The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvß6-integrin targeted radiotherapeutics.

In a recent paper in this journal (RSC Med. Chem., 2023, 14, 2429), we described an unusually strong impact of regiospecific exchange of phenylalanines by tyrosines in 10 gallium-68-labeled trimers of certain cyclic RGD peptides, c[XRGDLAXp(NMe)K] (X = F or Y), on non-specific organ uptakes. We found that there was, in part, no correlation of liver uptake with established polarity proxies, such as the octanol-water distribution coefficient (log D). Since this observation could not be explained straightforwardly, we suggested that the symmetry of the compounds had resulted in a synergistic interaction of certain components of the macromolecules. In the present work, we investigated whether a comparable effect also occurred for a series of 5 tetramers labeled with lutetium-177. We found that in contrast to the trimers, liver uptake of the tetramers was well correlated to their polarity, indicating that the unusual observations along the trimer series indeed was a unique feature, probably related to their particular symmetry. Since the Lu-177 labeled tetramers are also potential agents for treatment of a variety of αvß6-integrin expressing cancers, these were evaluated in mice bearing human lung adenocarcinoma xenografts. Due to their tumor-specific uptake and retention in biodistribution and SPECT imaging experiments, these compounds are considered a step forward on the way to αvß6-integrin-targeted anticancer agents. Furthermore, we noticed that the presence of tyrosines in general had a positive impact on the in vivo performance of our peptide multimers. In view of the fact that a corresponding rule was already proposed in the context of protein engineering, we argue in favor of considering peptide multimers as a special class of small or medium-sized proteins. In summary, we contend that the performance of peptide multimers is less determined by the in vitro characteristics (particularly, affinity and selectivity) of monomers, but rather by the peptides' suitability for the overall macromolecular design concept, and peptides containing tyrosines are preferred.

Sensitive Positron Emission Tomography Imaging of PD-L1 Expression in Human Breast and Lung Carcinoma Xenografts Using the Radiometalated Peptide Ga-68-TRAP-WL12.

Noninvasive imaging of the immune checkpoint protein programmed death ligand 1 (PD-L1; synonyms: CD274, B7-H1) holds great promise to improve patient selection and, thus, response rates for immune checkpoint therapy (ICT) with monoclonal antibodies targeting the PD1/PD-L1 axis. The PD-L1 specific peptide WL12 (cyclo(AcY-(NMe)A-N-P-H-L-Hyp-W-S-W(Me)-(NMe)Nle-(NMe)Nle-O-C)-G-NH2) was functionalized with the Gallium-68 chelator TRAP by means of click chemistry (CuAAC). The resulting conjugate TRAP-WL12 was labeled with Gallium-68 within 16 min, with approximately 90% radiochemical yield and 99% radiochemical purity, affording Ga-68-TRAP-WL12 with molar activities typically exceeding 100 MBq/nmol. This radiotracer was characterized by positron emission tomography (PET) imaging and ex vivo biodistribution in murine xenografts of nontransfected PD-L1 expressing tumor cell lines, MDA-MB-231 (human breast carcinoma), and H2009 (human lung adenocarcinoma). It showed a favorable biodistribution profile with rapid renal clearance and low background (tumor-to-blood ratio = 26.6, 3 h p.i.). Conjugation of the Ga-68-TRAP moiety to WL12 successfully mitigated the nonspecific uptake of this peptide in organs, particularly the liver. This was demonstrated by comparing Ga-68-TRAP-WL12 with the archetypical Ga-68-DOTA-WL12, for which tumor-to-liver ratios of 1.4 and 0.5, respectively, were found. Although immunohistochemistry (IHC) revealed a low PD-L1 expression in MDA-MB-213 and H2009 xenografts that corresponds well to the clinical situation, PET showed high tumor uptakes (6.6 and 7.3% injected activity per gram of tissue (iA/g), respectively) for Ga-68-TRAP-WL12. Thus, this tracer has the potential for routine clinical PD-L1 PET imaging because it detects even very low PD-L1 expression densities with high sensitivity and may open an avenue to replace PD-L1 IHC of biopsies as the standard means to select potential responders for ICT.

Complexity of αvß6-integrin targeting RGD peptide trimers: emergence of non-specific binding by synergistic interaction.

Multimerization is an established strategy to design bioactive macromolecules with enhanced avidity, which has been widely employed to increase the target-specific binding and uptake of imaging probes and pharmaceuticals. However, the factors governing the general biodistribution of multimeric probes are less well understood but are nonetheless decisive for their clinical application. We found that regiospecific exchange of phenylalanine by tyrosine (chemically equivalent to addition of single oxygen atoms) can have an unexpected, dramatic impact on the in vivo behavior of gallium-68 labeled αvß6-integrin binding peptides trimers. For example, introduction of one and two Tyr, equivalent to just 1 and 2 additional oxygens and molecular weight increases of 0.38% and 0.76% for our >4 kDa constructs, reduced non-specific liver uptake by 50% and 72%, respectively. The observed effect did not correlate to established polarity measures such as log D, and generally defies explanation by reductionist approaches. We conclude that multimers should be viewed not just as molecular combinations of peptides whose properties simply add up, but as whole entities with higher intrinsic complexity and thus a strong tendency to exhibit newly emerged properties that, on principle, cannot be predicted from the characteristics of the monomers used.

PET/CT imaging of head-and-neck and pancreatic cancer in humans by targeting the "Cancer Integrin" αvß6 with Ga-68-Trivehexin.

PURPOSE: To develop a new probe for the αvß6-integrin and assess its potential for PET imaging of carcinomas. METHODS: Ga-68-Trivehexin was synthesized by trimerization of the optimized αvß6-integrin selective cyclic nonapeptide Tyr2 (sequence: c[YRGDLAYp(NMe)K]) on the TRAP chelator core, followed by automated labeling with Ga-68. The tracer was characterized by ELISA for activities towards integrin subtypes αvß6, αvß8, αvß3, and α5ß1, as well as by cell binding assays on H2009 (αvß6-positive) and MDA-MB-231 (αvß6-negative) cells. SCID-mice bearing subcutaneous xenografts of the same cell lines were used for dynamic (90 min) and static (75 min p.i.) µPET imaging, as well as for biodistribution (90 min p.i.). Structure-activity-relationships were established by comparison with the predecessor compound Ga-68-TRAP(AvB6)3. Ga-68-Trivehexin was tested for in-human PET/CT imaging of HNSCC, parotideal adenocarcinoma, and metastatic PDAC. RESULTS: Ga-68-Trivehexin showed a high αvß6-integrin affinity (IC50 = 0.047 nM), selectivity over other subtypes (IC50-based factors: αvß8, 131; αvß3, 57; α5ß1, 468), blockable uptake in H2009 cells, and negligible uptake in MDA-MB-231 cells. Biodistribution and preclinical PET imaging confirmed a high target-specific uptake in tumor and a low non-specific uptake in other organs and tissues except the excretory organs (kidneys and urinary bladder). Preclinical PET corresponded well to in-human results, showing high and persistent uptake in metastatic PDAC and HNSCC (SUVmax = 10-13) as well as in kidneys/urine. Ga-68-Trivehexin enabled PET/CT imaging of small PDAC metastases and showed high uptake in HNSCC but not in tumor-associated inflammation. CONCLUSIONS: Ga-68-Trivehexin is a valuable probe for imaging of αvß6-integrin expression in human cancers.

There is a world beyond αvß3-integrin: Multimeric ligands for imaging of the integrin subtypes αvß6, αvß8, αvß3, and α5ß1 by positron emission tomography.

BACKGROUND: In the context of nuclear medicine and theranostics, integrin-related research and development was, for most of the time, focused predominantly on 'RGD peptides' and the subtype αvß3-integrin. However, there are no less than 24 known integrins, and peptides without the RGD sequence as well as non-peptidic ligands play an equally important role as selective integrin ligands. On the other hand, multimerization is a well-established method to increase the avidity of binding structures, but multimeric radiopharmaceuticals have not made their way into clinics yet. In this review, we describe how these aspects have been interwoven in the framework of the German Research Foundation's multi-group interdisciplinary funding scheme CRC 824, yielding a series of potent PET imaging agents for selective imaging of various integrin subtypes. RESULTS: The gallium-68 chelator TRAP was utilized to elaborate symmetrical trimers of various peptidic and non-peptidic integrin ligands. Preclinical data suggested a high potential of the resulting Ga-68-tracers for PET-imaging of the integrins α5ß1, αvß8, αvß6, and αvß3. For the first three, we provide some additional immunohistochemistry data in human cancers, which suggest several future clinical applications. Finally, application of αvß3- and αvß6-integrin tracers in pancreatic carcinoma patients revealed that unlike αvß3-targeted PET, αvß6-integrin PET is not characterized by off-target uptake and thus, enables a substantially improved imaging of this type of cancer. CONCLUSIONS: Novel radiopharmaceuticals targeting a number of different integrins, above all, αvß6, have proven their clinical potential and will play an increasingly important role in future theranostics.

Longitudinal follow-up and performance validation of an mRNA-based urine test (Xpert® Bladder Cancer Monitor ) for surveillance in patients with non-muscle-invasive bladder cancer.

OBJECTIVE: To evaluate the performance of the Xpert Bladder Cancer Monitor (Xpert; Cepheid, Sunnyvale, CA, USA) test as a predictor of tumour recurrence in patients with non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patients (n = 429) undergoing surveillance for NMIBC underwent Xpert, cytology, and UroVysion testing. Patients with a positive Xpert and a negative cystoscopy result (positive-negative [PN] group, n = 66) and a control group of double negative patients (negative Xpert and cystoscopy results [NN] group) were followed for 12 months (±90 days). RESULTS: Histology-confirmed recurrences were detected in 58 patients (13.5%). Xpert had an overall sensitivity of 60.3% and a specificity of 76.5%. The sensitivity for high-grade (HG) cancer was 87% with a negative predictive value (NPV) of 99%. Urine cytology showed an overall sensitivity of 23.2% (47.6% sensitivity for HG tumours) and a specificity of 88.3%. In the PN group, 32% (n = 21) developed a recurrence within 12 months, 11 of which were HG tumours. In the NN control group, 14% (n = 9) developed a recurrence and only two were HG tumours. The hazard ratio for developing recurrence in the PN group was 2.68 for all tumours and 6.84 for HG cancer. CONCLUSIONS: The Xpert test has a high sensitivity for detecting the recurrence of cancer and a high NPV for excluding HG cancer. In addition, the data suggest that patients with a positive Xpert assay in the setting of negative cystoscopy are at high risk for recurrence and need close surveillance.

Tracking a TGF-ß activator in vivo: sensitive PET imaging of αvß8-integrin with the Ga-68-labeled cyclic RGD octapeptide trimer Ga-68-Triveoctin.

PURPOSE: As a major activator of transforming growth factor ß (TGF-ß), the RGD receptor αvß8-integrin is involved in pathogenic processes related to TGF-ß dysregulation, such as tumor growth, invasion, and radiochemoresistance, metastasis and tumor cell stemness, as well as epithelial-mesenchymal transition. The novel positron emission tomography (PET) radiopharmaceutical Ga-68-Triveoctin for in vivo mapping of αvß8-integrin expression might enhance the prognosis of certain tumor entities, as well as support and augment TGF-ß-targeted therapeutic approaches. METHODS: Monomeric and trimeric conjugates of cyclo(GLRGDLp(NMe)K(pent-4-ynoic amide)) were synthesized by click chemistry (CuAAC), labeled with Ga-68, and evaluated in MeWo (human melanoma) xenografted SCID mice by means of PET and ex-vivo biodistribution. αvß8-integrin expression in murine tissues was determined by ß8-IHC. A human subject received a single injection of 173 MBq of Ga-68-Triveoctin and underwent 3 subsequent PET/CT scans at 25, 45, and 90 min p.i.. RESULTS: The trimer Ga-68-Triveoctin exhibits a 6.7-fold higher αvß8-integrin affinity than the monomer (IC50 of 5.7 vs. 38 nM, respectively). Accordingly, biodistribution showed a higher tumor uptake (1.9 vs. 1.0%IA/g, respectively) but a similar baseline upon blockade (0.25%IA/g for both). IHC showed an intermediate ß8-expression in the tumor while most organs and tissues were found ß8-negative. Low non-target tissue uptakes (< 0.4%IA/g) confirmed a low degree of unspecific binding. Due to its hydrophilicity (log D = - 3.1), Ga-68-Triveoctin is excreted renally and shows favorable tumor/tissue ratios in mice (t/blood: 6.7; t/liver: 6.8; t/muscle: 29). A high kidney uptake in mice (kidney-to-blood and -to-muscle ratios of 126 and 505, respectively) is not reflected by human PET (corresponding values are 15 and 30, respectively), which furthermore showed notable uptakes in coeliac and choroid plexus (SUVmean 6.1 and 9.7, respectively, 90 min p.i.). CONCLUSION: Ga-68-Triveoctin enables sensitive in-vivo imaging αvß8-integrin expression in murine tumor xenografts. PET in a human subject confirmed a favorable biodistribution, underscoring the potential of Ga-68-Triveoctin for mapping of αvß8-integrin expression in a clinical setting.

Click-Chemistry (CuAAC) Trimerization of an αv ß6 Integrin Targeting Ga-68-Peptide: Enhanced Contrast for in-Vivo PET Imaging of Human Lung Adenocarcinoma Xenografts.

αv ß6 Integrin is an epithelial transmembrane protein that recognizes latency-associated peptide (LAP) and primarily activates transforming growth factor beta (TGF-ß). It is overexpressed in carcinomas (most notably, pancreatic) and other conditions associated with αv ß6 integrin-dependent TGF-ß dysregulation, such as fibrosis. We have designed a trimeric Ga-68-labeled TRAP conjugate of the αv ß6 -specific cyclic pentapeptide SDM17 (cyclo[RGD-Chg-E]-CONH2 ) to enhance αv ß6 integrin affinity as well as target-specific in-vivo uptake. Ga-68-TRAP(SDM17)3 showed a 28-fold higher αv ß6 affinity than the corresponding monomer Ga-68-NOTA-SDM17 (IC50 of 0.26 vs. 7.4 nM, respectively), a 13-fold higher IC50 -based selectivity over the related integrin αv ß8 (factors of 662 vs. 49), and a threefold higher tumor uptake (2.1 vs. 0.66 %ID/g) in biodistribution experiments with H2009 tumor-bearing SCID mice. The remarkably high tumor/organ ratios (tumor-to-blood 11.2; -to-liver 8.7; -to-pancreas 29.7) enabled high-contrast tumor delineation in PET images. We conclude that Ga-68-TRAP(SDM17)3 holds promise for improved clinical PET diagnostics of carcinomas and fibrosis.

Clinical Validation of the Aptima Bacterial Vaginosis and Aptima Candida/Trichomonas Vaginitis Assays: Results from a Prospective Multicenter Clinical Study.

Infectious vaginitis due to bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and Trichomonas vaginalis accounts for a significant proportion of all gynecologic visits in the United States. A prospective multicenter clinical study was conducted to validate the performance of two new in vitro diagnostic transcription-mediated amplification nucleic acid amplification tests (NAATs) for diagnosis of BV, VVC, and trichomoniasis. Patient- and clinician-collected vaginal-swab samples obtained from women with symptoms of vaginitis were tested with the Aptima BV and Aptima Candida/Trichomonas vaginitis (CV/TV) assays. The results were compared to Nugent (plus Amsel for intermediate Nugent) scores for BV, Candida cultures and DNA sequencing for VVC, and a composite of NAAT and culture for T. vaginalis The prevalences of infection were similar for clinician- and patient-collected samples: 49% for BV, 29% for VVC due to the Candida species group, 4% for VVC due to Candida glabrata, and 10% for T. vaginalis Sensitivity and specificity estimates for the investigational tests in clinician-collected samples were 95.0% and 89.6%, respectively, for BV; 91.7% and 94.9% for the Candida species group; 84.7% and 99.1% for C. glabrata; and 96.5% and 95.1% for T. vaginalis Sensitivities and specificities were similar in patient-collected samples. In a secondary analysis, clinicians' diagnoses, in-clinic assessments, and investigational-assay results were compared to gold standard reference methods. Overall, the investigational assays had higher sensitivity and specificity than clinicians' diagnoses and in-clinic assessments, indicating that the investigational assays were more predictive of infection than traditional diagnostic methods. These results provide clinical-efficacy evidence for two in vitro diagnostic NAATs that can detect the main causes of vaginitis.

Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma.

BACKGROUND: Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test. METHODS: Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard. RESULTS: The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6-11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%-80%) and for stage I-III CRC, 64% (48%-77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%-82%). SIGNIFICANCE: The Epi proColon test is a simple, real-time PCR-based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.

Evaluation of a microarray-based genotyping assay for the rapid detection of cytochrome P450 2C19 *2 and *3 polymorphisms from whole blood using nanoparticle probes.

Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, *2 and *3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 *2 or *3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 *2 and *3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.

Rate of detection of high-risk HPV with two assays in women ≥ 30 years of age.

BACKGROUND: High-risk (HR) human papillomavirus (HPV) prevalence rates, as determined by the Cervista(®) HPV HR test, in women aged ≥30 years in a routine screening population have not been studied. OBJECTIVES: The primary objective of this study was to estimate HR HPV prevalence in women negative for intraepithelial lesion or malignancy (NILM) cytology using the CERVISTA HPV HR test. The study also compared HR HPV prevalence rates in women aged ≥30 years and NILM cytology using the CERVISTA HPV HR and Hybrid Capture(®) 2 (hc2) tests. STUDY DESIGN: A multi-center study was conducted to analyze HR HPV prevalence rates using the CERVISTA HPV HR test from residual ThinPrep(®) specimens. HR HPV positive rates were determined for hc2; percent agreement between the CERVISTA HPV HR and the hc2 tests were reported. RESULTS: HR HPV prevalence rates among women with NILM cytology were not statistically different between the CERVISTA HPV HR and hc2 tests (6.92% [98/1417] versus 5.93% [84/1417], respectively; P>0.05). The overall percent agreement between the tests was 95.3% (1351/1417; 95% confidence interval [CI]: 94.1-96.3; κ=0.61, 95% CI: 0.53-0.70). There were no statistically significant differences between tests across age groups or investigational sites. For both tests, there was a statistically significant decrease in HR HPV positive results as age increases (CERVISTA HPV HR, P=0.0009; hc2, P<0.0001). DISCUSSION: There is no statistically significant difference between HR HPV prevalence rates obtained with the CERVISTA HPV HR and hc2 tests in women aged ≥30 years with NILM cytology.

Analytical performance of Cervista HPV 16/18 genotyping test for cervical cytology samples.

BACKGROUND: Human papillomavirus (HPV) types 16 and 18 are the 2 most frequent types associated with cervical cancer. Identifying their presence or absence in cervical samples may assist in triaging women for subsequent management. The Cervista HPV 16/18 genotyping test specifically detects the presence of HPV 16 and 18 in ThinPrep cervical specimens. OBJECTIVES: The objective was to establish the analytical performance of the CERVISTA HPV 16/18 genotyping test. STUDY DESIGN: These studies were performed in support of a regulatory submission to the US Food and Drug Administration. Here we report the analytical sensitivity (limit of detection), accuracy compared to consensus L1 gene PCR/bi-directional sequencing, precision, reproducibility, and cross-reactivity (specificity) of the genotyping test. RESULTS: Analytical sensitivity for detection of HPV 16 and 18 ranged between 625 and 1250 copies/reaction for both types. When compared to PCR/sequencing for women with atypical squamous cells of undetermined significance cytology, the positive percent agreement was 94.1% (95% confidence interval [CI], 89.8-96.7) and the negative percent agreement was 85.7% (95% CI, 82.4-88.4). The test demonstrated high within-laboratory and inter-operator precision. Reproducibility within sites and between 3 testing sites resulted in 100% agreement with expected results (150 positive, 90 negative results). The genotyping test did not exhibit cross-reactivity to DNA from common low-risk HPV types and other microorganisms found in the human female reproductive tract. CONCLUSIONS: These analytical performance data support the use of CERVISTA HPV 16/18 genotyping test for the detection and differentiation of HPV 16 and 18 in ThinPrep cervical cytology specimens.

Real-time detection of influenza a, influenza B, and respiratory syncytial virus a and B in respiratory specimens by use of nanoparticle probes.

Seasonal epidemics of influenza and respiratory syncytial virus are responsible for significant morbidity and mortality worldwide. Infrequently, novel or reemergent strains of influenza A virus have caused rapid, severe global pandemics resulting in millions of fatalities. The ability to efficiently and accurately detect and differentiate respiratory viruses is paramount for effective treatment, infection control, and epidemiological surveillance. We evaluated the ability of two FDA-cleared nucleic acid-based tests, the semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus nucleic acid test SP (RVNAT(SP)) (Nanosphere Inc., Northbrook, IL) to detect influenza A virus, influenza B virus, and respiratory syncytial virus A and B (RSV A/B) from clinical nasopharyngeal swab specimens. Detection of viral RNA in both tests is based on nucleic acid amplification followed by hybridization to capture probes immobilized on a glass slide. A novel technology utilizing gold nanoparticle-conjugated probes is utilized to detect the presence of captured target DNA. This microarray-based approach to detection has proven to be more sensitive than the traditional culture/direct fluorescent-antibody assay (DFA) method for detecting RSV and influenza viruses in clinical specimens, including the novel 2009 H1N1 strain. Specifically, we report 98.0% sensitivity and 96.5% specificity for the VRNAT compared to culture/DFA. Further, the VRNAT detected virus in an additional 58% of specimens that were culture negative. These data were confirmed using bidirectional sequencing. Evaluation of the fully automated RVNAT(SP), which is built on the same detection technology as the VRNAT but contains an updated processor enabling complete automation, revealed the two tests to be functionally equivalent. Thus, the RVNAT(SP) is a fully automated sample-to-result test capable of reliable detection of select respiratory viruses directly from clinical specimens in 3.5 h.

Cervical cancer and human papillomavirus in indigenous Guyanese women.

OBJECTIVE: The purpose of this study was to determine the prevalence of cervical disease, human papillomavirus infection, and human papillomavirus (HPV) genotypes in indigenous villages of Guyana. STUDY DESIGN: This is a retrospective analysis of a clinical cervical cancer screening and treatment program: 2250 women underwent cytologic screening; 1423 women were concomitantly screened for HPV. HPV genotyping was performed in 45 women with high-grade dysplasia and in 9 women with cervical carcinoma. RESULTS: We found invasive cervical carcinoma in 0.80% of the women, cervical intraepithelial neoplasia II and III in 5.07% of the women, and a high-risk HPV infection rate in 19.3% of the women, all of which peaked between the ages of 20-30 years. Sixteen genotypes were detected in women with high-grade dysplasia or cancer: HPV 31, 25.0%; HPV 16, 22.7%; HPV 18, 13.6%. The rate of HPV 16 and 18 in cervical cancer was 55.50%. CONCLUSION: Indigenous Guyanese women have a high rate of cervical cancer and high-grade dysplasia, with an apparent predominance of HPV 16 and 18 in invasive cancer and overrepresentation of HPV 31 in high-grade dysplasia.

Analytical performance of the Investigational Use Only Cervista HPV HR test as determined by a multi-center study.

BACKGROUND: Any HPV test designed to be utilized in cervical cancer screening programs should be highly validated both analytically and clinically. OBJECTIVES: The Investigational Use Only (IUO) Cervista HPV HR test is designed to detect 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The analytical performance of the Cervista HPV HR test was characterized in a multi-center study. RESULTS: Analytical sensitivity for the 14 high-risk HPV types that the test is designed to detect ranged from 1,250 copies to 7,500 copies per reaction depending on HPV type. Accuracy compared to PCR with bi-directional sequencing was 91.4% [95% CI: 86.5 95.0%]. The reproducibility, when tested at three different testing centers, resulted in an overall inter-run reproducibility (between day/within site) agreement of 98.8% [1-sided 95% Confidence Lower Limit = 96.9%] and an overall inter-site reproducibility (between site) agreement of 98.7% [1-sided 95% Confidence Lower Limit = 97.9%]. The Cervista HPV HR test showed no cross-reactivity with DNA from seven non-oncogenic HPV types or 17 different infectious agents at up to 10(7) copies per reaction. CONCLUSIONS: The analytical performance of the Cervista HPV HR test demonstrates sufficient analytical performance for use in cervical cancer screening. As with any clinical laboratory test, analytical characteristics must be evaluated in light of the clinical performance of this assay.

Laboratory practice guidelines for detecting and reporting BCR-ABL drug resistance mutations in chronic myelogenous leukemia and acute lymphoblastic leukemia: a report of the Association for Molecular Pathology.

The BCR-ABL tyrosine kinase produced by the t(9;22)(q34;q11) translocation, also known as the Philadelphia chromosome, is the initiating event in chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Targeting of BCR-ABL with tyrosine kinase inhibitors (TKIs) has resulted in rapid clinical responses in the vast majority of patients with CML and Philadelphia chromosome+ ALL. However, long-term use of TKIs occasionally results in emergence of therapy resistance, in part through the selection of clones with mutations in the BCR-ABL kinase domain. We present here an overview of the current practice in monitoring for such mutations, including the methods used, the clinical and laboratory criteria for triggering mutational analysis, and the guidelines for reporting BCR-ABL mutations. We also present a proposal for a public database for correlating mutational status with in vitro and in vivo responses to different TKIs to aid in the interpretation of mutation studies.

Comparative evaluation of three JAK2V617F mutation detection methods.

The correlation of JAK2V617F with a proportion of chronic myeloproliferative disorders has generated numerous studies focused on the development of molecular-based assays for JAK2V617F detection. The current parallel study comparatively evaluated 3 JAK2V617F molecular detection methods. Genomic DNA from blood or bone marrow was assayed by 3 laboratories using allele-specific polymerase chain reaction (AS-PCR) or kit-based restriction fragment length polymorphism methods, which used polyacrylamide gel or capillary electrophoresis analysis. In addition, samples were sequenced in 2 of the laboratories. Results found 100% concordance among the 3 methods, with analytic sensitivities of 5% for both kit methods and 0.01% for AS-PCR. The kitbased assays detect JAK2V617F with equal sensitivity regardless of analysis method, and, despite greater sensitivity of AS-PCR, all 3 methods yielded 100% concordant results for this 36-sample set. Consistent with other reports, direct sequencing was insufficiently sensitive to serve as an initial diagnostic tool for JAK2V617F detection.