The Trifecta Study: Comparing Plasma Levels of Donor-derived Cell-Free DNA with the Molecular Phenotype of Kidney Transplant Biopsies.

BACKGROUND: The relationship between the donor-derived cell-free DNA fraction (dd-cfDNA[%]) in plasma in kidney transplant recipients at time of indication biopsy and gene expression in the biopsied allograft has not been defined. METHODS: In the prospective, multicenter Trifecta study, we collected tissue from 300 biopsies from 289 kidney transplant recipients to compare genome-wide gene expression in biopsies with dd-cfDNA(%) in corresponding plasma samples drawn just before biopsy. Rejection was assessed with the microarray-based Molecular Microscope Diagnostic System using automatically assigned rejection archetypes and molecular report sign-outs, and histology assessments that followed Banff guidelines. RESULTS: The median time of biopsy post-transplantation was 455 days (5 days to 32 years), with a case mix similar to that of previous studies: 180 (60%) no rejection, 89 (30%) antibody-mediated rejection (ABMR), and 31 (10%) T cell-mediated rejection (TCMR) and mixed. In genome-wide mRNA measurements, all 20 top probe sets correlating with dd-cfDNA(%) were previously annotated for association with ABMR and all types of rejection, either natural killer (NK) cell-expressed (e.g., GNLY, CCL4, TRDC, and S1PR5) or IFN-γ-inducible (e.g., PLA1A, IDO1, CXCL11, and WARS). Among gene set and classifier scores, dd-cfDNA(%) correlated very strongly with ABMR and all types of rejection, reasonably strongly with active TCMR, and weakly with inactive TCMR, kidney injury, and atrophy fibrosis. Active ABMR, mixed, and active TCMR had the highest dd-cfDNA(%), whereas dd-cfDNA(%) was lower in late-stage ABMR and less-active TCMR. By multivariate random forests and logistic regression, molecular rejection variables predicted dd-cfDNA(%) better than histologic variables. CONCLUSIONS: The dd-cfDNA(%) at time of indication biopsy strongly correlates with active molecular rejection and has the potential to reduce unnecessary biopsies. CLINICAL TRIAL REGISTRATION NUMBER: NCT04239703.

DENEB: Development of new criteria for curability after local excision of pathological T1 colorectal cancer using liquid biopsy.

According to the current international guidelines, high-risk patients diagnosed with pathological T1 (pT1) colorectal cancer (CRC) who underwent complete local resection but may have risk of developing lymph node metastasis (LNM) are recommended additional intestinal resection with lymph node dissection. However, around 90% of the patients without LNM are exposed to the risk of being overtreated due to the insufficient pathological criteria for risk stratification of LNM. Circulating tumor DNA (ctDNA) is a noninvasive biomarker for molecular residual disease and relapse detection after treatments including surgical and endoscopic resection of solid tumors. The CIRCULATE-Japan project includes a large-scale patient-screening registry of the GALAXY study to track ctDNA status of patients with stage II to IV or recurrent CRC that can be completely resected. Based on the CIRCULATE-Japan platform, we launched DENEB, a new prospective study, within the GALAXY study for patients with pT1 CRC who underwent complete local resection and were scheduled for additional intestinal resection with lymph node dissection based on the standard pathologic risk stratification criteria for LNM. The aim of this study is to explore the ability of predicting LNM using ctDNA analysis compared with the standard pathological criteria. The ctDNA assay will build new evidence to establish a noninvasive personalized diagnosis in patients, which will facilitate tailored/optimal treatment strategies for CRC patients.

Genetic Etiologies for Chronic Kidney Disease Revealed through Next-Generation Renal Gene Panel.

INTRODUCTION: Chronic kidney disease (CKD) is a major public health issue in the USA. Identification of monogenic causes of CKD, which are present in ∼10% of adult cases, can impact prognosis and patient management. Broad gene panels can provide unbiased testing approaches, which are advantageous in phenotypically heterogeneous diseases. However, the use and yield of broad genetic panels by nephrologists in clinical practice is not yet well characterized. METHODS: Renal genetic testing, ordered exclusively for clinical purposes, predominantly by general and transplant nephrologists within the USA, was performed on 1,007 consecutive unique patient samples. Testing was performed using a commercially available next-generation sequencing-based 382 gene kidney disease panel. Pathogenic (P) and likely pathogenic (LP) variants were reported. Positive findings included a monoallelic P/LP variant in an autosomal dominant or X-linked gene and biallelic P/LP variants in autosomal recessive genes. RESULTS: Positive genetic findings were identified in 21.1% (212/1,007) of cases. A total of 220 positive results were identified across 48 genes. Positive results occurred most frequently in the PKD1 (34.1%), COL4A5 (10.9%), PKD2 (10.0%), COL4A4 (6.4%), COL4A3 (5.9%), and TTR (4.1%) genes. Variants identified in the remaining 42 genes comprised 28.6% of the total positive findings, including single positive results in 26 genes. Positive results in >1 gene were identified in 7.5% (16/212) of cases. CONCLUSIONS: Use of broad panel genetic testing by clinical nephrologists had a high success rate, similar to results obtained by academic centers specializing in genetics.

Clinician-reported chorionicity and zygosity assignment using single-nucleotide polymorphism-based cell-free DNA: Lessons learned from 55,344 twin pregnancies.

OBJECTIVE: Prenatal chorionicity assessment relies on ultrasound, which can be confounded by many factors. Noninvasive assessment of zygosity is possible using single nucleotide polymorphism (SNP)-based cell-free DNA testing. Our objective was to determine the relationship between provider-reported chorionicity and SNP-cfDNA assignment of twin zygosity. METHODS: All twin pregnancy blood samples received by a reference laboratory between September 27, 2017 and September 8, 2021 were included. Chorionicity assignment was requested on the requisition, recorded as; monochorionic (MC), dichorionic, or "don't know". SNP-cfDNA zygosity results, monozygotic (MZ) or dizygotic (DZ), were correlated with chorionicity assignment. RESULTS: 59,471 twin samples (median gestational age = 12.0 weeks at draw) were received and analyzed; 55,344 (93.1%) received zygosity assignment. SNP-cfDNA reported 16,673 (30.1%) MZ and 38,671 (69.9%) as DZ. Provider-reported chorionicity was compared to the zygosity assignment for each case. Of 6283 provider-reported MC twins, 318 (5.1%) were reported as DZ using SNP-cfDNA. CONCLUSION(S): One in 20 suspected MC twin pregnancies were reported as DZ using SNP-cfDNA. Approximately 30% of 55,344 twin pregnancies were found to be MZ, including cases where chorionicity was unknown. SNP-cfDNA zygosity assessment is a useful adjunct assessment for twin pregnancies, particularly those reported as MC or without determined chorionicity.

CIRCULATE-Japan: Circulating tumor DNA-guided adaptive platform trials to refine adjuvant therapy for colorectal cancer.

Adjuvant chemotherapy has reduced the risk of tumor recurrence and improved survival in patients with resected colorectal cancer. Potential utility of circulating tumor DNA (ctDNA) prior to and post surgery has been reported across various solid tumors. We initiated a new type of adaptive platform trials to evaluate the clinical benefits of ctDNA analysis and refine precision adjuvant therapy for resectable colorectal cancer, named CIRCULATE-Japan including three clinical trials. The GALAXY study is a prospectively conducted large-scale registry designed to monitor ctDNA for patients with clinical stage II to IV or recurrent colorectal cancer who can undergo complete surgical resection. The VEGA trial is a randomized phase III study designed to test whether postoperative surgery alone is noninferior to the standard therapy with capecitabine plus oxaliplatin for 3 months in patients with high-risk stage II or low-risk stage III colon cancer if ctDNA status is negative at week 4 after curative surgery in the GALAXY study. The ALTAIR trial is a double-blind, phase III study designed to establish the superiority of trifluridine/tipiracil as compared with placebo in patients with resected colorectal cancer who show circulating tumor-positive status in the GALAXY study. Therefore, CIRCULATE-Japan encompasses both "de-escalation" and "escalation" trials for ctDNA-negative and -positive patients, respectively, and helps to answer whether measuring ctDNA postoperatively has prognostic and/or predictive value. Our ctDNA-guided adaptive platform trials will accelerate clinical development toward further precision oncology in the field of adjuvant therapy. Analysis of ctDNA status could be utilized as a predictor of risk stratification for recurrence and to monitor the effectiveness of adjuvant chemotherapy. ctDNA is a promising, noninvasive tumor biomarker that can aid in tumor monitoring throughout disease management.

Chr15q25 genetic variant (rs16969968) independently confers risk of lung cancer, COPD and smoking intensity in a prospective study of high-risk smokers.

IMPORTANCE: While cholinergic receptor nicotinic alpha 5 (CHRNA5) variants have been linked to lung cancer, chronic obstructive pulmonary disease (COPD) and smoking addiction in case-controls studies, their corelationship is not well understood and requires retesting in a cohort study. OBJECTIVE: To re-examine the association between the CHRNA5 variant (rs16969968 AA genotype) and the development of lung cancer, relative to its association with COPD and smoking. METHODS: In 9270 Non-Hispanic white subjects from the National Lung Screening Trial, a substudy of high-risk smokers were followed for an average of 6.4 years. We compared CHRNA5 genotype according to baseline smoking exposure, lung function and COPD status. We also compared the lung cancer incidence rate, and used multiple logistic regression and mediation analysis to examine the role of the AA genotype of the CHRNA5 variant in smoking exposure, COPD and lung cancer. RESULTS: As previously reported, we found the AA high-risk genotype was associated with lower lung function (p=0.005), greater smoking intensity (p<0.001), the presence of COPD (OR 1.28 (95% CI 1.10 to 1.49) p=0.0015) and the development of lung cancer (HR 1.41, (95% CI 1.03 to 1.93) p=0.03). In a mediation analyses, the AA genotype was independently associated with smoking intensity (OR 1.42 (95% CI 1.25 to 1.60, p<0.0001), COPD (OR 1.25, (95% CI 1.66 to 2.53), p=0.0015) and developing lung cancer (OR 1.37, (95% CI 1.03 to 1.82) p=0.03). CONCLUSION: In this large-prospective study, we found the CHRNA5 rs 16 969 968 AA genotype to be independently associated with smoking exposure, COPD and lung cancer (triple whammy effect).

Clinical experience with carrier screening in a general population: support for a comprehensive pan-ethnic approach.

PURPOSE: To present results from a large cohort of individuals receiving expanded carrier screening (CS) in the United States. METHODS: Single-gene disorder carrier status for 381,014 individuals was determined using next-generation sequencing (NGS) based CS for up to 274 genes. Detection rates were compared with literature-reported values derived from disease prevalence and carrier frequencies. Combined theoretical affected pregnancy rates for the 274 screened disorders were calculated. RESULTS: For Ashkenazi Jewish (AJ) diseases, 81.6% (4434/5435) of carriers identified did not report AJ ancestry. For cystic fibrosis, 44.0% (6260/14,229) of carriers identified had a variant not on the standard genotyping panel. Individuals at risk of being a silent spinal muscular atrophy carrier, not detectable by standard screening, comprised 1/39 (8763/344,407) individuals. For fragile X syndrome, compared with standard premutation screening, AGG interruption analysis modified risk in 83.2% (1128/1356) premutation carriers. Assuming random pairing across the study population, approximately 1/175 pregnancies would be affected by a disorder in the 274-gene screening panel. CONCLUSION: Compared with standard screening, NGS-based CS provides additional information that may impact reproductive choices. Pan-ethnic CS leads to substantially increased identification of at-risk couples. These data support offering NGS-based CS to all reproductive-aged women.

Correction: Clinical experience with carrier screening in a general population: support for a comprehensive pan-ethnic approach.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cell-free DNA fetal fraction in twin gestations in single-nucleotide polymorphism-based noninvasive prenatal screening.

OBJECTIVES: The performance of noninvasive prenatal screening (NIPS) for fetal aneuploidy in twin pregnancies is dependent on the amount of placentally derived cell-free DNA, the "fetal fraction (FF)," present in maternal plasma. We report FF values in monozygotic (MZ) and dizygotic (DZ) pregnancies. METHODS: We reviewed FF in pregnancies at 10 to 20 completed weeks gestational age based on single-nucleotide polymorphism (SNP)-based NIPS where zygosity was routinely established in twin pregnancies. The cohort included 121 446 (96.3%) singleton, 1454 (1.2%) MZ, and 3161 (2.5%) DZ pregnancies. For DZ twins, individual FFs were measured. RESULTS: Combined FF for DZ and MZ fetuses were 35% and 26% greater than singletons, respectively. The individual FF contributions from each fetus in DZ twins were, on average, 32% less than singletons. FF in DZ twin pairs were moderately correlated (Pearson correlation coefficient.66). When a threshold of 2.8% FF was applied to define uninterpretable results, 1.7% (2102/121 446) of singletons, 0.8% (11/1454) of MZ pairs, and 5.6% (178/3161) of DZ pairs were uninterpretable. CONCLUSION: For optimal aneuploidy NIPS in twin pregnancies, zygosity should be established and in DZ twins FF for both fetuses should be determined to identify those cases where results can be reliably interpreted.

Combining the use of a fetal fraction-based risk algorithm and probability of an informative redraw in noninvasive prenatal testing for fetal aneuploidy.

Some women undergoing noninvasive prenatal testing (NIPT) do not receive an informative result due to low fetal fraction (FF). A proportion of these are at increased risk for fetal trisomy 13, 18, or triploidy, while others have no change from their prior risk. Women with an initial uninformative NIPT need to be counseled about any such change in their risk for fetal abnormality and also the probability that a redraw will be informative. To help in the decision making, we reviewed a dataset of single nucleotide polymorphism-based NIPT with uninformative results where a redraw was received. Risk for trisomy 13, 18, or triploidy was evaluated using a fetal fraction-based risk (FFBR) algorithm. Risk-unchanged women were further analyzed using a regression model to determine the likelihood of an informative redraw. Of 2,644 women with an uninformative NIPT and a redraw, 1,147 (43.4%) were high risk for trisomy 13, 18, or triploidy. 1,497 (56.6%) were risk unchanged and, of these, 975 (65.1%) cases had an informative redraw (i.e., risks were available for 2,122 (80%) of those initially classified as uninformative). The regression model for the risk-unchanged cases provided a new table for predicting an informative redraw. Likelihood of a successful redraw was significantly (p < .001) dependent on the initial FF, maternal weight, and time between blood draws. We conclude that the FFBR algorithm and the predictive model for an informative redraw provide complementary additions in the management of women presented with an initially uninformative SNP-based NIPT due to low FF. We suggest approaches for the counseling and follow-up testing for women with an initially uninformative NIPT.

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Domains with highest heparan sulfate-binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function.

Signaling proteins, including bone morphogenetic proteins (BMPs), specifically interact with heparan sulfate (HS). These interactions regulate protein distribution and function and are largely mediated by domains rich in basic amino acids. The N-terminal region of BMP2 and BMP4 contains one such domain with a typical Cardin-Weintraub (CW) motif, but it is unclear whether the same occurs in BMP5, BMP6, and BMP7 that constitute a separate evolutionary subgroup. Peptides spanning the N-terminal domain of BMP2/4 interacted with substrate-bound HS with nanomolar affinity, but peptides spanning BMP5/6/7 N-terminal domain did not. We re-examined the entire BMP5/6/7 sequences and identified a novel CW-like motif at their C terminus. Peptides spanning this domain displayed high-affinity HS binding, but corresponding BMP2/4 C-terminal peptides did not, likely because of acidic or noncharged residue substitutions. Peptides pre-assembled into NeutrAvidin tetramers displayed the same exact binding selectivity of respective monomers but bound HS with greater affinity. Tests of possible peptide biological activities showed that the HS-binding N-terminal BMP2/4 and C-terminal BMP5/6/7 peptides stimulated chondrogenesis in vitro, potentially by freeing endogenous BMPs. Thus, HS interactions appear largely ascribable to domains at opposite ends of BMP2/4 versus BMP5/6/7, reiterating the evolutionary distance of these BMP subgroups and possible functional diversification.

Heparan sulfate antagonism alters bone morphogenetic protein signaling and receptor dynamics, suggesting a mechanism in hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a pediatric disorder caused by heparan sulfate (HS) deficiency and is characterized by growth plate-associated osteochondromas. Previously, we found that osteochondroma formation in mouse models is preceded by ectopic bone morphogenetic protein (BMP) signaling in the perichondrium, but the mechanistic relationships between BMP signaling and HS deficiency remain unclear. Therefore, we used an HS antagonist (surfen) to investigate the effects of this HS interference on BMP signaling, ligand availability, cell-surface BMP receptor (BMPR) dynamics, and BMPR interactions in Ad-293 and C3H/10T1/2 cells. As observed previously, the HS interference rapidly increased phosphorylated SMAD family member 1/5/8 levels. FACS analysis and immunoblots revealed that the cells possessed appreciable levels of endogenous cell-surface BMP2/4 that were unaffected by the HS antagonist, suggesting that BMP2/4 proteins remained surface-bound but became engaged in BMPR interactions and SMAD signaling. Indeed, surface mobility of SNAP-tagged BMPRII, measured by fluorescence recovery after photobleaching (FRAP), was modulated during the drug treatment. This suggested that the receptors had transitioned to lipid rafts acting as signaling centers, confirmed for BMPRII via ultracentrifugation to separate membrane subdomains. In situ proximity ligation assays disclosed that the HS interference rapidly stimulates BMPRI-BMPRII interactions, measured by oligonucleotide-driven amplification signals. Our in vitro studies reveal that cell-associated HS controls BMP ligand availability and BMPR dynamics, interactions, and signaling, and largely restrains these processes. We propose that HS deficiency in HME may lead to extensive local BMP signaling and altered BMPR dynamics, triggering excessive cellular responses and osteochondroma formation.

The Roles of Indian Hedgehog Signaling in TMJ Formation.

The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes. Uniquely, progenitor cells reside near the articular surface, proliferate, undergo chondrogenesis, and mature into hypertrophic chondrocytes. During the past decades, there has been a growing interest in the molecular mechanisms by which the TMJ develops and acquires its unique structural and functional features. Indian hedgehog (Ihh), which regulates skeletal development including synovial joint formation, also plays pivotal roles in TMJ development and postnatal maintenance. This review provides a description of the many important recent advances in Hedgehog (Hh) signaling in TMJ biology. These include studies that used conventional approaches and those that analyzed the phenotype of tissue-specific mouse mutants lacking Ihh or associated molecules. The recent advances in understanding the molecular mechanism regulating TMJ development are impressive and these findings will have major implications for future translational medicine tools to repair and regenerate TMJ congenital anomalies and acquired diseases, such as degenerative damage in TMJ osteoarthritic conditions.

Comparative analysis of colorimetric staining in skin using open-source software.

Colorimetric staining techniques such as immunohistochemistry (IHC), immunofluorescence (IF) and histochemistry (HC) provide useful information regarding the localization and relative amount of a molecule/substance in skin. We have developed a novel, straightforward method to assess colorimetric staining by combining features from two open-source software programs. As a proof of principle, we demonstrate the utility of this approach by analysing changes in skin melanin deposition during the radiation-induced tanning response of Yucatan mini-pigs. This method includes a visualization step to validate the accuracy of colour selection before quantitation to ensure accuracy. The data show that this method is robust and will provide a means to obtain accurate comparative analyses of staining in IHC/IF/HC samples.

Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries.

Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that, collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In doing so, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys and Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review, we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

The impact of a fourteen-gene molecular assay on physician treatment decisions in non-small-cell lung cancer.

BACKGROUND: Five-year survival in early-stage, non-squamous, non-small-cell lung cancer (NSCLC) remains poor compared with other solid tumors, even after complete resection. Post-operative management depends on prognostic staging to identify individuals at highest risk for death, and therefore with the greatest need for further intervention. A 14-gene quantitative RT-PCR test successfully differentiates stage I-III NSCLC patients who are at high-, intermediate-, or low-risk for 5-year mortality. This study assesses the impact of the assay's prognostic information on physician decisions regarding adjuvant chemotherapy. METHODS: We invited 115 physicians who ordered the test to participate in an on-line survey. The primary outcome measure was the proportion of patients with different pre- and post-test chemotherapy recommendations. RESULTS: Fifty-eight physicians (50 %) completed the survey on 120 stage I or II NSCLC patients. Ninety-one patients (76 %) had stage I lung cancer; 27 (23 %), 39 (33 %), and 54 (45 %) patients had low-, intermediate-, and high-risk scores, respectively. Physicians' chemotherapy recommendations were changed post-testing in 37 patients (30.8 %, 95 % CI 22.7-39.9 %). High-risk patients were more likely to have a change in treatment recommendation (44.4 %, 95 % CI 30.9-58.6 %) than low risk patients (3.7 %, 95 % CI 0.1-19.0 %); a substantial number of changes were observed in both stage I (33.0 %, 95 % CI 23.5-43.6 %) and stage II (24.1 %, 95 % CI 10.3-43.5 %). CONCLUSIONS: Our data show that the assay resulted in a significant impact on physician treatment decisions in early-stage NSCLC, and that the nature of treatment changes generally correlated with the test's assessment of risk.

Pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis.

Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.