions: High MVD in resected high-risk RCC patients is an independent prognostic, rather than predictive, biomarker of improved OS. Further studies should assess whether incorporating MVD into clinical models will enhance our ability to predict outcome and if low MVD can be used for selection of high-risk patients for adjuvant therapy trials. Clin Cancer Res; 24(1); 217–23. ©2017 AACR.

Supplementary Information and Table 1 from Classification of Breast Cancer Using Genetic Algorithms and Tissue Microarrays

Background Current interstitial lung disease (ILD) diagnostic guidelines assess criteria across clinical, radiologic and pathologic domains. Significant interobserver variation in histopathologic evaluation has previously been shown but the specific source of these discrepancies is poorly documented. We sought to document specific areas of difficulty and develop improved criteria that would reduce overall interobserver variation. Methods Using an internet-based approach, we reviewed selected images of specific diagnostic features of ILD histopathology and whole slide images of fibrotic ILD. After an initial round of review, we confirmed the presence of interobserver variation among our group. We then developed refined criteria and reviewed a second set of cases. Results The initial round reproduced the existing literature on interobserver variation in diagnosis of ILD. Cases which were pre-selected as inconsistent with usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF) were confirmed as such by multi-observer review. Cases which were thought to be in the spectrum of chronic fibrotic ILD for which UIP/IPF were in the differential showed marked variation in nearly all aspects of ILD evaluation including extent of inflammation and extent and pattern of fibrosis. A proposed set of more explicit criteria had only modest effects on this outcome. While we were only modestly successful in reducing interobserver variation, we did identify specific reasons that current histopathologic criteria of fibrotic ILD are not well defined in practice. Conclusions Any additional classification scheme must address interobserver variation in histopathologic diagnosis of fibrotic ILD order to remain clinically relevant. Improvements to tissue-based diagnostics may require substantial resources such as larger datasets or novel technologies to improve reproducibility. Benchmarks should be established for expected outcomes among clinically defined subgroups as a quality metric.

Purpose: Increased vascularity is a hallmark of renal cell carcinoma (RCC). Microvessel density (MVD) is one measurement of tumor angiogenesis; however, its utility as a biomarker of outcome is unknown. ECOG-ACRIN 2805 (E2805) enrolled 1,943 resected high-risk RCC patients randomized to adjuvant sunitinib, sorafenib, or placebo. We aimed to determine the prognostic and predictive role of MVD in RCC. Experimental Design: We obtained pretreatment primary RCC nephrectomy tissues from 822 patients on E2805 and constructed tissue microarrays. Using quantitative immunofluorescence, we measured tumor MVD as the area of CD34-expressing cells. We determined the association with disease-free survival (DFS), overall survival (OS), treatment arm, and clinicopathologic variables. Results: High MVD (above the median) was associated with prolonged OS for the entire cohort (P = 0.021) and for patients treated with placebo (P = 0.028). The association between high MVD and OS was weaker in patients treated with sunitinib or sorafenib (P = 0.060). MVD was not associated with DFS (P = 1.00). On multivariable analysis, MVD remained independently associated with improved OS (P = 0.013). High MVD correlated with Fuhrman grade 1–2 (P < 0.001), clear cell histology (P < 0.001), and absence of necrosis (P < 0.001) but not with gender, age, sarcomatoid features, lymphovascular invasion, or tumor size. Conclusions: High MVD in resected high-risk RCC patients is an independent prognostic, rather than predictive, biomarker of improved OS. Further studies should assess whether incorporating MVD into clinical models will enhance our ability to predict outcome and if low MVD can be used for selection of high-risk patients for adjuvant therapy trials. Clin Cancer Res; 24(1); 217–23. ©2017 AACR.

Purpura fulminans (PF) is associated with several infections and most commonly with meningococcemia. However, there are only a few reports of this entity in association with toxic shock syndrome toxin-1-producing Staphylococcus aureus. We report a 53-year-old man who presented with fever, progressive hemodynamic instability, multiorgan failure, and thrombocytopenia following lobectomy for a solitary lung metastasis from rectal adenocarcinoma. He developed progressive generalized eruption of nonblanching red, purple, and black macules, papules, and plaques on the trunk and extremities consistent with PF. He died on postadmission day 3. Autopsy examination revealed purulent pleural exudate, which grew toxic shock syndrome toxin-1-producing S. aureus. Premortem and autopsy skin biopsies demonstrated epidermal necrosis, subepidermal bullae, and fibrin thrombi within small cutaneous vessels with minimal perivascular lymphocytic inflammation and without accompanying vasculitis. With this case report, we would like to draw attention to the fact that staphylococcal toxic shock syndrome-associated PF may be highly underrecognized and much more common than reflected in the literature.

Single prolonged stress (SPS) is a rodent model of PTSD with high construct and face validity. Our previous studies revealed that early intervention with neuropeptide Y (NPY) delivered to the brain...

Aims Immunohistochemical stains have greatly improved the diagnostic accuracy of renal cell carcinoma (RCC) for primary and distant tumours. We evaluate a marker that has recently been incorporated in clinical practice, PAX-8, in primary and metastatic RCCs. Methods Two distinct tissue microarrays were used, one consisting of over 334 renal tumours, 294 with adjacent normal kidney and the other with 40 matched nephrectomy and metastatic sites of RCC. PAX-8 expression was assessed by a method of quantitative immunofluorescence. Results PAX-8 was positive in 96% (146/152) of normal renal tissue and 83% (227/272) of renal tumours. PAX-8 staining was positive in clear cell, papillary and chromophobe tumours in 80% (165/207), 95% (39/41) and 100% (6/6) of samples, respectively. Overall, intensity of PAX-8 expression was significantly higher in RCC metastatic sites than in the primary site (p=0.0047), however, in matched sites there was no statistically significant difference in the proportion of positive versus negative specimens (p=0.274). Conclusions As the role of molecular markers expands in the diagnostic algorithm, this study confirms that PAX-8 expression is a useful diagnostic marker for RCC. PAX-8 expression was found in the primary tumour and distant sites. Compared with normal tissue and other histological types, clear cell RCC has lower PAX-8 expression and is less frequently positive, therefore, the lack of expression does not exclude a tumour of renal origin.

// Eva Giesen 1,* , Lucia B. Jilaveanu 1,* , Fabio Parisi 2 , Yuval Kluger 2 , Robert L. Camp 2 and Harriet M. Kluger 1 1 Yale Cancer Center, Yale University School of Medicine, New Haven, CT, U.S 2 Department of Pathology, Yale University School of Medicine, New Haven, CT, U.S * These authors contributed equally to this work Correspondence: Harriet M. Kluger , email: // Keywords : Cancer Testis Antigens, drug targets, kidney cancer, clear cell carcinoma, adoptive cell therapy Received : April 15, 2014 Accepted : June 12, 2014 Published : June 13, 2014 Abstract Background: Novel immune therapies targeting tumor specific antigens are being developed. Our purpose was to determine expression of the cancer testes antigen NY-ESO-1 in renal cell carcinoma (RCC), as NY-ESO-1 targeting approaches, particularly adoptive cell therapy, have not been evaluated in this disease. Methods: We employed tissue microarrays containing >300 unique RCC cases and adjacent benign renal tissue to determine NY-ESO-1 expression using a quantitative immunofluorescence method. In addition, we studied NY-ESO-1 expression in 35 matched primary and metastatic RCC specimens to assess concordance between different tumor sites. Results: NY-ESO-1 was highly expressed in a subset of RCCs. Expression in primary RCC specimens was significantly higher than adjacent normal renal tissue (P

Activation of the c-Met pathway occurs in a range of malignancies, including papillary renal cell carcinoma (RCC). Its activity in clear cell RCC is less clear. We investigated c-Met expression and inhibition in a large cohort of RCC tumors and cell lines.c-Met protein expression was determined by automated quantitative analysis (AQUA) on a tissue microarray (TMA) constructed from 330 RCC tumors paired with adjacent normal renal tissue. c-Met expression and selective inhibition with SU11274 and ARQ 197 were studied in clear cell RCC cell lines.Higher c-Met expression was detected in all RCC subtypes than in the adjacent normal renal tissue (P0.0001). Expression was highest in papillary and sarcomatoid subtypes, and high-grade and stage tumors. Higher c-Met expression correlated with worse disease-specific survival [risk ratio = 1.36; 95% confidence interval (CI) 1.08-1.74; P = 0.0091] and was an independent predictor of survival, maintained in clear cell subset analyses. c-Met protein was activated in all cell lines, and proliferation (and colony formation) was blocked by SU11274 and ARQ 197.c-Met is associated with poor pathologic features and prognosis in RCC. c-Met inhibition demonstrates in vitro activity against clear cell RCC. Further study of ARQ 197 with appropriate biomarker studies in RCC is warranted.

Purpose: Measurement of autophagy in cancer and correlation with histopathologic grading or clinical outcomes has been limited. Accordingly, we investigated LC3B as an autophagosome marker by analyzing nearly 1,400 tumors from 20 types of cancer, focusing on correlations with clinical outcomes in melanoma and breast cancer. Experimental Design: Staining protocols were developed for automated quantitative analysis (AQUA) using antibodies versus LC3 isoform B (LC3B) and Ki-67. Clinically annotated breast and melanoma tissue microarrays (TMA) and a multitumor array were used. An AQUA program was developed to quantitate LC3B distribution in punctate and diffuse compartments of the cell. Results: LC3B staining was moderate to high in the large majority of tumors. The percentage of area occupied by punctate LC3B was elevated by 3- to 5-fold at high LC3B intensities. In breast cancer and melanoma TMAs, LC3B and Ki-67 showed strong correlations (P < 0.0001), and in multitumor TMAs, mitotic figures were most often seen in tumors with the highest LC3B expression (P < 0.002). In breast cancer, LC3B expression was elevated in node-positive versus node-negative primaries and associated with increased nuclear grade and shortened survival. In a melanoma TMA with no survival data, LC3B levels were highest in nodal, visceral, and cutaneous metastases. Conclusions: The results reveal a common expression of LC3B in malignancy and support emerging evidence that autophagy plays a significant role in cancer progression. High LC3B was associated proliferation, invasion and metastasis, high nuclear grade, and worse outcome. Thus, autophagy presents a key target of therapeutic vulnerability in solid tumors. Clin Cancer Res; 18(2); 370–9. ©2011 AACR.

We sought to determine whether lymph node ratio (LNR; defined as number of positive nodes/number of nodes dissected) provides additional prognostic information in node-positive breast cancer patients. Data from a cohort of 319 node-positive breast cancer patients diagnosed between 1956 and 1982 were analyzed for overall survival (OS) on the basis of current American Joint Committee on Cancer (AJCC) nodal staging versus LNR. In terms of AJCC categorization, 157 patients (49.2%) were pN1 (1–3 positive nodes), 97 (30.4%) were pN2 (4–9 positive nodes), and 65 (20.4%) were pN3 (≥10 positive nodes). In terms of LNR, 90 (28.2%) were low risk (LNR = 0.01–0.20), 119 (38.3%) were intermediate risk (LNR = 0.21–0.65), and 110 (34.5%) were high risk (LNR > 0.65). The median follow-up was 68.7 months. AJCC nodal status correlated with OS (median OS 85.9, 70.4, and 48.4 months for pN1–3, respectively, P = 0.018). LNR also correlated with OS (median OS 105.8, 72.2, and 48.4 months for the low-, intermediate-, and high-risk groups, respectively, P

Purpose: Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K. Methods: Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110α. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244. Results: Strong coexpression of mTOR and p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC50 values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage. Conclusions: mTOR and p110α are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors.

After lactation, weaning causes mammary epithelial cell (MEC) apoptosis. MECs express the plasma membrane calcium-ATPase 2 (PMCA2), which transports calcium across the apical surface of the cells into milk. Here we show that PMCA2 is down-regulated early in mammary involution associated with changes in MEC shape. We demonstrate that loss of PMCA2 expression raises intracellular calcium levels and sensitizes MECs to apoptosis. In contrast, overexpression of PMCA2 in T47D breast cancer cells lowers intracellular calcium and protects them from apoptosis. Finally, we show that high PMCA2 expression in breast cancers is associated with poor outcome. We conclude that loss of PMCA2 expression at weaning triggers apoptosis by causing cellular calcium crisis. PMCA2 overexpression, on the other hand, may play a role in breast cancer progression by conferring resistance to apoptosis.

Purpose As a result of the questionable risk-to-benefit ratio of adjuvant therapies, stage II melanoma is currently managed by observation because available clinicopathologic parameters cannot identify the 20% to 60% of such patients likely to develop metastatic disease. Here, we propose a multimarker molecular prognostic assay that can help triage patients at increased risk of recurrence. Methods Protein expression for 38 candidates relevant to melanoma oncogenesis was evaluated using the automated quantitative analysis (AQUA) method for immunofluorescence-based immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a cohort of 192 primary melanomas collected during 1959 to 1994. The prognostic assay was built using a genetic algorithm and validated on an independent cohort of 246 serial primary melanomas collected from 1997 to 2004. Results Multiple iterations of the genetic algorithm yielded a consistent five-marker solution. A favorable prognosis was predicted by ATF2 ln(non-nuclear/nuclear AQUA score ratio) of more than –0.052, p21WAF1 nuclear compartment AQUA score of more than 12.98, p16INK4A ln(non-nuclear/nuclear AQUA score ratio) of ≤ −0.083, β-catenin total AQUA score of more than 38.68, and fibronectin total AQUA score of ≤ 57.93. Primary tumors that met at least four of these five conditions were considered a low-risk group, and those that met three or fewer conditions formed a high-risk group (log-rank P < .0001). Multivariable proportional hazards analysis adjusting for clinicopathologic parameters shows that the high-risk group has significantly reduced survival on both the discovery (hazard ratio = 2.84; 95% CI, 1.46 to 5.49; P = .002) and validation (hazard ratio = 2.72; 95% CI, 1.12 to 6.58; P = .027) cohorts. Conclusion This multimarker prognostic assay, an independent determinant of melanoma survival, might be beneficial in improving the selection of stage II patients for adjuvant therapy.

BACKGROUND: Assessment of outcome using a single prognostic or predictive marker is the current basis of targeted therapy, but is inherently limited by its simplicity. Multiplexing has provided better classification, but has only been done quantitatively using RNA or DNA. Automated quantitative analysis is a new technology that allows quantitative in situ assessment of protein expression. The authors hypothesized that multiplexed quantitative measurement of ErbB receptor family proteins may allow better prediction of outcome. METHODS: The authors quantitatively assessed the expression of 6 proteins in 4 subcellular compartments in 676 patients using breast carcinoma tissue microarrays. Then using Cox proportional hazards modeling and unsupervised hierarchical clustering, they assessed the prognostic value of the expression singly and multiplexed. RESULTS: Epidermal growth factor receptor (EGFR), HER-2, and HER-3 expression were associated with decreased survival. Multivariate analysis showed high HER-2 and HER-3 expression maintained independence as prognostic markers. Hierarchical clustering of expression data defined a small class enriched for HER-2 expression with 40% 10-year survival, compared with 55% using conventional methods. Clustering also revealed a similarly poor-prognostic subgroup coexpressing EGFR and HER-3 (but low for estrogen receptor, progesterone receptor, and HER-2) with a 42% 10-year survival. CONCLUSIONS: This work shows that the combined analysis of protein expression improved prognostic classification, and that multiplexed models were superior to any single-marker–based method for prediction of 10-year survival. These methods illustrate a protein-based, multiplexed approach that could more accurately identify patients for targeted therapies. Cancer 2009. © 2009 American Cancer Society.

We constructed a large-scale functional network model in Drosophila melanogaster built around two key transcription factors involved in the process of embryonic segmentation. Analysis of the model allowed the identification of a new role for the ubiquitin E3 ligase complex factor SPOP. In Drosophila , the gene encoding SPOP is a target of segmentation transcription factors. Drosophila SPOP mediates degradation of the Jun kinase phosphatase Puckered, thereby inducing tumor necrosis factor (TNF)/Eiger–dependent apoptosis. In humans, we found that SPOP plays a conserved role in TNF-mediated JNK signaling and was highly expressed in 99% of clear cell renal cell carcinomas (RCCs), the most prevalent form of kidney cancer. SPOP expression distinguished histological subtypes of RCC and facilitated identification of clear cell RCC as the primary tumor for metastatic lesions.

Background: Sorafenib, a multitarget kinase inhibitor, inhibits members of the mitogen-activated protein kinase (MAPK) pathway and receptor tyrosine kinases, including vascular endothelial growth factor receptor 2 (VEGF-R2). Sorafenib, carboplatin, and paclitaxel (SCP) has antitumor activity in melanoma patients, but no association was found between response and activating B-Raf V600E mutations. We assessed the expression of sorafenib targets in SCP-treated patient specimens and evaluated the association with response and progression-free survival. Experimental Design: Using automated quantitative analysis, we quantified the expression of VEGF-R1, VEGF-R2, VEGF-R3, fibroblast growth factor receptor 1, platelet-derived growth factor receptor β, c-Kit, B-Raf, C-Raf, meiosis-specific serine/threonine protein kinase 1, and extracellular regulated kinase 1/2 (ERK1/2) in pretreatment specimens from 46 patients. Furthermore, we assessed ERK1/2 expression in 429 archival melanomas. Results: VEGF-R2 expression was significantly higher in patients with a complete or partial response (P = 0.0435), whereas ERK1/2 was higher in patients who did not respond (P = 0.0417). High ERK1/2 was an independent predictor of poor survival. High ERK1/2 was associated with decreased survival in the archival melanoma cohort, suggesting that high ERK1/2-expressing tumors are biologically more aggressive. All of the six patients with both high VEGF-R2 and low ERK1/2 responded to SCP. Conclusions: High VEGF-R2 expression is associated with response to SCP in melanoma, whereas high ERK1/2 is associated with resistance. Collection of specimens from SCP-treated melanoma patients in a cooperative group phase III trial comparing this regimen with the chemotherapy alone is ongoing, and confirmation of these findings is necessary. These markers might be useful for predicting response to sorafenib when given with other chemotherapies and in other diseases, resulting in the possible elimination of unnecessary treatment of patients unlikely to respond.

This year, 2008, marks the 10-year anniversary of the development of the modern tissue microarray (TMA). During the last decade, the use of TMAs has grown steadily and accounts for a small but increasing percentage of all cancer biomarker studies performed. The growing popularity of TMA-based studies attests to their benefits in the discovery and validation of new biomarkers. This review will focus on these benefits, but also on the faults of TMAs and the challenges of TMA studies that have been overcome in the last decade. We will also discuss the role of TMAs in the latest revolution in cancer treatment, the use of targeted drug therapy.

Context.—Increased thymidylate synthase expression is a marker for decreased survival in colorectal cancer.Objective.—Thymidylate synthase localizes to both the nucleus and cytoplasm, but how the relationship of these expression levels affects colon cancer outcome has yet to be determined.Design.—Using AQUA, we assessed prognosis of thymidylate synthase expression as a function of subcellular localization in 2 retrospective cohorts of colorectal carcinoma. We used the first cohort (n = 599) as a training set, subsequently validating optimal expression cut points in the second cohort (n = 447).Results.—A significant association between decreased 5-year disease-specific survival and increased nuclear expression (16% decreased survival [72% to 56%] for the top 60% of nuclear-expressing tumors [P < .001]) and cytoplasmic expression (12% decreased survival [70% to 58%] for the top 54% of cytoplasmic-expressing tumors [P = .02]) was observed for the training set. A higher nuclear to cytoplasmic ratio also correlated significantly with decreased survival (15% decreased survival [66% to 51%] for the top 25% of tumors [P < .001]). Applying these findings to the validation set, as a function of time to recurrence, only the ratio (P = .03 [expression ratio]; P = .18 [nuclear]; P = .71 [cytoplasmic]) showed a significant association with decreased time to recurrence. Additionally, the expression ratio significantly added to the prognostic value given by the primary tumor pathologic classification and nodal status.Conclusions.—These data suggest the relationship of nuclear to cytoplasmic thymidylate synthase expression, given as a ratio of continuous AQUA scores, to be a strong predictor of colon cancer survival.

Kallikrein-related peptidases, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. In the present study, we sought to determine the prognostic value of kallikrein-related peptidase 6 (KLK6) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. A tissue array composed of 150 advanced stage ovarian cancers, uniformly treated with surgical debulking followed by platinumpaclitaxel combination chemotherapy, was constructed. For evaluation of KLK6 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). Mean follow-up time of the cohort was 34.35 months. One hundred and thirty-five of 150 cases had sufficient tissue for AQUA analysis. In univariate survival analysis, low tumor KLK6 expression was associated with better outcome for overall survival over 3 years (P = 0.019). There was no association between tumor KLK6 expression and progression-free survival (P = 0.128). In multivariate survival analysis, adjusting for well-characterized prognostic variables, low tumor KLK6 expression level was one of the most significant predictor variable for overall survival (95% confidence interval, 1.19–3.50; P = 0.009). High tumor KLK6 protein expression is associated with inferior patient outcome in ovarian cancer. KLK6 may represent a promising disease biomarker and therapeutic target in ovarian cancer. (Cancer Sci 2008; 99: 2224–2229)

Recent development of antiangiogenic therapy for renal cell carcinoma (RCC) has significantly improved the treatment of these often refractory tumors. However, not all patients respond to therapy and assays for predicting outcome are needed. As a first step, we analyzed a retrospective cohort of tumors and assessed the ability of VEGF and VEGF receptors (VEGF-R1, -R2 and -R3) to classify tumors. We analyzed tissue microarrays containing 330 RCCs using a novel method of automated quantitative analysis of VEGF and VEGF-R expression by fluorescent immunohistochemistry. Expression of markers was separately quantified within three tissue components: tumor cells, endothelial cells and adjacent normal epithelium. VEGF and VEGF receptors were tightly coexpressed both within tumors and within adjacent normal cells (all P-values

Purpose Amplified in breast cancer (AIB1 or SRC-3) is an estrogen receptor coregulatory protein that together with other co-activators like transcription intermediary factor 2 (TIF2) and nuclear receptor co-repressor (NCoR), is implicated in estrogen signaling pathway and estrogen regulated tumor progression. We investigated the prognostic significance of AIB1, TIF2 & NCoR protein expression breast tissue microarray (TMA), and studied the relationship of coregulatory proteins to prognostic biomarkers like estrogen (ER), progesterone (PR) & HER2/neu and between coregulatory proteins. Methods: AIBI, TIF2 & NCoR were studied by fluorescent immunohistochemical staining of a TMA with 670 breast cancer specimens, using AQUA software. Result: Using Cox univariate survival analyses, high AIB1 expression was associated with poor patient outcome (P = 0.002), while no association was noted for TIF2 (P = 0.376) & NCoR (P = 0.12). When subclassified by nodal or ER status, AIB1 was not prognostic in the node positive and ER positive subsets. However, in the ER negative and node negative subsets, high AIB1 expression was associated with poor patient outcome (P = 0.02 and P = 0.007 respectively). AIB1 retained its independent association with survival by multivariate analyses (P = 0.028). There was significant positive correlation between AIB1 and ER and PR status and with other cofators (TIF2 and NCoR) but not with HER2/neu status. Conclusion: High AIB1 expression was predictive of worse overall survival in our study, suggesting that AIB1 may be critical in breast carcinogenesis.

HSP90 chaperones molecules critical for cell survival and malignant progression, including mutated B-raf. HSP90-targeting agents are in clinical trials. No large studies have been conducted on expression of HSP90 in melanomas.Tissue microarrays containing 414 nevi, 198 primary and 270 metastatic melanomas were assessed using our automated quantitative analysis (AQUA) method of in situ protein measurement; we use S-100 to define pixels as melanocytes (tumor mask) within the array spot, and measure HSP90 expression within the mask using Cy5-conjugated antibodies.HSP90 expression was higher in melanomas than nevi (P0.0001) and higher in metastatic than primary specimens (P0.0001). No association was seen between high HSP90 expression and survival in the primary or metastatic patient subsets. In primary melanomas, high HSP90 expression was associated with higher Clark level (P = 0.0167) and increased Breslow depth (P0.0001).HSP90 expression was significantly higher in tumors than nevi and was associated with disease progression, indicating that it might be a valuable drug target in melanoma, as well as a useful diagnostic marker. Prospective studies are needed to confirm the diagnostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.

Purpose The variability in scoring of immunohistochemistry, whether a result of true heterogeneity or artifacts in preparation, has led to decreased reliability in companion diagnostics and the recommendation for new standards (eg, the American Society of Clinical Oncology/College of American Pathologists [ASCO-CAP] guidelines). The basis of this problem is the amount of tissue required to be representative of an entire tumor. Because protein expression on tissue microarrays (TMAs) can be rigorously measured and one 0.6-mm spot is equivalent to two to three high-power fields, we used TMAs to assess levels of heterogeneity and to determine optimal representation as a function of outcome. Patients and Methods We analyzed estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER-2) expression in two cohorts (n = 676 and n = 152) on a series of four to five separate TMA cores and assessed heterogeneity by linear regression analysis. Minimum, average, and maximum scores were generated for each set, which were then assessed for prognostic and predictive value. Results Each marker shows some heterogeneity, but average r values between 0.7 and 0.8 are seen between TMA spots. Analysis for prognostic value shows that the highest maximum score (of five spots) is the most prognostic for ER, whereas a high HER-2 minimum score is most prognostic for poor outcome and most predictive of response to trastuzumab. Conclusion These results suggest that the representivity required for each biomarker may be a function of its role in tumorigenesis. Furthermore, these results provide scientific basis for the ASCO-CAP guidelines for assessment of HER-2 expression but perhaps suggest that the 30% figure is still too conservative.

Although it is known that small intestinal carcinoids are derived from enterochromaffin (EC) cells, these cells remain poorly characterized and little is known of the growth regulatory mechanisms of these neuroendocrine cells. Down-regulation or loss of the transforming growth factor-beta1 (TGFbeta1) cytostatic program and activation of TGFbeta-mediated transcriptional networks is associated with uncontrolled growth and metastasis in other neural tumors, glioblastomas. Whether this phenomenon is common to small intestinal carcinoid tumors was investigated.The effects of TGFbeta1 on cultured normal EC cells (isolated by FACS sorting) and the neoplastic EC cell line, KRJ-I, was assessed using the MTT assay. The TGFbetaRII transcript and protein were identified in tumor cells and the effects of TGFbeta1 on SMAD2 phosphorylation and nuclear translocation quantified. The time-dependent response of SMAD4, SMAD7, c-Myc, and P21(WAF1/CIP1) protein expression and c-Myc and p21(WAF1/CIP1) transcript was measured in response to TGFbeta1 and the transcript expression of candidate downstream targets, MTA1 and E-cadherin, were assessed.TGFbeta1 inhibited normal EC cell proliferation (IC(50) = 17 pM) but stimulated neoplastic EC cell proliferation (EC(50) = 22 pM). In tumor cells, significantly decreased transcript (P.01) of TGFbetaRII was identified, but no receptor mutations were identified and protein expression was evident. TGFbeta1 (1 ng/mL) resulted in SMAD2 phosphorylation and7% nuclear expression compared with 93% in normal EC cells. In neoplastic cells, TGFbeta1 (1 ng/mL) caused a decrease in SMAD4 (16%, P.05), whereas SMAD7 and c-Myc transcript and protein were respectively increased21% (P.05) and approximately 40% (P.002). TGFbeta1 (1 ng/mL) also decreased p21(WAF1/CIP1) transcript by 60% (P.001) and protein that was undetectable at 24 hours. Expression of the downstream targets of the c-Myc pathway, MTA1, was increased (20%) and E-cadherin decreased (30%).The neoplastic EC cell is characterized by loss of TGFbeta-1-mediated growth inhibition and, similar to glioblastomas, utilizes the TGFbeta system to induce gene responses associated with growth promotion (c-Myc and the ERK pathway), invasion (E-cadherin), and metastasis (MTA1).

The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors. [Cancer Res 2007;67(7):2932–7]

Expression of Met, the Hepatocyte Growth Factor receptor, has been shown to have prognostic value in numerous types of cancer including breast, gastric, cervical and head and neck carcinomas. However, traditional analyses of expression have shown variable results and a lack of reproducibility. The AQUA® system is a method of quantitative in situ analysis of protein expression that allows the assessment of reproducibility of both antibodies and assay conditions. Here, we illustrate the necessity for antibody validation when assaying the prognostic value of a potential biomarker. Using five antibodies to the intracellular domain of the Met receptor and 10 cell line controls, we quantitatively assess reproducibility of protein expression. We show that many antibodies are not reproducible at a quantitative level from lot to lot or assay to assay, suggesting new criteria for antibody validation. We also build upon past literature addressing the prognostic value of Met in a cohort of 640 cases of invasive breast cancer on a tissue microarray. We show that high levels of expression of nuclear Met, as determined by antibodies to the intracellular domain and defined as nuclear by subcellular compartmental analysis, is associated with shorter disease-specific survival in breast cancer.

Background: Several lines of laboratory evidence support a role of persistent activation of Akt pathway in oropharyngeal squamous cell carcinoma (OSCC) progression. Loss of phosphatase PTEN is one of the proposed mechanisms of Akt activation. We sought to determine the prognostic significance of Akt activation in a cohort of patients with OSCC as well as the association between phosphorylated (activated) Akt and PTEN levels. Methods: Using a novel system of in situ quantitative protein expression analysis (AQUA), we studied the protein expression levels of phosphorylated Akt (p-Akt) and PTEN on a tissue microarray. The array included 79 OSCCs with a mean follow-up of 36 months. Results: Patients with tumors expressing low tumor p-Akt levels had lower 5-year local recurrence rates (5% versus 38%). Additionally, these patients had improved 5-year overall survival rates (45% versus 27%). This survival effect was likely due to disease recurrence, as there was no difference in death without recurrence between low- and high-expressing groups. In adjusted analysis, tumor p-Akt expression was a strong predictor of local recurrence. A significant inverse relationship was found between nuclear p-Akt and nuclear PTEN: Tumors with high nuclear p-Akt had low nuclear PTEN and vice versa. Conclusions: Akt activation in OSCC is associated with adverse patient outcome, indicating that Akt is a promising molecular target in OSCC. PTEN loss may be one of the mechanisms of Akt activation in OSCC. (Cancer Epidemiol Biomarkers Prev 2007;16(3):553–8)

Mastomys enterochromaffin-like (ECL) cell proliferation is initially gastrin driven, but once neoplasia develops, cells become gastrin autonomous. We hypothesized that CCN2 (CTGF), a mitogenic growth factor, may regulate ECL cell proliferation. A Mastomys GeneChip database was examined (dCHIP) to identify CCN2 expression levels. CCN2 in normal and tumor ECL cell preparations obtained using FACS (100 nM acridine orange) was examined by real-time PCR. CCN2 protein was identified in mucosal and ECL cell preparations by immunohistochemistry. Short-term cultured cells were stimulated with either CCN2 or CCN2 + EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor PD-98059 (0.1–100 μM) was assessed in terms of CCN2 (1 ng/ml)-mediated proliferation and ERK1/2 phosphorylation. CCN2 transcript and protein was then examined in clinical gastric carcinoids. The ccn2 transcript was upregulated in tumor samples compared with the normal mucosa (+2.36-fold, P < 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (>98% pure) normal ECL cells but was elevated in tumor ECL cell fractions (41.3 ± 10.7-fold). Immunostaining of the Mastomys gastric mucosa and FACS preparations confirmed that CCN2 protein was present in ECL tumors but not in normal ECL cells. Neither CCN2 nor CCN2 + EGF stimulated normal ECL cell proliferation. CCN2 stimulated tumor proliferation (EC50 ∼0.01 ng/ml); EGF significantly augmented ( P < 0.01) CCN2-induced tumor cell proliferation (EC50 = 20 pg/ml). PD-98059 inhibited CCN2-induced proliferation (−12 ± 3%, P < 0.05) and ERK1/2 phosphorylation (−34 ± 5%, P < 0.05) in tumor cells. In clinical samples, both CCN2 transcript and protein were elevated in gastrin-autonomous carcinoids ( P < 0.02) compared with the normal mucosa. In conclusion, CCN2 may be a proliferative regulator of Mastomys ECL neoplastic proliferation once these cells become autonomous of gastrin regulation. Identification of CCN2 in gastric carcinoid tissue may be useful both as an indicator of ECL cell transformation and may define gastrin autonomy, a criteria of gastric carcinoid malignancy.

Purpose: The cellular inhibitor of apoptosis protein (cIAP) is regarded as an important prognostic biomarker in cancer. Here, we sought to determine the prognostic value of cIAP protein levels in epithelial ovarian cancer using a novel method of compartmentalized in situ protein analysis. Methods: A tissue array composed of 150 advanced-stage ovarian cancers, treated with surgical debulking followed by platinum/paclitaxel–based combination chemotherapy, was constructed. For evaluation of protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis. Results: The mean follow-up time for the entire cohort was 34.4 months. Patients with tumors bearing high cIAP membranous expression had a 3-year survival rate of 31% compared with 73% for patients with low cIAP expressing tumors (P = 0.0020). In multivariable analysis, adjusting for well-characterized prognostic variables, low membranous cIAP expression level was the only significant prognostic factor for overall survival. Conclusions: Our results indicate that cIAP protein levels have prognostic value in ovarian cancer patients. Modulation of cIAP levels may improve clinical outcome in ovarian cancer. (Cancer Epidemiol Biomarkers Prev 2006;15(6):1179-83)

BACKGROUND Measures of vascular endothelial growth factor (VEGF) expression in pancreatic cancer typically have been qualitative or semiquantitative. The objective of this study was to use a series of algorithms called AQUA that quantitatively assesses protein expression on tissue microarrays (TMAs) to compare in situ expression of VEGF and its primary receptors, VEGF receptor 1 (FLT-1) and VEGF receptor 1 (FLK-1), on a pancreatic cancer TMA. METHODS TMAs were constructed by arraying 1.5-mm cores from 76 samples of pancreatic adenocarcinoma (1996-2002) that were obtained from the archives of the Yale Department of Pathology. The staining for AQUA was similar to standard immunohistochemistry and involved antigen retrieval and the application of primary antibodies, but with epifluorescence detection. Slides were counterstained with 4′,6-diamidino-2-phenylindole for nuclear visualization and cytokeratin for membrane visualization. The primary antibodies used were VEGF, FLT-1, FLK-1, and cytokeratin. RESULTS Disease stage was highly prognostic for outcome, as expected. Total amounts of VEGF and its receptors were assessed within the tumor mask and were divided into quartiles. Kaplan–Meier survival curves showed that VEGF and FLK-1 were not associated clearly with outcome. However, the expression of FLT-1 was correlated significantly, and the patients who had tumors with the lowest expression FLT-1 levels had the worst survival (P = .0038). In multivariate analysis, FLT-1 expression was an independent prognostic factor for overall survival (P = .0044). CONCLUSIONS VEGF and its 2 principal receptors were expressed to varying degrees in tumors of the pancreas. A significant association was found between low expression of FLT-1 and both poor prognosis and advanced stage, suggesting that tumor expression of this VEGF receptor is a marker of less aggressive disease. Cancer 2006. © 2006 American Cancer Society.

Current techniques to define gastric neoplasia are limited but molecular genetic signatures can categorize tumors and provide biological rationale for predicting clinical behavior. We identified three gene signatures: Chromogranin A (CgA), MAGE-D2 (adhesion), and MTA1 (metastasis) that define gastrointestinal (GI) carcinoids and hypothesize that their expression can delineate gastric neoplasia. This strategy provides a molecular basis to define neuroendocrine gastric carcinoids (GCs), neuronal stromal tumors (GISTs), or epithelial cell (gastric adenocarcinomas [GCAs])-derived tumors.Total RNA was isolated from 38 GCs: Type I/II (n = 7), Type III/IV (n = 6), GISTs (n = 12), GCAs (n = 13), and normal mucosa (n = 12). Quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR) gene expression was quantified against glyseraldehyde-3-phosphate dehydrogenase (GAPDH) and CgA and MTA1 protein expression levels were analyzed by immunohistochemical analyses of a gastric neoplasia microarray.CgA was elevated in Type I/II (10-fold; P.01) and Type III/IV (100-fold, P.005), decreased in GISTs (100-fold, P.03), and unchanged in GCAs. MAGE-D2 was 5-10-fold elevated (P.05) in Type III/IV, GISTs, and GCAs but not in Type I/II tumors. MTA1 (5-fold, P.01) was elevated in GCs (Type III/IVI/II, P.05), in GISTs (4-fold, P.05), and GCAs. CgA protein levels were elevated in GCs (P.005) but not in GISTs and GCAs. MTA1 levels were elevated in all tumors (P.02) compared with normal, and especially with tumor invasion (P.05).CgA discriminates GCs from other gastric neoplasms; overexpression of MAGE-D2 and MTA1 differentiate Type III/IV from Type I/II GCs. GISTs share similar expression patterns with Type III/IV GCs but have decreased CgA. MTA1 is a marker of tumor invasion.

Background: Disparate results in the immunohistochemistry literature regarding the relationship between biomarker expression and patient outcome decrease the credibility of tissue biomarker studies. We investigated whether some of these disparities result from subjective optimization of an tibody concentration. Methods: We used the automated quantitative analysis (AQUA) system and various concentrations of antibodies against HER2 (1 : 500 to 1 : 8000 dilutions), p53 (1 : 50 to 1 : 800 dilutions), and estrogen receptor (ER; 1 : 100 and 1 : 1000 dilutions) to assess expression of HER2 and p53 in a tissue microarray containing specimens from 250 breast cancer patients with long-term survival data available. HER2 expression in the tissue microarray was also assessed by conventional immunohistochemistry. Relative risk (RR) of disease-specifi c mortality was assessed for every cutpoint with the X-tile program. Cumulative disease-specifi c survival was assessed by the Kaplan – Meier method. All statistical tests were two-sided. Results: For HER2 and p53 and an optimal cutpoint, when a high antibody concentration (i.e., 1 : 500 dilution) was used with the AQUA system, low expression was associated with poorer survival than high expression; however, when a low antibody concentration (i.e., 1 : 8000 dilution) was used, high expression was associated with poorer survival. For example, for a 1 : 8000 dilution of HER2 antibody and high expression defi ned as the top 15% of HER2 expression, high HER2 expression was associated with increased disease-specifi c mortality (RR = 1.98, 95% confi dence interval [CI] = 1.21 to 3.23; P = .007), compared with low expression. However, for a 1 : 500 dilution of HER2 antibody and high expression defi ned as the top 85% of HER2 expression, high HER2 expression was associated with decreased disease-specifi c mortality (RR = 0.47, 95% CI = 0.29 to 0.76; P = .002), compared with low HER2 expression. Conclusions: Biomarker antibody concentration appears to dramatically affect the apparent relationship between biomarker expression and outcome. [J Natl Cancer Inst 2005;97:1808 – 15]