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Background:Anti-EGFR antibody, cetuximab, improves overall survival (OS) in K-ras wild-type chemotherapy-refractory colorectal cancer. Epidermal growth factor receptor ligand epiregulin (EREG) gene expression may further predict cetuximab benefit.Methods:Tumour samples from a phase III clinical trial of cetuximab plus best supportive care (BSC) vs BSC alone (CO.17) were analysed for EREG mRNA gene expression. Predictive effects of high vs low EREG on OS and progression-free survival (PFS) were examined for treatment-biomarker interaction.Results:Both EREG and K-ras status were ascertained in 385 (193 cetuximab, 192 BSC) tumour samples. Within the high EREG and K-ras wild-type status ('co-biomarker')-positive group (n=139, 36%), median PFS was 5.4 vs 1.9 months (hazard ratio (HR) 0.31; P<0.0001), and median OS was 9.8 vs 5.1 months (HR 0.43; P<0.001) for cetuximab vs BSC, respectively. In the rest (n=246, 64%), PFS (HR 0.82; P=0.12) and OS (HR 0.90; P=0.45) were not significantly different. Test for treatment interaction showed a larger cetuximab effect on OS (HR 0.52; P=0.007) and PFS (HR 0.49; P=0.001) in the co-biomarker-positive group.Conclusion:In pre-treated K-ras wild-type status colorectal cancer, patients with high EREG gene expression appear to benefit more from cetuximab therapy compared with low expression. Epiregulin as a selective biomarker requires further evaluation. [ABSTRACT FROM AUTHOR]

Context: Patients with metastatic colorectal cancer who have KRAS codon 12- or KRAS codon 13-mutated tumors are presently excluded from treatment with the anti-epidermal growth factor receptor monoclonal antibody cetuximab.Objective: To test the hypothesis that KRAS codon 13 mutations are associated with a better outcome after treatment with cetuximab than observed with other KRAS mutations.Design, Setting, and Patients: We studied the association between KRAS mutation status (p.G13D vs other KRAS mutations) and response and survival in a pooled data set of 579 patients with chemotherapy-refractory colorectal cancer treated with cetuximab between 2001 and 2008. Patients were included in the CO.17, BOND, MABEL, EMR202600, EVEREST, BABEL, or SALVAGE clinical trials or received off-study treatment. Univariate and multivariate analyses, adjusting for possible prognostic factors and data set, were performed. The effect of the different mutations was studied in vitro by constructing isogenic cell lines with wild-type KRAS, p.G12V, or p.G13D mutant alleles and treating them with cetuximab.Main Outcome Measures: The main efficacy end point was overall survival. Secondary efficacy end points were response rate and progression-free survival.Results: In comparison with patients with other KRAS-mutated tumors, patients with p.G13D-mutated tumors (n = 32) treated with cetuximab had longer overall survival (median, 7.6 [95% confidence interval {CI}, 5.7-20.5] months vs 5.7 [95% CI, 4.9-6.8] months; adjusted hazard ratio [HR], 0.50; 95% CI, 0.31-0.81; P = .005) and longer progression-free survival (median, 4.0 [95% CI, 1.9-6.2] months vs 1.9 [95% CI, 1.8-2.8] months; adjusted HR, 0.51; 95% CI, 0.32-0.81; P = .004). There was a significant interaction between KRAS mutation status (p.G13D vs other KRAS mutations) and overall survival benefit with cetuximab treatment (adjusted HR, 0.30; 95% CI, 0.14-0.67; P = .003). In vitro and mouse model analysis showed that although p.G12V-mutated colorectal cells were insensitive to cetuximab, p.G13D-mutated cells were sensitive, as were KRAS wild-type cells.Conclusions: In this analysis, use of cetuximab was associated with longer overall and progression-free survival among patients with chemotherapy-refractory colorectal cancer with p.G13D-mutated tumors than with other KRAS-mutated tumors. Evaluation of cetuximab therapy in these tumors in prospective randomized trials may be warranted. [ABSTRACT FROM AUTHOR]

Context.—: Trastuzumab deruxtecan (T-DXd), a human epidermal growth factor receptor 2 (HER2)-targeted therapy, has demonstrated durable anticancer activity in patients with advanced, metastatic HER2 (also known as ERBB2)-mutant (HER2m) non-small cell lung cancer (NSCLC) who have limited treatment options and poor prognosis., Objective.—: To analytically validate and assess the clinical utility of the Oncomine Dx Target (ODxT) Test as a companion diagnostic to identify patients with HER2m NSCLC., Design.—: Tumor samples from patients in DESTINY-Lung01 and DESTINY-Lung02 were retrospectively analyzed alongside commercially procured samples using the ODxT test and compared to the assays used for screening in these clinical trials., Results.—: Positive percent agreement (PPA) and negative percent agreement (NPA) between the ODxT Test and TruSight Tumor 170 assay when testing DESTINY-Lung01 and commercially procured samples met prespecified thresholds (PPA and NPA ≥90%) for analytical accuracy (100% and 99.1%). The ODxT Test results were highly concordant with clinical trial assays (CTAs) used in DESTINY-Lung01 (PPA and NPA, 98.0% and 100%) and DESTINY-Lung02 (PPA and NPA, 96.7% and 100%) to identify activating HER2 mutations in tumor samples. Confirmed objective response rates were similar between patients with HER2m tumors identified by the ODxT Test and by CTAs in DESTINY-Lung01 (58.3% and 54.9%) and DESTINY-Lung02 (53.6% and 53.8%). Response duration was 12.0 and 9.3 months for patients identified by the ODxT Test and CTAs, respectively, in DESTINY-Lung01., Conclusions.—: The ODxT Test detected HER2 mutations in NSCLC with high analytical and clinical accuracy and identified HER2m populations with response rates similar to populations identified by CTAs, supporting clinical utility of the ODxT Test to inform treatment decisions for HER2m NSCLC., Competing Interests: Karnoub, Pereira, and Shiga are employees of Daiichi Sankyo, Inc. Qi, Feng, and Khambata-Ford are employees of and hold stock and stock options in Daiichi Sankyo, Inc. Ha and Velasco Roth are employees of Thermo Fisher Scientific. Smit has received consulting fees paid to his institution from Daiichi Sankyo, Inc, and has participated in advisory boards for Daiichi Sankyo, Inc. Y. Goto has received research grants paid to his clinical trial group or institution from Daiichi Sankyo, Inc, speaker payments or honoraria from Thermo Fisher Scientific, and has participated in advisory boards for Daiichi Sankyo, Inc. K. Goto has received research grants paid to his institution from Daiichi Sankyo, Inc, speaker payments or honoraria from Daiichi Sankyo, Inc, and Thermo Fisher Scientific, and has participated in advisory boards for Daiichi Sankyo, Inc. De Langen has no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

In DESTINY-Breast04 (DB-04), safety and efficacy of HER2-targeted antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) in previously treated HER2-low unresectable/metastatic breast cancer were established. This manuscript describes the analytical validation of PATHWAY Anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody (PATHWAY HER2 (4B5)) to assess HER2-low status and its clinical performance in DB-04. Preanalytical processing and tissue staining parameters were evaluated to determine their impact on HER2 scoring. The recommended antibody staining procedure provided the optimal tumor staining, and deviations in cell conditioning and/or antibody incubation times resulted in unacceptable negative control staining and/or HER2-low status changes. Comparisons between antibody lots, kit lots, instruments, and day-to-day runs showed overall percent agreements (OPAs) exceeding 97.9%. Inter-laboratory reproducibility showed OPAs of ≥97.4% for all study endpoints. PATHWAY HER2 (4B5) was utilized in DB-04 for patient selection using 1340 tumor samples (59.0% metastatic, 40.7% primary, (0.3% missing data); 74.3% biopsy, 25.7% resection/excisions). Overall, 77.6% (823/1060) of samples were HER2-low by both central and local testing, with the level of concordance differing by sample region of origin and collection date. In DB-04, the efficacy of T-DXd over chemotherapy of physician's choice was consistent, regardless of the characteristics of the sample used (primary or metastatic, archival, or newly collected, biopsy or excision/resection). These results demonstrate that PATHWAY HER2 (4B5) is precise and reproducible for scoring HER2-low status and can be used with multiple breast cancer sample types for reliably identifying patients whose tumors have HER2-low expression and are likely to derive clinical benefit from T-DXd., (© 2023. The Author(s).)

Background: Pembrolizumab monotherapy is a standard-of-care treatment for the first- and second-line treatment of advanced non-small cell lung cancer with programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) values ≥ 50% and ≥ 1%, respectively. PD-L1 testing with the PD-L1 immunohistochemistry (IHC) 22C3 pharmDx companion assay has been validated on tumor tissue with the Dako Autostainer Link 48 (ASL48). 22C3 anti-PD-L1 antibody-based laboratory-developed tests (LDTs) compatible with other autostainers and cytology samples are essential to support pembrolizumab treatment decisions across institutions globally., Methods: ASL48 and BenchMark Ultra LDTs were optimized for the evaluation of cytology samples through comparisons with cell lines with known PD-L1 expression levels (strong, moderate, and negative). The PD-L1 TPS was then evaluated for 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30) with these LDTs. Biopsy and cytology LDT TPS values were also compared with a subset of biopsy samples (n = 37) evaluated with the PD-L1 IHC 22C3 pharmDx assay on the ASL48., Results: Intraclass correlation coefficients of 0.884 to 0.898 were observed for biopsy samples versus cytology samples with the ASL48 and BenchMark Ultra LDTs. Concordance was high, regardless of the TPS cut point (<1% vs ≥ 1% and <50% vs ≥ 50%), sample type (pleural effusion vs bronchial wash), or tumor histology (adenocarcinoma vs squamous cell carcinoma). Concordance was high for each LDT versus the PD-L1 IHC 22C3 pharmDx assay., Conclusions: ASL48 and BenchMark Ultra 22C3 antibody concentrate-based LDTs have been validated for PD-L1 testing in cytology samples, and they will support reliable, high-quality PD-L1 testing across regions globally. Cancer Cytopathol 2018;126:264-74. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

[This corrects the article DOI: 10.1371/journal.pone.0183023.].

Background: For non-small cell lung cancer (NSCLC), treatment with pembrolizumab is limited to patients with tumours expressing PD-L1 assessed by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx (Dako, Inc.) companion diagnostic test, on the Dako Autostainer Link 48 (ASL48) platform. Optimised protocols are urgently needed for use of the 22C3 antibody concentrate to test PD-L1 expression on more widely available IHC autostainers., Methods: We evaluated PD-L1 expression using the 22C3 antibody concentrate in the three main commercially available autostainers Dako ASL48, BenchMark ULTRA (Ventana Medical Systems, Inc.), and Bond-III (Leica Biosystems) and compared the staining results with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Several technical conditions for laboratory-developed tests (LDTs) were evaluated in tonsil specimens and a training set of three NSCLC samples. Optimised protocols were then validated in 120 NSCLC specimens., Results: Optimised protocols were obtained on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. Significant expression of PD-L1 was obtained on tissue controls with the Leica Bond-III autostainer when high concentrations of the 22C3 antibody were used. It therefore was not tested on the 120 NSCLC specimens. An almost 100% concordance rate for dichotomized tumour proportion score (TPS) results was observed between TPS ratings using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Interpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform., Conclusion: Availability of standardized protocols for determining PD-L1 expression using the 22C3 antibody concentrate on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant manner.

Background: Kirsten rat sarcoma virus (KRAS) wild-type status determined using a locked nucleic acid (LNA)-mediated quantitative polymerase chain reaction (qPCR) clamping assay (LNA assay) predicted response to therapy in the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) study. A companion KRAS diagnostic tool has been developed for routine clinical use (QIAGEN therascreen kit) (QIAGEN Manchester Ltd, Manchester, UK). We wanted to assess the concordance between the validated US Food and Drug Administration (FDA)-approved therascreen assay and the LNA assay in determining the KRAS status of a subset of patients enrolled in the CRYSTAL study., Patients and Methods: DNA extracted from paraffin-embedded tumor sections was tested for KRAS status using the therascreen assay. Efficacy data from the CRYSTAL study were assessed to determine if the overall survival (OS) hazard ratio for cetuximab in patients identified as having KRAS wild-type status using the therascreen assay was equivalent to that in patients identified as KRAS wild-type using the LNA assay. This was determined by assessing if the concordance between the therascreen assay and the LNA assay met the minimum threshold (prespecified as 0.8) to achieve a significant difference in the OS hazard ratio in favor of the cetuximab + FOLFIRI (5-fluorouracil, leucovorin [folinic acid], irinotecan) arm in the KRAS wild-type population as identified using the therascreen assay., Results: Of the 148 samples determined to be KRAS wild-type (therascreen assay), 141 (95.3%) samples were also KRAS wild-type (LNA assay) and 7 samples (4.7%) were KRAS mutant (LNA assay). The prespecified primary concordance measure p was 141/148 = 0.953 (95% confidence interval [CI], 0.905-0.981). The concordance was statistically significantly higher than the prespecified threshold of 0.8 for concordance between the therascreen assay and the LNA assay. Consistent with the concordance exceeding the prespecified threshold, the OS hazard ratio (cetuximab + FOLFIRI arm vs. FOLFIRI arm) in the KRAS wild-type population, determined by the therascreen assay, supported a significant benefit for cetuximab (ie, the 95% CI excluded 1) and was comparable to the OS hazard ratio observed in the CRYSTAL study KRAS wild-type population (LNA assay) even after adjustment for potentially confounding baseline variables., Conclusion: These results support the utility of the therascreen assay for identifying patients who may benefit from cetuximab therapy for metastatic colorectal cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Context: The therascreen KRAS RGQ polymerase chain reaction kit is being developed as a companion diagnostic to aid clinicians, through detection of KRAS mutations, in the identification of patients with metastatic colorectal cancer (mCRC) who are more likely to benefit from cetuximab., Objectives: To assess whether KRAS mutation status, determined by using the therascreen KRAS kit, is a predictive marker of cetuximab efficacy., Design: Tissue samples were obtained from patients with mCRC treated on the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) CO.17 phase 3 study of cetuximab plus best supportive care (BSC) versus BSC alone. Tumor DNA samples were assessed for the presence of KRAS mutations by using the therascreen KRAS kit. Efficacy and safety were assessed to determine whether mutation status was predictive of outcomes. Results.-Evaluable samples were available from 453 patients (79.2%) enrolled in the NCIC CTG CO.17 trial. The KRAS wild-type subset represented 54.1% (245 of 453) of the evaluated population. Median overall survival of patients with KRAS wild-type tumors was 8.6 months among those who received cetuximab plus BSC and 5.0 months among patients who received BSC alone (hazard ratio [HR], 0.63; P = .002). Among patients with KRAS mutant mCRC, no meaningful difference in overall survival was observed between arms (HR, 0.91; P = .55). These results are consistent with a previous report that analyzed patient tumor samples by using bidirectional sequencing., Conclusions: These data support the utility of the therascreen KRAS kit as a means of selecting patients who may benefit from cetuximab therapy.

Purpose: This study examined potential correlations between markers related to the insulin-like growth factor-1 receptor (IGF-1R) pathway and clinical benefit from the anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in metastatic colorectal cancer (mCRC)., Experimental Design: Gene expression profiles for 70 pretreatment specimens from metastatic lesions of patients with chemorefractory mCRC receiving cetuximab monotherapy were analyzed using 74 predefined Gene-Chip probesets representing 33 unique IGF-1R pathway markers to determine correlations with progression-free survival (PFS) and disease control rate., Results: Higher IGF-1R, higher GRB(7), and lower INSIG(2) expression were associated with longer PFS with cetuximab in univariate analyses, particularly in patients with wild-type K-Ras tumors: median, 122 versus 60 days (P = 0.01), 122 versus 57 days (P = 0.011), and 57 versus 156 days (P < 0.0001), favoring higher IGF-1R, higher GRB(7), and lower INSIG(2) expression, respectively. Lower IGF-1 expression was associated with a PFS benefit with cetuximab, whereas lower IGFBP(3) and INSR expression levels showed trends for a PFS benefit. Lower INSIG(2) expression (vs. higher expression) was associated with greater PFS in the high epiregulin-expressing group (P = 0.001), but not in the low-expressing cohort suggesting an effect independent from the previously reported effect of epiregulin expression. Lower INSIG(2) expression was also associated with higher disease control rate in the overall population (51.4% vs. 11.4%; P = 0.001) and wild-type K-Ras subset (76.2% vs. 18.2%; P < 0.0001)., Conclusions: These results suggest that markers of the IGF-1R pathway may play a role in predicting benefit from cetuximab therapy in mCRC. Additional clinical studies are warranted to validate these findings., (©2012 AACR.)

One approach to delivering cost-effective healthcare requires the identification of patients as individuals or subpopulations that are more likely to respond to an appropriate dose and/or schedule of a therapeutic agent, or as subpopulations that are less likely to develop an adverse event (i.e., personalized or stratified medicine). Biomarkers that identify therapeutically relevant variations in human biology are often only uncovered in the later stage of drug development. In this article, the Industry Pharmacogenomics Working Group provides, for regulatory consideration, its perspective on the rationale for the conduct of what is commonly referred to as the prospective-retrospective analysis (PRA) of biomarkers. Reflecting on published proposals and materials presented by the US FDA, a decision tree for generating robust scientific data from samples collected from an already conducted trial to allow PRA is presented. The primary utility of the PRA is to define a process that provides robust scientific evidence for decision-making in situations where it is not necessary, nor practical or ethical to conduct a new prospective clinical study.

The anti-EGF receptor monoclonal antibody cetuximab provides a case study for the development of predictive biomarkers in oncology. The identification and validation of KRAS mutation status as a predictor of lack of benefit from cetuximab in metastatic colorectal cancer provides an important first step. However, KRAS mutation status does not appear to be predictive of cetuximab benefit in advanced non-small-cell lung cancer, illustrating the necessity for separate biomarker validation to occur across tumor types. Numerous candidate biomarkers have been suggested based on noncontrolled exploratory analyses, but they require validation in sufficiently sized controlled studies. Key pending issues include distinguishing markers predictive of treatment benefit from those prognostic of disease outcome, selecting the best specimen for analysis (determining the tissue type and collection site, as well as the sample matrix type); and optimizing and standardizing assay technology and scoring systems, particularly for markers expressed over a continuous dynamic range.

PURPOSE The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is efficacious in multiple tumor types. Patient selection with markers predictive of benefit may enhance its therapeutic index. This retrospective, correlative analysis of the phase III trial BMS099 of cetuximab in advanced non-small-cell lung cancer (NSCLC) was conducted to identify molecular markers for the selection of patients most likely to benefit from cetuximab. METHODS In BMS099, 676 chemotherapy-naïve patients with stage IIIB (pleural effusion) or stage IV NSCLC of any histology or EGFR expression status were randomly assigned to taxane/carboplatin (T/C) with or without cetuximab. Biomarkers analyzed included K-Ras and EGFR mutations by direct sequencing, EGFR protein expression by immunohistochemistry (IHC), and EGFR gene copy number by fluorescent in situ hybridization (FISH). Relationships between biomarker status and progression-free survival (PFS), overall survival (OS), and overall response rate (ORR) were assessed by log-rank tests per treatment arm for treatment-specific effects and across the total evaluable population. Results Tumor samples were available from 225 randomly assigned patients. K-Ras mutations were found in 17% of evaluable patients (35 of 202 patients), EGFR mutations were found in 10% (17 of 166 patients), EGFR positivity by IHC was found in 89% (131 of 148 patients), and FISH positivity was found in 52% (54 of 104 patients). No significant associations were found between biomarker status and PFS, OS, and ORR in the treatment-specific analyses. CONCLUSION In contrast with colorectal cancer, and within the limitations of the data set, efficacy parameters did not appear to correlate with K-Ras mutation status or with any of the EGFR-related biomarkers evaluated. Additional exploratory analyses are essential to identify predictive markers and to optimize patient selection for cetuximab therapy in NSCLC.

Purpose: This multicenter study evaluated the antitumor activity of cetuximab, an immunoglobulin G1 antibody directed at the epidermal growth factor receptor (EGFR), in metastatic colorectal carcinoma (CRC) refractory to irinotecan, oxaliplatin, and a fluoropyrimidine. It also evaluated the safety, pharmacokinetics, immunokinetics, and biologic determinants of activity., Patients and Methods: Patients with metastatic CRC, whose tumors demonstrated EGFR immunostaining and were refractory to irinotecan, oxaliplatin, and fluoropyrimidines, were treated with cetuximab at a loading dose of 400 mg/m2 followed by 250 mg/m2 weekly. An independent review committee (IRC) reviewed responses. Blood was collected for cetuximab pharmacokinetics and to detect antibodies to cetuximab. EGFR gene sequencing of the tyrosine kinase domain and gene copy number assessments were performed., Results: The response rates in 346 patients, as determined by the investigators and IRC, were 12.4% (95% CI, 9.1 to 16.4) and 11.6% (95% CI, 8.4 to 16.4). The median progression-free survival (PFS) and survival times were 1.4 months (95% CI, 1.4 to 2.1) and 6.6 months (95% CI, 5.6 to 7.6), respectively. An acneiform rash occurred in 82.9% of patients; grade 3 rash was observed in 4.9%. Response and survival related strongly to the severity of the rash. In contrast, clinical benefit did not relate to EGFR immunostaining. EGFR tyrosine kinase domain mutations were not identified, and EGFR gene copy number did not relate to response or PFS, but to survival (P = .03)., Conclusion: Cetuximab is active and well tolerated in metastatic CRC refractory to irinotecan, oxaliplatin, and fluoropyrimidines. The severity of rash was related to efficacy. Neither EGFR kinase domain mutations nor EGFR gene amplification appear to be essential for response to cetuximab in this setting.

Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences. We also observed enrichment for putative promoter regions of genes predicted in at least two annotation databases and for clones overlapping with CpG islands. Functional validation of randomly selected clones enriched by this method showed that a large fraction of these putative promoters can drive the expression of a reporter gene in transient transfection experiments. This method promises to be a useful genome-wide function-based approach that can complement existing methods to look for promoters.