PurposeTo determine whether (1) immunohistochemical (IHC) HER2 status (ie, 2+ or 3+), (2) degree of fluorescence in situ hybridization (FISH) amplification according to (2a) HER2/CEP17 ratio or (2b) HER2 gene copy number, or (3) polysomy significantly influenced clinical outcome for patients with human epidermal growth factor receptor 2 (HER2) –positive breast cancer enrolled in the Herceptin Adjuvant trial of trastuzumab versus no trastuzumab administered after completion of chemotherapy.Patients and MethodsIHC and/or FISH analyses were performed locally and required central confirmation as indicating HER2 positivity for trial entry. FISH data from the central HER2 analysis on patients in the 1-year trastuzumab and no trastuzumab arms were assessed in relation to disease-free survival (DFS) after a median 2 years of follow-up.ResultsCentral FISH results were available for 2,071 (61%) of the 3,401 patients randomized to the 2 arms. Among patients with FISH-positive disease, (1) the hazard ratios for trastuzumab versus no trastuzumab were 0.56 (95% CI, 0.32 to 0.99) for locally IHC2+ cases (n = 340) and 0.80 (95% CI, 0.40 to 1.61) for centrally IHC2+ cases (n = 299). There was no significant prognostic relationship between (2a) HER2 FISH ratio, (2b) HER2 copy number, or (3) polysomy and DFS in the control arm or predictive relationship defining differential benefit from trastuzumab.ConclusionThere was no evidence for reduced benefit of trastuzumab in HER2 IHC2+FISH+ cases. The degree of HER2 amplification does not influence prognosis or benefit from adjuvant trastuzumab in patients treated with prior adjuvant chemotherapy.

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.

The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and pathways to fulfil their anti-apoptotic role. In the search for novel IAP-interacting proteins we identified the neurotrophin receptor-interacting MAGE homologue (NRAGE) as being able to bind to the avian IAP homologue ITA. This interaction requires the RING domain of ITA. NRAGE additionally coimmunoprecipitates with XIAP. When overexpressed in 32D cells NRAGE augments interleukin-3 withdrawal induced apoptosis, possibly through binding endogenous XIAP. Moreover, NRAGE is able to overcome the anti-apoptotic effect of Bcl-2.

In the search for physiological substrates of MAPKactivated protein (MAPKAP) kinases, we identified the basic helix-loop-helix (bHLH) transcription factor E47as an interaction partner of chromosome 3p kinase (3pK) and MAPKAP-K2 (MK2). The E2A protein E47 is known to be involved in the regulation of tissue-specific gene expression and cell differentiation. E47 is a phosphoprotein, and we identified 3pK and MK2 as E47 kinases in vitro. Furthermore, the expression of either kinase results in a repression of the transcriptional activity of E47 on an E-box containing promoter. In summary, the MAPK-activated protein kinases 3pK and MK2 were identified to form an assembly with the bHLH protein E47 suggesting that these kinases are regulators of E47 activity and E47-dependent gene expression.Chromosome 3p kinase(3pK) 1 (21) and MK2 belong to a family of serine/threonine kinases that are activated by members of the mitogen-activated protein kinase (MAPK) family. 3pK, also known as MAPKAP kinase 3 (1) is unique in that it was shown to be activated by mitogenic inducers such as serum/TPA through the Raf/MEK/ERK cascade as well as by stress-inducing agents that are activators of stress-induced MAPK cascades, thereby leading to the phosphorylation of 3pK by either JNK/SAPK or p38 (2). Thus, the kinase is targeted by the corresponding cascade depending on the extracellular stimulus (2). Although the upstream activation pathways of 3pK are well documented, little is known about its downstream effectors.A close homologue of 3pK, MK2, is involved in stress response mediated through p38. Among the substrates of MK2 are the small heat shock proteins (Hsp27/25) (3, 4) and transcription factors such as CREB (5) and SRF (6).The basic helix-loop-helix (bHLH) transcription factors E12 and E47 are encoded by the E2A gene and are generated by differential splicing of E12-and E47-specific bHLH-encoding exons (7,8). These E-proteins are characterized by their ability to bind to the consensus DNA sequence CANNTG, referred to as E-box, either as homodimers in B-cells or as heterodimers with tissue-specific Class II HLH proteins in other cell types (9, 10). Initially identified in B-cells as immunoglobulin enhancerbinding proteins, the E2A gene products classified as Class I HLH proteins are also involved in cell differentiation, lineage commitment, and the expression of many tissue-specific genes (11). Through phosphorylation, DNA binding and transactivation of a variety of transcription factors are regulated (12). For example, the dimerization of myogenin with E2A products was shown to enhance phosphorylation of myogenin, thereby reducing the transcriptional activity of myogenin, suggesting that phosphorylation of this myocyte enhancer factor (MEF) negatively interferes with muscle-specific gene expression (13). The DNA-binding activity of MyoD homodimers but not MyoD-E12 heterodimers was inhibited by phosphorylation (14). Johnson et al. (16) demonstrated that overexpression of casein kinase II (CKII) increased the transcriptional activit...

Despite recent advances in chemotherapy, the prognosis for patients with advanced gastric cancer (GC) or gastroesophageal junction cancer remains poor. Human epidermal growth factor receptor 2 (HER2) is a novel target for biologic therapy in metastatic GC. We analyzed the association between HER2 overexpression and the clinicopathologic characteristics of advanced GC. Formalin-fixed, paraffin-embedded tumor samples were collected from patients with stage III or to IV (M(0)) GC who subsequently underwent curative surgery followed by adjuvant chemotherapy with 5-fluorouracil and cisplatin. All the samples were analyzed for HER2 status by immunohistochemistry (IHC) and fluorescence in situ hybridization. Of 142 samples analyzed, 7.1% scored IHC 2+ and 8.6% scored IHC 3+, whereas 9.3% were HER2-amplified. Of HER2-amplified cases, 76.9% (10/13) scored IHC 3+, showing the correlation between HER2 amplification and overexpression (P=0.01). HER2 IHC 3+ cases were more common in the intestinal-type tumors compared with diffuse-type tumors (16.7% vs. 5.1%, respectively; P=0.049), and a nonsignificant trend was observed using fluorescence in situ hybridization (14.3% vs. 9.2%, respectively; P=0.399). HER2 gene amplification was more frequent in stage IV (M(0)) than stage III disease (15.4% vs. 4.0%, respectively; P=0.037). Interestingly, HER2-amplified disease was more common than nonamplified disease in patients with nodal stage 3 tumors (76.9% vs. 38.6%, respectively; P=0.009); a similar pattern was observed using IHC. HER2 overexpression correlated with nodal stage, and a lymph node ratio greater than 0.5 was more common in HER2-amplified tumors than HER2-nonamplified tumors (69.2% vs. 43.3%, respectively; P=0.086). These findings suggest that further investigations of adjuvant therapy with HER2-targeted therapy for advanced GC are warranted.

Personalized health care (PHC) is an evolving field of medicine aimed at providing the right therapy to the right patient at the right time. This approach often incorporates the use of companion diagnostics (CDx) assays that provide information essential for the safe and effective use of the corresponding drug. In addition to oncology, many other therapy areas, such as cardiovascular, neurological, and infectious and inflammatory diseases, may benefit from PHC, owing to disease complexity and heterogeneity. Furthermore, although most U.S. Food and Drug Administration–approved CDx are based on molecular‐based technologies, immunoassays can provide a significant contribution to the evolution of CDx in patient management. In this review we discuss how the incorporation of biomarker immunoassays into routine diagnostic testing may allow early and definitive detection of Alzheimer's disease and enable population enrichment in clinical trials. In addition, we will describe how biomarker‐based CDx immunoassays have potential utility for stratifying patients with asthma based on their potential response to therapy and for selecting treatment according to phenotypic profile. Continued research into the underlying disease pathology and development of accurate and reliable diagnostic assays may ensure that PHC becomes the future standard for many indications.

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15057 Background: Accurate HER2 testing is required to identify patients eligible for treatment with trastuzumab (Herceptin®). HER2 positivity is reported as 6–35% in gastric cancer (GC). This range is due to small sample sets and differing methods of evaluation or scoring. A specific HER2-testing process was established for the Phase III ToGA trial, which is evaluating trastuzumab added to chemotherapy in HER2-positive advanced GC. Methods: A validation study was completed to standardise IHC (HercepTest™) and FISH (PharmDx™) protocols, and to establish a scoring system specific for GC (M Hofmann et al. ASCO Gastrointestinal Cancers Symposium 2006. Abstract no. 24). Tumour samples for ToGA were then centrally tested by both IHC and FISH to identify patients eligible for enrolment. Results: To date, 1024 tumour samples have been assessed (243 HER2 positive and 781 HER2 negative) giving an overall HER2-positivity rate of 23.7%. Both IHC and FISH results are available for 960 patients, with 87% concordance. Differences were largely due to FISH-positive cases that were IHC 0/1+. HER2 positivity differed significantly by histological subtype: 36% in intestinal, 7% in diffuse and 23% in mixed. HER2 positivity also varied according to the site of the tumour: 36% (8/22) for gastro-oesophageal junction tumours and 21% (60/291) for gastric tumours. Sample numbers were very small so these results must be treated with caution. The HER2- positivity rate was similar in specimens obtained by biopsy (168/689; 24%) and surgery (71/322; 22%). Conclusions: Using validated methodology and based on the large sample set from the ongoing ToGA trial, the HER2-positivity rate observed in advanced GC is as high as in breast cancer: ∼24%. The first efficacy data from ToGA are expected in 2009. No significant financial relationships to disclose.