Global Ring Study to Investigate the Comparability of Total Assay Performance of Commercial Claudin 18 Antibodies for Evaluation in Gastric Cancer.

Claudin 18.2 (CLDN18.2), the dominant isoform of CLDN18 in gastric tissues, is a highly specific tight junction protein of the gastric mucosa with variably retained expressions in gastric and gastroesophageal junction cancers. Additionally, CLDN18.2-targeted treatment with zolbetuximab, in combination with chemotherapy, has recently been assessed in 2 phase-III studies of patients with HER2-negative, locally advanced, unresectable, or metastatic gastric or gastroesophageal junction adenocarcinoma. These trials used the investigational VENTANA CLDN18 (43-14A) RxDx immunohistochemistry (IHC) assay on the Ventana BenchMark platform to identify patients eligible for CLDN18.2-targeted treatment. We report the findings of a global ring study evaluating the analytical comparability of concordance of the results of 3 CLDN18 antibodies (Ventana, LSBio, and Novus) stained on 3 IHC-staining platforms (Ventana, Dako, and Leica). A tissue microarray (TMA), comprising 15 gastric cancer cases, was stained by 27 laboratories across 11 countries. Each laboratory stained the TMAs using at least 2 of the 3 evaluated CLDN18 antibodies. Stained TMAs were assessed and scored using an agreed IHC-scoring algorithm, and the results were collated for statistical analysis. The data confirmed a high level of concordance for the VENTANA CLDN18 (43-14A; Ventana platform only) and LSBio antibodies on both the Dako and Leica platforms, with accuracy, precision, sensitivity, and specificity rates all reaching a minimum acceptable ≥85% threshold and good-to-excellent levels of concordance as measured by Cohen's kappa coefficient. The Novus antibody showed the highest level of variability against the reference central laboratory results for the same antibody/platform combinations. It also failed to meet the threshold for accuracy and sensitivity when used on either the Dako or Leica platform. These results demonstrated the reliability of IHC testing for CLDN18 expression in gastric tumor samples when using commercially available platforms with an appropriate methodology and primary antibody selection.

Salivary duct carcinomas can be classified into luminal androgen receptor-positive, HER2 and basal-like phenotypes.

AIMS: The aim of this study was to devise a molecular classification for salivary duct carcinomas (SDCs) based on the similarities between SDCs and breast carcinomas and on characteristics of the microarray-based gene expression profiling-defined molecular subtypes of breast cancer. METHODS AND RESULTS: Forty-two pure salivary duct carcinomas, 35 of which contained an in-situ component as defined by histological review and/or immunohistochemical analysis, were stained with antibodies for oestrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin (CK) 5/6. Based on these markers, tumours were classified into HER2, luminal androgen receptor-positive, basal-like, luminal and indeterminate phenotype. Analysis revealed that 16.7%, 69%, 4.8%, 9.5% and 0% were of HER2, luminal androgen receptor-positive, basal-like, indeterminate and luminal phenotype, respectively. The in-situ and invasive components displayed the same molecular subtype in all but one case. CONCLUSION: Salivary duct carcinomas can be classified into molecular subgroups approximately equivalent to those in the breast. We also report on the existence of a subgroup of bona fide pure salivary duct carcinomas that have a 'basal-like' phenotype. Understanding the phenotypic complexity of SDCs may help to expedite the identification of novel therapeutic targets for these aggressive tumours.

Cost-Effectiveness of PD-L1 Testing in Non-Small Cell Lung Cancer (NSCLC) Using In Vitro Diagnostic (IVD) Versus Laboratory-Developed Test (LDT).

INTRODUCTION: Accurate PD-L1 testing for non-small cell lung cancer (NSCLC) maximizes the benefits of immune checkpoint inhibitor (ICI) drugs like pembrolizumab. False negative test results deny ICI treatments to eligible patients, worsening clinical and economic outcomes, while false positives increase costs by using ICI treatments without their benefits. This study evaluates the cost-effectiveness of PD-L1 testing with an in vitro diagnostic (IVD) compared to a laboratory-developed test (LDT) for allocating patients with NSCLC to treatment with either pembrolizumab or chemotherapy using the German healthcare system as a model. METHODS: We developed a decision analytical model to evaluate the cost-effectiveness of PD-L1 testing with a regulatory body approved IVD compared to an LDT from the national German healthcare payer (statutory health insurance system) perspective. Accuracy of PD-L1 testing was based on data from two independent proficiency testing programs. The 1-year model was based on outcomes data from the KEYNOTE-024 clinical trial and treatment patterns reflecting current German practices. RESULTS: IVDs produced accurate PD-L1 testing results in 93% (752/811) of tested cases compared to 73% (492/672) with LDTs. Most misclassifications concerned false negatives, occurring in 21% of LDTs vs 7% of IVDs. Total costs of the IVD group (48,878 €) were 196 € higher than the LDT group (48,682 €). These costs incorporate testing, first- and second-line therapy, managing treatment-related grade 3+ adverse events (AEs), and end-of-life costs for those who died within the year. Total effectiveness (percentage of patients successfully diagnosed and prescribed the correct therapy per German treatment guidelines) was 19 percentage points higher for the IVD group (88%) compared to the LDT group (69%). These differences in costs and effects lead to an incremental cost-effectiveness ratio (ICER) of 1057 €. CONCLUSION: Compared to LDT technology, on-label IVD use for PD-L1 testing is only slightly more costly and substantially more effective for aligning patients with PD-L1-positive NSCLC with ICI therapy according to German practice guidelines. Given these findings, changes to testing and reimbursement policies may be considered to maximize patient outcomes in NSCLC.

A Consortium for Analytic Standardization in Immunohistochemistry.

CONTEXT.­: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.­: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.­: Literature review and published external quality assurance data. CONCLUSIONS.­: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.

UK NEQAS ICC & ISH Ki-67 Data Reveal Differences in Performance of Primary Antibody Clones.

We examined data from 374 laboratories staining for Ki-67 as part of external quality assessment over 8 runs between 2013 and 2017 (total data sets=2601). One of 5 primary antibodies was used for 94.8% of submissions, with MIB-1 (Agilent Dako) comprising 58.8% of the total. Examining assessment score as a continuous variable showed the 30-9 (Ventana) and K2 (Leica Biosystems) clones were associated with the highest mean scores (17.0; 95% confidence interval, 16.8-17.2 and 16.3; 95% confidence interval, 15.9-16.6, respectively). Stain quality was not significantly different between them. Both were associated with significantly better staining compared with MIB-1 (Agilent Dako), MM1 (Leica Biosystems), and SP6 from various suppliers (P<0.05). Similarly, categorical assessment of "Good" versus "Not good" staining quality showed that the 30-9 and K2 clones were both significantly associated with "Good" staining (both P<0.001). Other methodological parameters were examined for significant primary antibody-specific effects; none were seen for 30-9, K2, or SP6. The MM1 clone was more likely to be associated with good quality staining when it was used with Leica Biosystems sourced antigen retrieval, detection, and platform, all statistically significant at P<0.01. MIB-1 was more likely to be associated with good quality staining results when it was used with Agilent Dako antigen retrieval, detection, and staining platforms (P<0.0001), and less likely at the same significance level when used with Leica Biosystems reagents and equipment. The data presented here show the importance of not just primary antibody choice but also matching that choice to other methodological factors.

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer.

INTRODUCTION: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear. METHODS: We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain. RESULTS: Most brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors. CONCLUSIONS: Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.

Genomic and immunohistochemical analysis of adenosquamous carcinoma of the breast.

Breast adenosquamous carcinomas are rare tumours characterized by well-developed gland formation intimately admixed with solid nests of squamous cells immersed in a highly cellular spindle cell stroma. A low-grade variant has been described that is associated with a better prognosis. Here we studied five cases of adenosquamous carcinomas to determine their genetic profiles and to investigate whether the spindle cell component of these cancers could at least in part stem from the glandular/epithelial components. Five adenosquamous carcinomas of the breast were subjected to (1) immunohistochemical analysis, (2) microdissection and genetic analysis with a high-resolution microarray comparative genomic hybridization platform, and (3) chromogenic in situ hybridization. All cases displayed a triple-negative immunophenotype, consistently expressed 'basal' keratins and showed variable levels of epidermal growth factor receptor expression. Microarray comparative genomic hybridization analysis of two of the cases revealed multiple low-level gains and losses affecting several chromosomal arms. Case 1 displayed gains of the whole of chromosome 7, and case 2 harboured a focal, high-level amplification of 7p12, encompassing the epidermal growth factor receptor gene, which was associated with strong and intense membranous epidermal growth factor receptor expression. Chromogenic in situ hybridization revealed that the genetic features found in the epithelial cells were also present in a minority of the spindle cells of the stromal component, in particular in those near the epithelial clusters, indicating that some of the spindle cells are clonal and derived from the epithelial component of the tumour. In conclusion, breast adenosquamous carcinomas are triple-negative cancers that express 'basal' keratins. These tumours harbour complex genetic profiles. Some of the spindle cells in adenosquamous carcinomas are derived from the epithelial component, suggesting that adenosquamous carcinomas may also be part of the group of metaplastic breast carcinomas with spindle cell metaplastic elements.

Genome-wide transcriptomic profiling of microdissected human breast tissue reveals differential expression of KIT (c-Kit, CD117) and oestrogen receptor-alpha (ERalpha) in response to therapeutic radiation.

The pathogenesis of late normal tissue fibrosis after high-dose ionizing radiation involves multiple cell types and signalling pathways but is not well understood. To identify the molecular changes occurring after radiotherapy, paired normal tissue samples were collected from the non-irradiated breast and from the treated breast of women who had undergone curative radiotherapy for early breast cancer months or years previously. As radiation may induce distinct transcriptional changes in the different components of the breast, laser capture microdissection and gene expression microarray profiling were performed separately for epithelial and stromal components and selected genes were validated using immunohistochemistry. In the epithelial compartment, a reduction of KIT (c-Kit; CD117) and a reciprocal increase in ESR1 (oestrogen receptor-alpha, ERalpha) mRNA and protein levels were seen in irradiated compared to non-irradiated samples. In the stromal compartment, extracellular matrix genes including FN1 (fibronectin 1) and CTGF (connective tissue growth factor; CCN2) were increased. Further investigation revealed that c-Kit and ERalpha were expressed in distinct subpopulations of luminal epithelial cells. Interlobular c-Kit-positive mast cells were also increased in irradiated cases not showing features of post-radiation atrophy. Pathway analysis revealed 'cancer, reproductive system disease and tumour morphology' as the most significantly enriched network in the epithelial compartment, whereas in the stromal component, a significant enrichment for 'connective tissue disorders, dermatological diseases and conditions, genetic disorder' and 'cancer, tumour morphology, infection mechanism' networks was observed. These data identify previously unreported changes in the epithelial compartment and show altered expression of genes implicated in late normal tissue injury in the stromal compartment of normal breast tissue. The findings are relevant to both fibrosis and atrophy occurring after radiotherapy for early breast cancer.

Mixed micropapillary-ductal carcinomas of the breast: a genomic and immunohistochemical analysis of morphologically distinct components.

Micropapillary carcinomas (MPCs) can present as a rare histological special type of breast cancer; however, this histological type is more frequently found admixed with invasive ductal carcinomas of no special type (IDC-NSTs). We have previously demonstrated that pure MPCs constitute a distinct entity at the morphological and genetic levels. Here, we sought to determine whether mixed MPCs have genomic aberrations similar to those found in pure MPCs, and to investigate whether the distinct morphological components of MPCs harbour different genetic aberrations. Using high-resolution microarray comparative genomic hybridization (aCGH), we profiled a series of 10 MPCs of mixed histology and 20 IDC-NSTs matched for grade and oestrogen receptor (ER) status. In addition, we generated tissue microarrays containing a series of 24 pure and 40 mixed MPCs and performed immunohistochemical analysis with ER, progesterone receptor (PR), Ki-67, HER2, cytokeratin (CK) 5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1 and E-cadherin antibodies. In situ hybridization was employed to evaluate the prevalence of HER2, TOP2A, EGFR, CCND1, MYC and FGFR1 gene amplification. Our results demonstrate that mixed MPCs harbour similar patterns of genomic aberrations and phenotype (82.5% luminal and 17.5% HER2) compared to pure MPCs. A comparison between the distinct morphological components of mixed MPCs in a pairwise fashion revealed that both components harbour strikingly similar genomic profiles. When compared to grade- and ER-matched IDC-NSTs, mixed MPCs significantly more frequently harboured amplification of multiple regions on 8q (adjusted Fisher's p value < 0.05). Furthermore, mixed MPCs displayed higher proliferative rates than grade- and ER-matched IDC-NSTs. Our results suggest that micropapillary differentiation in breast cancer may identify a subgroup of more aggressive ER-positive breast carcinomas, even in those featuring a mixed histology, and that mixed MPCs are more closely related to pure MPCs than to IDC-NSTs.

PPM1D is a potential therapeutic target in ovarian clear cell carcinomas.

PURPOSE: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer. EXPERIMENTAL DESIGN: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers. RESULTS: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas. CONCLUSION: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.

External quality assessment demonstrates that PD-L1 22C3 and SP263 assays are systematically different.

PD-L1 inhibitors are part of first line treatment options for patients with advanced non-small cell lung cancer. PD-L1 immunohistochemistry (IHC) assays act as either a companion or a complementary diagnostic. The purpose of this study is to describe the experience of external quality assurance (EQA) provider UK NEQAS ICC and ISH with the comparison of different PD-L1 assays used in daily practice. Three EQA rounds (pilot, run A and run B) were carried out using formalin fixed paraffin embedded samples with sample sets covering a range of epitope concentrations, including 'critical samples' near to clinical threshold cut-offs. An expert panel (n = 4) evaluated all returned slides simultaneously and independently on a multi-header microscope together with the participants own in-house control material. The tonsil sample was evaluated as 'acceptable' or 'unacceptable', and for the other samples the percentage of PD-L1 stained tumour cells were estimated in predetermined categories (<1%, 1 to <5%, 5 to <10%, 10 to <25%, 25 to <50%, 50 to <80%, 80 to 100%). In the pilot and the two subsequent runs the number of participating laboratories was 43, 69 and 76, respectively. The pass rate for the pilot run was 67%; this increased to 81% at run A and 82% at run B. For two 'critical samples', in runs A and B, 22C3 IHC had significantly higher PD-L1 expression than SP263 IHC (p < 0.001), whilst the PD-L1 scores for the other six samples were similar for all assays. In run A the laboratory developed tests (LDTs) using 22C3 scored lower than the commercial 22C3 tests (p = 0.01). After the initial testing, improvement in performance of PD-L1 IHC is shown for approved and LDT PD-L1 assays. Equivalency of approved PD-L1 22C3 and SP263 assays cannot be assumed as the scores cross the clinically relevant thresholds of 1% and 50% PD-L1 expression.

Distribution and significance of caveolin 2 expression in normal breast and invasive breast cancer: an immunofluorescence and immunohistochemical analysis.

BACKGROUND: The aims of this study were to define the distribution of caveolin 2 (CAV2) in frozen and formalin fixed, paraffin embedded (FFPE) normal breast samples and the significance of CAV2 expression in breast cancer. METHODS: Caveolin 2 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry and immunofluorescence, in conjunction with antibodies to define luminal epithelial cells (oestrogen receptor and cytokeratin 8/18) and myoepithelial/ basal cells (cytokeratins 14 and 5/6, p63 and smooth muscle actin). CAV2 expression was also immunohistochemically analysed in two independent cohorts of invasive breast carcinomas (n = 245 and n = 418). RESULTS: In normal breast, CAV2 was expressed in myoepithelial cells, endothelial cells, fibroblasts and adipocytes. Luminal epithelial cells showed no or only negligible staining. CAV2 expression was observed in 9.6% of all breast cancers and was strongly correlated with high histological grade, lack of oestrogen receptor, progesterone receptor and cyclin D1 expression, and positivity for epidermal growth factor receptor, basal markers, p53 expression, and high proliferation index. Furthermore, CAV2 expression was significantly associated with basal-like immunophenotype and proved to be a prognostic factor for breast cancer-specific survival on univariate analysis. CONCLUSION: Our results demonstrate that CAV2 is preferentially expressed in basal-like cancers and is associated with poor prognosis. Further in vitro studies are required to determine whether CAV2 has oncogenic properties or is only a surrogate marker of basal-like carcinomas.

Breast cancer biomarkers in clinical testing: analysis of a UK national external quality assessment scheme for immunocytochemistry and in situ hybridisation database containing results from 199 300 patients.

We describe a collated data set of results from clinical testing of breast cancers carried out between 2009 and 2016 in the United Kingdom and Republic of Ireland. More than 199 000 patient biomarker data sets, together with clinicopathological parameters were collected. Our analyses focused on human epidermal growth factor receptor-2 (HER2), oestrogen receptor (ER) and progesterone receptor (PR), with the aim of the study being to provide robust confirmatory evidence on known associations in these biomarkers and to uncover new data on previously undescribed or unconfirmed associations, thus strengthening the evidence-base in clinical breast cancer testing. Overall, 13.1% of tumours were HER2-positive; 10.6% in ER-positive tumours, and 25.5% in ER-negative tumours. Higher rates of HER2 positivity were significantly associated with patient age <56 years versus age ≥56 years, symptomatic versus screen-detected tumours, testing of involved axillary node versus primary breast cancer, invasive ductal carcinoma (not otherwise specified) versus other histological types, higher histological grade, increasing tumour size, increasing nodal involvement, ER-negative versus ER-positive tumour status, PR-negative versus PR-positive tumour status. Where ER status was known, 82.7% of tumours were ER-positive; 80.9% in women age <56 years, and 83.6% in those age ≥56 years (ER-positive cut-off ≥1.0% positive tumour cells or equivalent). Where PR status was known, 64.9% of tumours were PR-positive; 65.8% in women age <56 years, and 64.4% in women age ≥56 years (PR-positive cut off ≥10.0% or equivalent). These analyses of clinical test results provide contemporary benchmarking data for HER2, ER and PR positive rates.

Neuregulin3 alters cell fate in the epidermis and mammary gland.

BACKGROUND: The Neuregulin family of ligands and their receptors, the Erbb tyrosine kinases, have important roles in epidermal and mammary gland development as well as during carcinogenesis. Previously, we demonstrated that Neuregulin3 (Nrg3) is a specification signal for mammary placode formation in mice. Nrg3 is a growth factor, which binds and activates Erbb4, a receptor tyrosine kinase that regulates cell proliferation and differentiation. To understand the role of Neuregulin3 in epidermal morphogenesis, we have developed a transgenic mouse model that expresses Nrg3 throughout the basal layer (progenitor/stem cell compartment) of mouse epidermis and the outer root sheath of developing hair follicles. RESULTS: Transgenic females formed supernumerary nipples and mammary glands along and adjacent to the mammary line providing strong evidence that Nrg3 has a role in the initiation of mammary placodes along the body axis. In addition, alterations in morphogenesis and differentiation of other epidermal appendages were observed, including the hair follicles. The transgenic epidermis is hyperplastic with excessive sebaceous differentiation and shows striking similarities to mouse models in which c-Myc is activated in the basal layer including decreased expression levels of the adhesion receptors, alpha6-integrin and beta1-integrin. CONCLUSION: These results indicate that the epidermis is sensitive to Nrg3 signaling, and that this growth factor can regulate cell fate of pluripotent epidermal cell populations including that of the mammary gland. Nrg3 appears to act, in part, by inducing c-Myc, altering the proliferation and adhesion properties of the basal epidermis, and may promote exit from the stem cell compartment. The results we describe provide significant insight into how growth factors, such as Nrg3, regulate epidermal homeostasis by influencing the balance between stem cell renewal, lineage selection and differentiation.

Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.

PURPOSE: The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. EXPERIMENTAL DESIGN: Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. RESULTS: WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. CONCLUSIONS: These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.

Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype.

PURPOSE: To investigate the proportion of breast cancers arising in patients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients. EXPERIMENTAL DESIGN: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin. RESULTS: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls. CONCLUSION: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.

Expression profiling of purified normal human luminal and myoepithelial breast cells: identification of novel prognostic markers for breast cancer.

The normal duct-lobular system of the breast is lined by two epithelial cell types, inner luminal secretory cells and outer contractile myoepithelial cells. We have generated comprehensive expression profiles of the two normal cell types, using immunomagnetic cell separation and gene expression microarray analysis. The cell-type specificity was confirmed at the protein level by immunohistochemistry in normal breast tissue. New prognostic markers for survival were identified when the luminal- and myoepithelial-specific molecules were evaluated on breast tumor tissue microarrays. Nuclear expression of luminal epithelial marker galectin 3 correlated with a shorter overall survival in these patients, and the expression of SPARC (osteonectin), a myoepithelial marker, was an independent marker of poor prognosis in breast cancers as a whole. These data provide a framework for the interpretation of breast cancer molecular profiling experiments, the identification of potential new diagnostic markers, and development of novel indicators of prognosis.

ALK Immunohistochemistry in NSCLC: Discordant Staining Can Impact Patient Treatment Regimen.

INTRODUCTION: Diagnostic immunohistochemistry (IHC) is increasingly accepted as a screening method for anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements in NSCLC. We have sought to establish an ongoing robust external quality assessment process to gauge quality of anaplastic lymphoma kinase (ALK) IHC, which can have an impact on interpretation of patient samples. METHODS: Unstained tissue and cell line samples were distributed on a quarterly basis to participating laboratories from 30 countries. Participants stained the slide using their routine diagnostic ALK IHC method and returned the slide along with their in-house control and methodology details. Slides were assessed by a team of pathologists and scientists. RESULTS: Overall, there was a mean pass rate of 83% (range 71%-98%), with 38 variations in staining protocol. Methods included the following: the Roche D5F3 assay (65% of users, pass rate 93%); Novocastra 5A4 (15% of users, pass rate 65%); Cell Signaling Technology D5F3 (7% of users, pass rate 91%), and Dako ALK1 (5% of users, pass rate 50%). Choice of methodology directly affected final interpretation of distributed ALK-positive and ALK-negative NSCLC cases, which were correctly identified by 89% and 88% of participants, respectively. Antibody detection method was a contributing factor in false-negative staining results. The choice of laboratory controls was found to be unsuitable, and as such, in-house control recommendations are also provided. CONCLUSIONS: ALK IHC is a robust screening technique, but there is concern that some diagnostic laboratories are using inadequate staining methods, which has a direct impact on final interpretation. External assessment helps provide laboratories with continued confidence in their ALK IHC testing.