Droplet digital polymerase chain reaction offers an improvisation over conventional immunohistochemistry and fluorescent in situ hybridization for ascertaining Her2 status of breast cancer.

BACKGROUND: Droplet digital polymerase chain reaction (DDPCR) is a recent modality for detecting Her2 expression which is quantitative, cheaper, easier to standardize, and free from interobserver variation. PURPOSE: The purpose of this study is to incorporate DDPCR in the current diagnostic paradigm with clinical benefit. MATERIALS AND METHODS: Fifty-four consecutive patients were tested by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and DDPCR. With FISH result as gold standard, receiver operating characteristic curves for DDPCR ratio were analyzed to label Her2-negative, equivocal, and positive cases as DDPCR score 1, 2, and 3, respectively. Proportion of patients labeled unequivocally as Her2 positive or negative was defined to have "clinically benefitted" from the test. Drawing parallel to inter-relationships between DDPCR, IHC, and FISH in the test cohort, four diagnostic pathways were defined - (1) initial IHC followed by FISH, (2) initial DDPCR followed by FISH, (3) initial IHC followed by DDPCR followed by FISH, and (4) initial DDPCR followed by IHC followed by FISH. RESULTS: Clinical benefit of DDPCR as an initial test in the test cohort was 57%, while it was 65% if used as a second-line test among those with an initial inconclusive IHC result. Sensitivity analysis in the simulation cohort revealed that if DDPCR cost was ≤0.6 times the cost of IHC, then a three-step pathway with DDPCR upfront would near certainly prove most cost beneficial. If DDPCR cost was >0.6 but ≤2 times the cost of IHC, then a three-step pathway with DDPCR as second-line test had a higher probability to prove most cost beneficial. If DDPCR cost was >2 times the cost of IHC, then conventional pathway had a higher probability to prove most cost-effective. CONCLUSION: Incorporating DDPCR in the current clinical diagnostic paradigm has the potential to improve its cost-effectiveness and benefit.

The detection of primary and secondary EGFR mutations using droplet digital PCR in patients with nonsmall cell lung cancer.

BACKGROUND: We share our experience of using droplet digital polymerase chain reaction (DdPCR) in liquid biopsy specimens for detecting primary and secondary epidermal growth factor receptor (EGFR) mutations among patients with nonsmall-cell lung cancer who had tissue biopsy initially analyzed for del19, L858R and T790M. MATERIALS AND METHODS: Three groups of patients were chosen: Group 1: patients positive for EGFR mutation (del 19 or L858R) by conventional tissue biopsy that were treatment naïve, Group 2: patients positive for EGFR mutation (del 19 or L858R) by conventional tissue biopsy with acquired resistance to tyrosine kinase inhibitor (TKI) therapy, documented by radiology, and Group 3: no known EGFR mutation detected on primary tissue biopsy and treatment naive. RESULTS: One hundred and thirty-three patients were included in the study. Group 1 had 40 cases, of which 21 (52.5%) and 19 (47.5%) were positive for del19 and L858R mutations, respectively, by tissue biopsy. DdPCR detected primary mutation in all but 5 cases. DdPCR additionally found four patients to have T790M mutation. Group 2 had 73 cases and DdPCR detected T790M mutation in 39 (53.4%) cases. Liquid biopsy also picked the original primary mutation in 56/73 cases. Secondary tissue biopsy for T790M mutation status was performed in 11 patients and while it detected mutation in 2 out of 11 cases, DdPCR detected the same in 7 cases, thus providing significantly superior yield (46% difference, McNemar's test, P value 0.063). Tissue biopsy additionally detected c-MET amplification in a patient who had T790M mutation on liquid biopsy. Group 3 had 20 patients and none were falsely positive for EGFR mutation on liquid biopsy. Overall, DdPCR had a Cohen's kappa of 0.82 (standard error 0.074, 95% CI 0.68-0.97) indicating "very good agreement" with conventional tissue biopsy. CONCLUSION: DdPCR demonstrated 87.5% sensitivity and 100% specificity in detecting primary EGFR mutations in patients who were treatment naïve with overall positive and negative predictive value of 100% and 80%, respectively. DdPCR demonstrated T790M mutation postprogression on TKI therapy in 53.4% patients.

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

BACKGROUND: IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. METHODS: 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. FINDINGS: 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost-benefit provided the cost of RT-PCR was 0.1 times the cost of IHC. CONCLUSION: RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.

ROS1 rearrangement and response to crizotinib in Stage IV non-small cell lung cancer.

BACKGROUND: The frequency of ROS1 rearrangement in non-small cell lung cancers has been reported from 1.6% to 2.3%. MATERIALS AND METHODS: We examined 105 lung adenocarcinoma patients for ROS1 rearrangement which were negative for EGFR and anaplastic lymphoma kinase. Clinical characteristics of ROS1 rearranged patients and their responses to crizotinib therapy were studied. RESULTS: Of the 105 patients, three cases were positive for ROS1 rearrangement by fluorescence in situ hybridization analysis. All of them showed heterogeneous pattern. All the 3 ROS1-positive patients were females in their forties and started on crizotinib. All of them responded to treatment. One of them developed resistance after 3 months. Another one showed marked systemic response but central nervous system lesions progressed. The third case is doing well till date with inactive lesions on positron emission tomography scan. CONCLUSIONS: The frequency of ROS1 rearrangement is low in non-small cell lung carcinoma, but their diagnosis offers patients an opportunity to receive highly effective targeted therapies.