FDA-approved Assay Research Articles

Profile of the MP Diagnostics HTLV Blot 2.4 test: a supplemental assay for the confirmation and differentiation of antibodies to HTLV-1 and HTLV-2

ABSTRACTAs the first US FDA-approved assay for supplemental HTLV testing, the MP Diagnostics HTLV Blot 2.4 is an effective and efficient method for confirming and differentiating HTLV type infection in repeatedly reactive samples. Novel and patented antigens added increased sensitivity in identifying specimens from infected individuals while differentiating those from uninfected individuals with false reactivity.

Challenges & Perspectives of Immunotherapy Biomarkers & The HistoOncoImmune™ Methodology

ABSTRACTThe confluence of the immunology and oncology tributaries has precipitated a biomarker turbulence that is novel to the cancer community. On the heels of reversing the dismal outcomes for numerous patients suffering from terminal disease, it has also left behind deep pools of biomarker questions – which ones, when to use them, how to use them and what cells express them – to name just a few. The tumor microenvironment, an arena potentially housing a complex mix of immune infiltrators in the midst of tumor cells, is a wildly heterogeneous terrain, one that severely challenges our single biomarker model. This article examines the recently FDA-approved biomarker assays for PD-1 therapeutics, considers the technical shortcomings of the single biomarker model, scans alternate biomarker technologies and proposes a model that may allow investigators a method to systematically address these complexities.

Chromogenic In Situ Hybridisation Test for Breast Cancer Patients with Equivocal IHC Results--a Study from Iran.

HER2/neu overexpression on cell membranes of breast cancer cells is due to HER2/neu gene amplification and it is important to identify potential candidates for anti HER2 therapy with trastuzumab. IHC, FISH and CISH are standard FDA approved assays currently used to determine HER2 status in routine practice. The aim of this study was to determine HER2 gene amplification, using the CISH method in breast carcinoma samples which had IHC +2 reactions. This study was conducted from 2008- 2010 using 334 consecutive breast carcinoma samples referred from local laboratories to Mehr Hospital. CISH assays were performed for all cases, and IHC tests were also done for determining efficacy and accuracy of local labs. HER2 status in local IHC tests was compared with central IHC and CISH results. Of 334 breast cancer patients, 16 were negative for HER2 IHC (0, +1), 201 cases were equivocal (+2), and 31 positive (+3). Of 334 referral cases, 88 were CISH positive (26.3%) and 246 were CISH negative (73.7%). Of 201 IHC +2 cases, HER2 gene amplification was observed in 42 cases (kappa: 0.42). A 29.9% concordance was found between local IHC and central IHC. Sensitivity and specificity of local IHC were 90% and 53.8%, respectively. Low accuracy of IHC results in local labs was associated with the following factors: using former FDA-approved criteria for HER2 interpretation, utilizing non-validated kits, and lack of any quality assurance program. Therefore, following the new 2014 ASCO/CAP guideline and comprehensive quality assurance should be implemented to ensure accuracy of HER2 testing.

Clinical Implications of Variant ALK FISH Rearrangement Patterns

Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small-cell lung cancer (NSCLC), identifying patients who can gain dramatic benefit from ALK kinase inhibitors. Assay interpretation can be technically challenging, and either splitting of the 5' and 3' probes or loss of the 5' probe constitute rearrangement. We hypothesized that there may be clinical differences depending on rearrangement pattern on FISH. An IRB-approved database of NSCLC patients at Dana-Farber Cancer Institute was queried for ALK rearrangement. Clinical characteristics and response to crizotinib were reviewed. Immunohistochemistry (IHC) and targeted next-generation sequencing (NGS) were obtained when available. Of 1614 NSCLC patients with ALK testing, 82 patients (5.1%) had ALK rearrangement by FISH: 30 patients with split signals, 25 patients with 5' deletion, and 27 patients with details unavailable. Patients with 5' deletion were older (p = 0.01) and tended to have more extensive smoking histories (p = 0.08). IHC was positive for ALK rearrangement in all 27 patients with FISH split signals, whereas three of 21 patients with FISH 5' deletion had negative IHC (p = 0.05). Targeted NGS on two of three cases with discordant FISH and IHC results did not identify ALK rearrangement, instead finding driver mutations in EGFR and KRAS. Patients with 5' deletion treated with crizotinib had a smaller magnitude of tumor response (p = 0.03). Patients with 5' deletion on ALK FISH harbor features less typical of ALK-rearranged tumors, potentially indicating that some cases with this variant are false positives. Corroborative testing with IHC or NGS may be beneficial.

Abstract 4250: 5′ deletion on ALK break-apart FISH and risk of false positive results

Abstract Purpose: Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC), identifying patients (pts) who may benefit from ALK tyrosine kinase inhibitors (TKIs). A case is considered positive for rearrangement either based on split 3′ and 5′ signals or loss of the 5′ signal (5′ deletion). We hypothesized that 5′ deletion on FISH may not be completely specific for presence of rearrangement, making the assay vulnerable to false positive results. Methods: An institutional IRB-approved database of NSCLC pts was queried for those with ALK-rearrangements. Clinical characteristics and response to ALK TKIs were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were obtained to further characterize ALK status. Results: Of 1614 NSCLC pts with ALK testing, 97 (6.0%) patients had abnormal ALK FISH: 30 with split signals, 24 with 5′ deletion, 3 with 3′ deletion, 1 with atypical rearrangement, and 39 with details unavailable. Pts with 5′ deletion were older (p = 0.01), had a more extensive smoking history (p = 0.03), and were more likely to harbor mutations in EGFR or KRAS (2 cases vs 0). Pts with 5′ deletion treated with ALK TKIs had a trend toward less tumor shrinkage (p = 0.07). ALK IHC and FISH were discordant in 3 of 21 pts with 5′ deletion and 0 of 26 pts with split signals (p = 0.03). NGS was concordant with ALK IHC, but not FISH, in 2 of 7 samples with 5′ deletions. All three testing modalities were concordant in 3 samples with split signals. Conclusions: NSCLC pts with 5′ deletion on ALK FISH have clinical and molecular features less typical of ALK-rearranged tumors, suggesting an unappreciated risk of false positives with this assay. While the majority of patients with 5′ deletion on ALK FISH harbor a true rearrangement and respond to ALK TKI, confirming these cases with IHC or NGS could reduce the false positive rate. Table 1.Clinicopathologic Features of NSCLC patients with positive ALK FISHSplit Signals (N = 30)5″ Deletion (N = 24)p-valueAge at Diagnosis - yearsp = 0.01MedianRange22-8228-76Age Distribution - No. (%)<40517%14%40-491033%28%50-59827%1042%60-69413%833%>70310%313%Race - No. (%)p = 0.03Caucasian2480%2188%Asian27%28%Other413%14%Smoking History - No. (%)p = 0.03Never Smoker1963%1042%Light Smoker (<10py)620%729%Heavy Smoker517%625%Presence of Co-MutationsEGFR00%14%KRAS00%14%None30100%2292%ALK Immunohistochemistryp = 0.03Positive26100%1886%Negative00%314%Next-Generation SequencingPositive3100%571%Negative00%229% Citation Format: Xin Gao, Lynette M. Sholl, Mizuki Nishino, Jennifer Heng, Pasi A. Janne, Geoffrey R. Oxnard. 5′ deletion on ALK break-apart FISH and risk of false positive results. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2015-4250

Detection of Acute HIV-1 Infection by RT-LAMP.

A rapid, cost-effective diagnostic test for the detection of acute HIV-1 infection is highly desired. Isothermal amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for the development of a rapid nucleic acid amplification test (NAAT) because they are quick, easy to perform and do not require complex, dedicated equipment and laboratory space. In this study, we assessed the ability of the HIV-1 RT-LAMP assay to detect acute HIV infection as compared to a representative rapid antibody test and several FDA-approved laboratory-based assays. The HIV-1 RT-LAMP assay detected seroconverting individuals one to three weeks earlier than a rapid HIV antibody test and up to two weeks earlier than a lab-based antigen/antibody (Ag/Ab) combo enzyme immunoassay (EIA). RT-LAMP was not as sensitive as a lab-based qualitative RNA assay, which could be attributed to the significantly smaller nucleic acid input volume. To our knowledge, this is the first demonstration of detecting acute HIV infection using the RT-LAMP assay. The availability of a rapid NAAT, such as the HIV-1 RT-LAMP assay, at the point of care (POC) or in laboratories that do not have access to large platform NAAT could increase the percentage of individuals who receive an acute HIV infection status or confirmation of their HIV status, while immediately linking them to counseling and medical care. In addition, early knowledge of HIV status could lead to reduced high-risk behavior at a time when individuals are at a higher risk for transmitting the virus.

P-A7 Comparison of HIV-1 Drug Resistance Intrepretations Software

Laboratories offering HIV-1 genotyping testing require reliable and up-to-date software and database solutions to optimally analyse Sanger sequences and generate clinical report drug resistance interpretations. In this study, we evaluated 2 downstream analysis software applications; the newly FDA-registered Data Processing Module (DPM v1.0) from ABL which connects to several external and validated contents, and the ViroScore-HIV [VS] software (for Research Use Only), which offers an end to end solution, from FASTA files to clinical reporting, including alignments and base-calling. Results (NRTI, NNRTI and PI drug resistance determinations) were compared with the respective determinations obtained from TG-HIV (GuideLines v.17.0). On a total of 51 samples, the agreement between the 2 methodologies (DPM and VS) was very good (>99.9%) but only 12% shown equivalent interpretation profiles between TruGene TG, with Geno2Pheno (G2P) and Stanford HIVdb (SD). Drug resistance interpretations comparisons (excluding AZT, DDI, D4T and RPV) performed between TG and SD; TG and G2P; SD and G2P gave a median agreement of 92.2, 82.9, 81% respectively with 93.4; 79.6; 85.3% for the PIs, 87.5; 81.9; 80.7% for the NRTIs and 90.1; 82.9 and 79.5% for the NNRTIs. Compared to TruGene, 56.9%; 56.9% of the samples showed at least 1 moderate positive drug resistance switch (Sensitive [S] to Intermediate [I] or I to Resistant [R]), 5.9%; 7.8% a high positive switch (S to R), 33.3%; 43.1% a moderate negative switch (I to S; R to I) and 9.8%; 29.4% a high negative switch (R to S) for SD and G2P respectively suing both platforms. This study demonstrated usability and robustness of 2 HIV genotyping software applications for clinical and R/D labs involved in HIV-1 genotyping. Differences of drug resistance interpretations were due to lack up-to-date guidelines in TruGene software, a common issue present in FDA approved assay. DPM software could be easily applied as a valuable interpretation tool helping physician to select the best therapy for their patients.

Abstract 4675: Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis

Abstract BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

Abstract 3278: The effect of cold ischemia time on protein expression in breast cancer tissues

Abstract Background: HER2 testing is a critical tool for determining the molecular subtypes of breast tumors. However there are various factors which may influence the accuracy of HER2 immunohistochemistry (IHC) results. Those factors have been addressed by the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) and outlined in the updated 2013 guideline. The ASCO/CAP guidelines advise limiting the tissues cold ischemia time as much as possible specifically to no more than one hour. A tissue's cold ischemia time is defined as the time from the tissues removal from the patient to the initiation of fixation. Our institution works with several collaborators for whom postoperative tissue banking is performed for the majority of breast resection specimens. In most cases, the procedure of tissue excision takes 30-60 minutes. However, in some cases cold ischemia time is increased more than 60 minutes. Presently, there is limited information available on the effect of cold ischemia on HER2 IHC staining. In this study, we compared HER2 IHC results for breast tumor specimens to determine the effect of prolonged cold ischemia time on protein expression. HER2 status was confirmed by the in-situ hybridization method (FISH). Design: In this study we evaluated HER2 status using two different FDA-approved FISH and an FDA-approved IHC assay in a large cohort of invasive breast carcinomas with differing cold ischemia time in pathology laboratories at the University of Chicago (U of C) and in Ibadan, Nigeria (IMRAT). Cold ischemia time was used to stratify the samples into two groups 1-3 h (n=27 U of C cases) and >3h (n=79 MRAT cases). Both FISH and IHC were evaluated using ASCO/CAP scoring criteria. Tissue microarrays (TMAs) including duplicate 1.0-mm cores of invasive breast carcinoma were constructed utilizing formalin-fixed, paraffin-embedded tissue of 106 breast cancer patients, who had surgery at the U of C and IMRAT. HER2 status determination was performed on the TMAs utilizing IHC with the HercepTest (DAKO, USA), and FDA-approved FISH assay (PathVysions, Abbott Molecular, USA). Results: Only 3.7% of the cases examined in the 1-3 h cold ischemia time group had a discrepancy in the HER2 IHC and FISH result. However, within the >3 h cold ischemia time cohort a total of 7.6 % were identified with a minor discrepancy between the HER2 IHC and FISH results. Conclusions: In this study, we compared the effect of cold ischemia time on standard HER2 testing methodologies including FISH and IHC. Comparison of FISH and IHC showed that IHC did not identify several FISH HER2-amplified cases at >3h cold ischemia times. These findings are important in consideration of specimen preparation and could have an effect on specimen handling and fixation guidelines. Citation Format: Galina F. Khramtsova, Andrey I. Khramtsov, Lise Sveen, Abayomi Odetunde, Oyinlolu Olorunsogo Adeyanju, Olufunmilayo I. Olopade, Oluwasola A. Olayiwola. The effect of cold ischemia time on protein expression in breast cancer tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3278. doi:10.1158/1538-7445.AM2014-3278

BNP and NT-proBNP as prognostic markers in persons with acute decompensated heart failure: a systematic review.

A systematic review was undertaken to examine the evidence for B-type natriuretic peptides (BNP and NT-proBNP) as independent predictors of mortality, morbidity, or combined mortality and morbidity outcomes in persons with acute decompensated heart failure (ADHF). Electronic databases (Medline(®), Embase™, AMED, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and CINAHL) were searched from 1989 to June 2012. Reference lists of included articles, systematic reviews, and the gray literature were also searched. English language studies were eligible if they included subjects with ADHF and measured BNP/NT-proBNP using FDA approved assays. Standardized forms were used to select studies, extract data, and assess risk of bias. Seventy-nine studies, ranging over followup intervals from 14days to 7years, evaluating levels of BNP (n=38), NT-proBNP (n=35), or both (n=6) were eligible. The majority of studies predicted mortality outcomes for admission BNP/NT-proBNP levels, with fewer studies evaluating serial, change from admission, or discharge levels. In general, higher levels of admission BNP or NT-proBNP predicted greater risk for all outcomes. Decreased levels post-admission predicted decreased risk. Overall, these studies were rated as having moderate risk of bias. This systematic review shows that BNP and NT-proBNP are independent predictors of mortality (all-cause and cardiovascular) in ADHF despite different cutpoints, time intervals, and prognostic models. Findings for morbidity and composite outcomes were less frequently evaluated and showed inconsistency. Further research is required to assess cutpoints for admission, serial measurements, change following admission, and discharge levels to assist clinical decision-making.

Prediction of clinical outcomes using B-type natriuretic peptides in the general population: a systematic review.

The use of B-type natriuretic peptides to predict outcomes in general populations has been investigated in a number of primary studies. A previous systematic review considering natriuretic peptides in cardiovascular disease included a subgroup of general population studies, which suggested an association with a number of clinical outcomes. We electronically searched Medline, Embase, AMED, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and CINAHL for English-language articles published between 1989 and mid-2012. We utilized trained reviewers and standardized forms to screen articles for inclusion and extract data from included articles. All included studies (n=7) were summarized in narrative and tabular form. A general population was defined as one that was randomly selected from a community setting where no specific inclusion or exclusion criteria were specified. The seven included studies all used FDA approved assays for NT-proBNP. The range of clinical outcomes and heterogeneity did not allow for meta-analysis. The hazard ratios for predicting outcomes in the included studies ranged from 1.0 to 4.1 (all p values <0.05). The discrimination statistics reported in four studies all demonstrated statistically significant improvements in predicting outcomes. NT-proBNP is associated with heart failure, all-cause and cardiovascular mortality, and other combined cardiovascular events in a general unselected population. The discrimination statistics suggest modest improvements in risk stratification. No prospective studies exist to demonstrate the clinical utility of using B-type natriuretic peptides to predict clinical outcomes in a general population.

Use of BNP and NT-proBNP for the diagnosis of heart failure in the emergency department: a systematic review of the evidence.

Our purpose was to determine the test performance characteristics of BNP and NT-proBNP in the diagnosis of heart failure for patients presenting to an emergency department or urgent care center. We searched Medline, Embase, AMED, Cochrane, Cochrane Database of Systematic Reviews, and CINAHL for English-language articles published between 1989 and June 2012. Studies were limited to those using FDA-approved assays. We examined test performance at three pre-specified cutpoints (manufacturers' suggested, researchers' optimal, and lowest) and considered the effect of age, gender, ethnicity and renal function. We used the QUADAS-2 tool to examine risk of bias and applicability, and the AHRQ Methods Guide to assess the strength of evidence. Seventy-six articles met our inclusion criteria, 37 examined BNP, 25 examined NT-proBNP, and 14 examined both. Pooled sensitivity and specificity for BNP at the three pre-specified cutpoints were 95, 91, and 95% (sensitivity) and 55, 80, and 67% (specificity), respectively. For NT-proBNP, sensitivity and specificity at the same cutpoints were 91, 90, and 96% (sensitivity) and 67, 74, and 55% (specificity). Both BNP and NT-proBNP perform well to rule out, but less well to rule in, the diagnosis of heart failure among persons presenting to emergency departments or urgent care centers. Both BNP and NT-proBNP levels are positively associated with age and negatively associated with renal function. However, the effect of these factors with respect to selecting optimal cutpoints is unclear. For BNP, 100pg/mL appears to be a consensus cutpoint. No clear consensus has emerged for NT-proBNP, but the age-adjusted cutpoints of 450pg/mL for <50years, 900pg/mL for 50-75years and 1,800pg/mL for >75years appear promising and merit greater scrutiny and validation.

Creating a Molecular Atmosphere, Phase 5

As mentioned in previous articles in this series, product inserts are available with most FDA-approved assays. These product inserts contain information about the product as well as explain processes and procedures approved by the manufacturer of the assay. Included in these procedures are quality control (QC) recommendations. Some FDA-approved assays have QC material included in the packaging of test materials; others suggest from whom to purchase the quality control material and provide recommendations on how to use it. For all of the assays that we perform on the Infinity (Thermo Fisher Scientific, Waltham, MA) instrument, we routinely perform external QC on new lots and new shipments. All of these assays are FDA-approved, qualitative, and include a built-in internal control for every patient test that is performed. Because the internal control is present, we are not required to perform external QC procedures with every patient specimen. The external controls used for these assays are purchased from a manufacturer and can be used directly or after a simple dilution is made. The assays we perform on the SmartCycler (Cepheid, Sunnyvale, CA) are laboratory developed; therefore, control materials are not commercially available in ready-to-use form. Clinical Laboratory Standards Institute (CLSI) and College of American Pathologist (CAP) guidelines suggest that laboratorians ensure that a control is present throughout the entirety of a patient testing procedure. If an internal control cannot be present from start to finish, external-control material must be included in every patient-testing procedure. For herpes simplex virus (HSV) external control, we purchase stock virus from a manufacturer. We grow the virus in shell vials, in a similar manner that we would use for controls when setting up HSV cultures. This growth of virus in shell vials takes anywhere from 3 to 5 days to yield a 90% or greater cytopathic effect (CPE). … [↵][1]* To whom correspondence should be addressed. E-mail: stephanie.e.wallace{at}osfhealthcare.org [1]: #xref-corresp-1-1

Mycoplasma genitalium – The missing pathogen?

M. genitalium has become an established cause of sexually transmitted urethritis and cervicitis, and there is increasing evidence that it may cause upper genital tract disease in women. Detection by nucleic acid amplification tests is currently the only diagnostic method available, but no FDA approved assays are currently available, and the CE marked tests suffer from limited clinical evaluation. In most settings, M. genitalium infections explain 15-25% of symptomatic non-gonococcal urethritis, but as diagnosis of the infection is not routinely carried out, treatment is usually syndromic. A few randomized trials have evaluated treatment of M. genitalium, and have compared only doxycycline 200 mg daily for 7 days with a 1 g single dose of azithromycin. Together with results from open trials, it is obvious that doxycycline is inefficient in eradicating M. genitalium, showing eradication rates around 35%. The eradication rate after azithromycin 1 g single dose is significantly better, but differs greatly between studies. Thus, older studies appear to have higher eradication rates than the most recent one, where no significant difference between azithromycin and doxycycline could be demonstrated. Furthermore, a lower eradication rate is reported in studies from centres where azithromycin has been used as the primary treatment for chlamydial and idiopathic urethritis and cervicitis, suggesting that selection for macrolide resistance may be rapid and widespread. At present, the only second line antibiotic that has been shown to have a high activity against macrolide resistant M. genitalium is moxifloxacin. However, this drug is significantly more expensive and has a less favourable safety profile than macrolides, and multidrug resistant infections have emerged, primarily in patients with contact to South East Asia. Consequently, there is an urgent need for clinical trials with possible alternative drugs. Such trials should preferably also address the treatment efficacy in chlamydial and idiopathic urethritis and cervicitis as a single treatment covering these conditions would be advantageous.

Will the Requirement by the US FDA to Simultaneously Co-Develop Companion Diagnostics (CDx) Delay the Approval of Receptor Tyrosine Kinase Inhibitors for RTK-Rearranged (ROS1-, RET-, AXL-, PDGFR-α-, NTRK1-) Non-Small Cell Lung Cancer Globally?

The discovery of anaplastic lymphoma kinase (ALK) rearrangement in non-small cell lung cancer (NSCLC) in 2007 and the approval of crizotinib for the treatment of advanced ALK-rearranged NSCLC in 2011 represents a landmark in the development of targeted oncology therapy. The approval of crizotinib was accompanied simultaneously by the approval of the Vysis (Abbott Molecular) break-apart fluorescence in situ hybridization (FISH) test as the companion diagnostic (CDx) test to detect ALK rearrangement. Pfizer, the manufacturer of crizotinib, sponsored the screening of thousands of patients and the standardization of the ALK FISH test as part of the approval process for crizotinib, a first in class ALK inhibitor. Many pharmaceutical companies are now using the Food and Drug Administration (FDA)-approved ALK FISH assay to enroll patients onto trials for their own respective ALK inhibitors. In essence they are “piggybacking” on the FDA-approved ALK FISH assay without having to pay for the development of a CDx, nor screening for ALK-rearranged NSCLC patients in the protocols because screening for ALK rearrangement is now the standard of care in NSCLC after the approval of crizotinib. Since 2007, rearrangement in more receptor tyrosine kinases (RTKs) such as ROS1, RET, AXL, PDGFR-α, and NTRK1 have been discovered in NSCLC but the incidence of each subtype of RTK-rearranged NSCLC is quite rare. Crizotinib has now demonstrated significant clinical activity in ROS1-rearranged NSCLC patients. Whether crizotinib will gain official FDA approval for use in ROS1-rearranged NSCLC, on the other hand, remains unclear as there is no test for ROS1-rearrangement currently being developed to support US FDA approval as a CDx. This may be due in part to the fact that the full cost associated with the development of a pre-market approved-approved CDx must be borne by the company seeking the first drug approval in a new indication. Given the low incidence of ROS1-rearrangement in NSCLC, and the availability of crizotinib in most countries, a more cost-effective way is for crizotinib to gain compendium listing for ROS1-rearranged NSCLC in treatment guidelines. However, without a formal indication from the FDA, a drug cannot be marketed for off label use, it is unlikely that payers public or private will routinely pay for molecular testing for ROS1-rearrangement in NSCLC let alone reimburse off label use of crizotinib. Similarly, several marketed tyrosine kinase inhibitors (TKIs) in the US (sorafenib, sunitinib, vandetanib, cabozantinib, regorafenib) are potent RET inhibitors in vitro. It does not make sense for any one pharmaceutical company to shoulder the full cost of developing a particular CDx for RET-rearranged NSCLC where, once approved, it may be used by other pharmaceutical companies to gain addition labeling approval for their own RET inhibitors. Thus, the requirement by the US FDA that a specific CDx have to be co-developed and standardized for each of the molecular subtype of NSCLC as part of the drug approval process, while prudent, may have the un-intended consequence of deterring clinical development of these TKIs in these very rare molecular subsets of NSCLC. While we all march to the drumbeat of precision cancer medicine, the stringent requirement of co-development CDx for each molecular subtype of solid tumor may inadvertently make this goal substantially more difficult to achieve.

Cobas® 4800 HPV Test, a real-time polymerase chain reaction assay for the detection of human papillomavirus in cervical specimens

Cervical cancer screening incorporating high-risk human papillomavirus (HPV) detection has become the preferred screening strategy in some countries and is increasingly more widespread in other countries with organized or opportunistic screening programs. Given knowledge that high-risk HPV genotypes differ in their oncogenic potential, commercial HPV assays with genotyping capabilities have been developed and have garnered attention in the recent literature. The cobas® 4800 HPV Test is a qualitative multiplex assay that provides specific genotyping information for HPV types 16 and 18, while concurrently detecting 12 other high-risk HPV genotypes as a pooled result. It is currently the only clinically validated, US FDA-approved assay with this capability. Since HPV types 16 and 18 have been designated as conferring the greatest risk for cervical disease, their detection may prove useful in guiding patient management.

A novel approach for rapid detection of bacterially contaminated platelet concentrates via sensitive measurement of microbial DNA polymerase activity

Transfusion of bacterially contaminated platelet concentrates (PCs) can result in serious health consequences for the affected patient. Before being released from blood banking facilities, PCs are routinely screened for bacterial contamination by culture-based tests. However, culture-based PC screening methods require extended holding and incubation periods and are prone to false-negative results due to sampling error. Screening PCs closer to the time of transfusion using rapid point-of-issue tests represents an alternative approach; however, FDA-approved assays generally suffer from a lack of sensitivity. Presented herein is the feasibility of a novel approach toward rapid, sensitive, and universal detection of bacterially contaminated PCs via selective measurement of microbial DNA polymerase activity. This approach is achieved using a differential cell lysis procedure in combination with enzymatic template generation and amplification (termed ETGA-PC assay). Serial dilution spiking experiments revealed an approximate sensitivity of 30 to 200 colony-forming units (CFUs)/mL (mean, 85 CFUs/mL) for Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. An additional 22 clinically relevant strains of bacteria were also detected below 200 CFUs/mL after spiking into PC aliquots. Furthermore, the ETGA-PC assay was able to accurately monitor the presence and growth of seven clinically relevant bacterial species that were spiked into PC units. Together, the data presented here demonstrate that the ETGA-PC assay is a feasible approach for rapid and sensitive detection of bacterially contaminated PCs. Experiments, aimed at simplification and/or automation of the assay procedure, are under way.

The Aptima HPV Assay Fulfills the Cross-Sectional Clinical and Reproducibility Criteria of International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening

The Aptima HPV assay (Hologic Gen-Probe, San Diego, CA) is an FDA-approved assay for detecting human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types. This study evaluated the clinical performance of the Aptima HPV assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+), relative to the high-risk HPV GP5+/GP6+ PCR, in a cross-sectional clinical equivalence analysis using the noninferiority score test with cervical samples from population-based screening, i.e., 69 cervical scraping samples from women with CIN2+ and 843 from women without evidence of CIN2+. In addition, intralaboratory reproducibility over time and interlaboratory agreement of the Aptima HPV assay results were assessed with another set of 548 cervical samples. The Aptima HPV assay showed a clinical sensitivity for CIN2+ of 94.2% (95% confidence interval [CI], 85.5 to 97.8%) and a clinical specificity for CIN2+ of 94.5% (95% CI, 92.8 to 95.9%); by comparison, these figures were 97.1% (95% CI, 89.1 to 99.3%) (67/69 samples) and 93.6% (95% CI, 91.7 to 95.0%) (785/839 samples), respectively, for GP5+/GP6+ PCR. The clinical sensitivity and specificity of the Aptima HPV assay were noninferior to those of GP5+/GP6+ PCR (P = 0.039 and 0.00016, respectively). In addition, high reproducibility of the Aptima HPV assay, as reflected by the intralaboratory reproducibility over time of 96.0% (95% CI, 94.4 to 97.3%) (526/548 samples; kappa = 0.89) and interlaboratory agreement of 96.7% (95% CI, 95.4 to 98.1%) (531/548 samples; kappa = 0.91), was found. Altogether, these data show that the Aptima HPV assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements for cervical screening. Longitudinal data are needed to ensure that the long-term negative predictive value of this mRNA assay is similar to those of validated HPV DNA tests.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Comparison of the FDA-Approved CDC DENV-1-4 Real-Time Reverse Transcription-PCR with a Laboratory-Developed Assay for Dengue Virus Detection and Serotyping

Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.