Clinical variability at the mild end of BRAT1-related spectrum: Evidence from two families with genotype-phenotype discordance.

Biallelic mutations in the BRAT1 gene, encoding BRCA1-associated ATM activator 1, result in variable phenotypes, from rigidity and multifocal seizure syndrome, lethal neonatal to neurodevelopmental disorder, and cerebellar atrophy with or without seizures, without obvious genotype-phenotype associations. We describe two families at the mildest end of the spectrum, differing in clinical presentation despite a common genotype at the BRAT1 locus. Two siblings displayed nonprogressive congenital ataxia and shrunken cerebellum on magnetic resonance imaging. A third unrelated patient showed normal neurodevelopment, adolescence-onset seizures, and ataxia, shrunken cerebellum, and ultrastructural abnormalities on skin biopsy, representing the mildest form of NEDCAS hitherto described. Exome sequencing identified the c.638dup and the novel c.1395G>A BRAT1 variants, the latter causing exon 10 skippings. The p53-MCL test revealed normal ATM kinase activity. Our findings broaden the allelic and clinical spectrum of BRAT1-related disease, which should be suspected in presence of nonprogressive cerebellar signs, even without a neurodevelopmental disorder.

Evaluation of the Relationships Between Computed Tomography Features, Pathological Findings, and Prognostic Risk Assessment in Gastrointestinal Stromal Tumors.

OBJECTIVES: The aim of this study was to correlate computed tomography (CT) findings with pathology in gastrointestinal stromal tumors (GISTs). METHODS: A retrospective evaluation of CT images of 44 patients with GISTs was performed. Computed tomography findings analyzed were location, size, margins, degree and pattern of contrast enhancement, angiogenesis, necrosis, signs of invasion, peritoneal effusion, peritoneal implants, surface ulceration, and calcifications.Associations between CT features and mitotic rate, Miettinen classes of risk, lesions size, and among CT features were investigated. χ Test and Fisher test were performed. RESULTS: Mitotic rate was associated with margins (P = 0.016) and with adjacent organ invasion (P = 0.043). Pattern of contrast enhancement (P = 0.002), angiogenesis (P = 0.006), necrosis (P = 0.006), invasion of adjacent organs (P = 0.011), and margins (P = 0.006) were associated with classes of risk. Several associations (P < 0.05) between lesion size and CT features and among all the investigated CT features were found. CONCLUSIONS: Computed tomography features could reflect GIST biology being associated with the mitotic rate and with classes of risk.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.

Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages.

Gadoxetic acid-enhanced MRI for hepatocellular carcinoma and hypointense nodule observed in the hepatobiliary phase.

PURPOSE: The aim of our study was to evaluate the diagnostic accuracy of gadoxetic acid-enhanced magnetic resonance (MR) imaging both in the detection of hepatocellular carcinoma (HCC) and precancerous lesions and in the assessment of their evolution. MATERIALS AND METHODS: A retrospective study was undertaken on 56 patients with chronic liver disease and suspected liver lesions. We evaluated the number, size and signal intensity of the nodules on dynamic and hepatobiliary MR images. Follow-up studies were carried out every 3 months. Statistical analysis was performed using the Fisher's exact test. RESULTS: A total of 120 nodules were identified in 41 patients. Of these, 92/120 nodules (76.6%; mean diameter 18.4 mm) showed the typical HCC vascular pattern: 90/92 nodules appeared hypointense and 2/92 were hyperintense on hepatobiliary phase images. An additional 28/120 hypointense, nonhypervascular nodules (23.3%; mean diameter 11 mm) were detected on hepatobiliary phase images, 15 of which showed hypointensity also on the equilibrium phase images. During the 3- to 12-month follow-up, 14/28 nodules (mean diameter 13.3 mm) developed the typical vascular pattern of HCC. CONCLUSIONS: Gadoxetic acid-enhanced MR imaging is useful for detecting HCC as well as hypovascular nodules with potential progression to HCC. Lesions measuring more than 10 mm in diameter are at higher risk of developing into HCC (p = 0.0128).

Concise review: stem cell-derived erythrocytes as upcoming players in blood transfusion.

Blood transfusions have become indispensable to treat the anemia associated with a variety of medical conditions ranging from genetic disorders and cancer to extensive surgical procedures. In developed countries, the blood supply is generally adequate. However, the projected decline in blood donor availability due to population ageing and the difficulty in finding rare blood types for alloimmunized patients indicate a need for alternative red blood cell (RBC) transfusion products. Increasing knowledge of processes that govern erythropoiesis has been translated into efficient procedures to produce RBC ex vivo using primary hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. Although in vitro-generated RBCs have recently entered clinical evaluation, several issues related to ex vivo RBC production are still under intense scrutiny: among those are the identification of stem cell sources more suitable for ex vivo RBC generation, the translation of RBC culture methods into clinical grade production processes, and the development of protocols to achieve maximal RBC quality, quantity, and maturation. Data on size, hemoglobin, and blood group antigen expression and phosphoproteomic profiling obtained on erythroid cells expanded ex vivo from a limited number of donors are presented as examples of the type of measurements that should be performed as part of the quality control to assess the suitability of these cells for transfusion. New technologies for ex vivo erythroid cell generation will hopefully provide alternative transfusion products to meet present and future clinical requirements.

Proliferation state and polo-like kinase1 dependence of tumorigenic colon cancer cells.

Tumor-initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor-initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo-like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer-initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133(+) colon cancer cells leading to complete growth arrest of colon cancer stem cell-derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133(+) cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor-initiating cells. Quiescent CD133(+) cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor-initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRß, TGFß, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

Mild Neurological Phenotype Associated with Hypomorphic Variants in the Ataxia-Telangiectasia Mutated Gene.

Background: Ataxia-telangiectasia (A-T) is a progressive multisystemic neurodegenerative disease. The phenotypic spectrum includes conditions (variant A-T) with mild, late-onset, and atypical clinical presentations characterized by the prevalence of dyskinetic rather than ataxic features. Cases: We describe the clinical presentations of 3 siblings with early-onset truncal ataxia without obvious neurological deterioration or biological markers of classic A-T phenotype. We performed functional and genetic evaluation of 3 siblings with very mild neurological phenotype. Genetic evaluation with a next-generation sequencing panel for genes causative of cerebellar ataxia detected 2 known ATM gene variants, missense c.9023G>A p.(Arg3008His), and leaky splicing c.1066-6T>G variants. Functional studies showed mildly reduced ATM expression and residual kinase activity in the probands compared with healthy controls. Conclusions: These results suggest the importance of investigating ATM variants even in the presence of clinical and biological atypical cases to ensure specific therapeutic regimens and oncological surveillance in these patients.

Breast cancer stem cells: a new target for therapy.

Normal adult tissue stem cells awake from a dormant state to grow, differentiate, and regenerate damaged tissue. They also travel in the circulation and colonize distant organs at sites undergoing tissue repair. These same traits are utilized or co-opted by metastatic cancer cells. The cancer stem cell theory proposes that tumors emerge from a subpopulation of cancer cells that possess stem cell properties. This theory has profound implications for therapy. A small number of cancer stem cells may lie dormant following conventional therapy and tumor remission, only to re-emerge and regenerate the entire recurrent cancer. Consequently, it has been proposed that targeting cancer stem cells is the only way to obtain durable cancer treatment responses. Several strategies for targeting cancer stem cells have been proposed. Nevertheless, a number of issues must be investigated and resolved before effective treatments targeting cancer stem cells can enter clinical testing.

Functional Classification of the ATM Variant c.7157C>A and In Vitro Effects of Dexamethasone.

Most of the ATM variants associated with Ataxia Telangiectasia are still classified as variants with uncertain significance. Ataxia Telangiectasia is a multisystemic disorder characterized by "typical" and "atypical" phenotypes, with early-onset and severe symptoms or with late-onset and mild symptoms, respectively. Here we classified the c.7157C > A ATM variant found in homozygosity in two brothers of Lebanese ethnicity. The brothers presented with an atypical phenotype, showing less than 50% of the positive criteria considered for classification. We performed several in silico analyses to predict the effect of c.7157C > A at the DNA, mRNA and protein levels, revealing that the alteration causes a missense substitution in a highly conserved alpha helix in the FAT domain. 3D structural analyses suggested that the variant might be pathogenic due to either loss of activity or to a structural damage affecting protein stability. Our subsequent in vitro studies showed that the second hypothesis is the most likely, as indicated by the reduced protein abundance found in the cells carrying the variant. Moreover, two different functional assays showed that the mutant protein partially retains its kinase activity. Finally, we investigated the in vitro effect of Dexamethasone showing that the drug is able to increase both protein abundance and activity. In conclusion, our results suggest that the c.7157C > A variant is pathogenic, although it causes an atypical phenotype, and that dexamethasone could be therapeutically effective on this and possibly other missense ATM variants.

Inhibition of the mTOR pathway and reprogramming of protein synthesis by MDM4 reduce ovarian cancer metastatic properties.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer.

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-ß5, Survivin, TGF-ß, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools.

Variants of uncertain significance in the era of high-throughput genome sequencing: a lesson from breast and ovary cancers.

The promising expectations about personalized medicine have opened the path to routine large-scale sequencing and increased the importance of genetic counseling for hereditary cancers, among which hereditary breast and ovary cancers (HBOC) have a major impact. High-throughput sequencing, or Next-Generation Sequencing (NGS), has improved cancer patient management, ameliorating diagnosis and treatment decisions. In addition to its undeniable clinical utility, NGS is also unveiling a large number of variants that we are still not able to clearly define and classify, the variants of uncertain significance (VUS), which account for about 40% of total variants. At present, VUS use in the clinical context is challenging. Medical reports may omit this kind of data and, even when included, they limit the clinical utility of genetic information. This has prompted the scientific community to seek easily applicable tests to accurately classify VUS and increase the amount of usable information from NGS data. In this review, we will focus on NGS and classification systems for VUS investigation, with particular attention on HBOC-related genes and in vitro functional tests developed for ameliorating and accelerating variant classification in cancer.

Interplay between Endoplasmic Reticulum (ER) Stress and Autophagy Induces Mutant p53H273 Degradation.

The unfolded protein response (UPR) is an adaptive response to intrinsic and external stressors, and it is mainly activated by the accumulation of misfolded proteins at the endoplasmic reticulum (ER) lumen producing ER stress. The UPR signaling network is interconnected with autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins in order to survive bioenergetic stress and/or induce cell death. Oncosuppressor TP53 may undergo inactivation following missense mutations within the DNA-binding domain (DBD), and mutant p53 (mutp53) proteins may acquire a misfolded conformation, often due to the loss of the DBD-bound zinc ion, leading to accumulation of hyperstable mutp53 proteins that correlates with more aggressive tumors, resistance to therapies, and poorer outcomes. We previously showed that zinc supplementation induces mutp53 protein degradation by autophagy. Here, we show that mutp53 (i.e., Arg273) degradation following zinc supplementation is correlated with activation of ER stress and of the IRE1α/XBPI arm of the UPR. ER stress inhibition with chemical chaperone 4-phenyl butyrate (PBA) impaired mutp53 downregulation, which is similar to IRE1α/XBPI specific inhibition, reducing cancer cell death. Knockdown of mutp53 failed to induce UPR/autophagy activation indicating that the effect of zinc on mutp53 folding was likely the key event occurring in ER stress activation. Recently discovered small molecules targeting components of the UPR show promise as a novel anticancer therapeutic intervention. However, our findings showing UPR activation during mutp53 degradation indicate that caution is necessary in the design of therapies that inhibit UPR components.

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors.

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.

Renal cancer: new models and approach for personalizing therapy.

BACKGROUND: Clear cell RCC (ccRCC) accounts for approximately 75% of the renal cancer cases. Surgery treatment seems to be the best efficacious approach for the majority of patients. However, a consistent fraction (30%) of cases progress after surgery with curative intent. It is currently largely debated the use of adjuvant therapy for high-risk patients and the clinical and molecular parameters for stratifying beneficiary categories. In addition, the treatment of advanced forms lacks reliable driver biomarkers for the appropriated therapeutic choice. Thus, renal cancer patient management urges predictive molecular indicators and models for therapy-decision making. METHODS: Here, we developed and optimized new models and tools for ameliorating renal cancer patient management. We isolated from fresh tumor specimens heterogeneous multi-clonal populations showing epithelial and mesenchymal characteristics coupled to stem cell phenotype. These cells retained long lasting-tumor-propagating capacity provided a therapy monitoring approach in vitro and in vivo while being able to form parental tumors when orthotopically injected and serially transplanted in immunocompromised murine hosts. RESULTS: In line with recent evidence of multiclonal cancer composition, we optimized in vitro cultures enriched of multiple tumor-propagating populations. Orthotopic xenograft masses recapitulated morphology, grading and malignancy of parental cancers. High-grade but not the low-grade neoplasias, resulted in efficient serial transplantation in mice. Engraftment capacity paralleled grading and recurrence frequency advocating for a prognostic value of our developed model system. Therefore, in search of novel molecular indicators for therapy decision-making, we used Reverse-Phase Protein Arrays (RPPA) to analyze a panel of total and phosphorylated proteins in the isolated populations. Tumor-propagating cells showed several deregulated kinase cascades associated with grading, including angiogenesis and m-TOR pathways. CONCLUSIONS: In the era of personalized therapy, the analysis of tumor propagating cells may help improve prediction of disease progression and therapy assignment. The possibility to test pharmacological response of ccRCC stem-like cells in vitro and in orthotopic models may help define a pharmacological profiling for future development of more effective therapies. Likewise, RPPA screening on patient-derived populations offers innovative approach for possible prediction of therapy response.

Preclinical rationale for TGF-ß inhibition as a therapeutic target for the treatment of myelofibrosis.

To assess the role of abnormal transforming growth factor-beta (TGF-ß) signaling in the pathogenesis of primary myelofibrosis (PMF), the effects of the TGF-ß receptor-1 kinase inhibitor SB431542 on ex vivo expansion of hematopoietic cells in cultures from patients with JAK2V617+-polycythemia vera (PV) or PMF (JAK2V617F+, CALRpQ365f+, or unknown) and from normal sources (adult blood, AB, or cord blood, CB) were compared. In cultures of normal sources, SB431542 significantly increased by 2.5-fold the number of progenitor cells generated by days 1-2 (CD34+) and 6 (colony-forming cells) (CB) and that of precursor cells, mostly immature erythroblasts, by days 14-17 (AB and CB). In cultures of JAK2V617F+-PV, SB431542 increased by twofold the numbers of progenitor cells by day 10 and had no effect on that of precursors cells by days 12-17 (∼fourfold increase in all cases). In contrast, SB431542 had no effect on the number of either progenitor or precursor cells in cultures of JAK2V617F+ and CALR pQ365fs+ PMF. These ontogenetic- and disease-specific effects were associated with variegation in the ability of SB431542 to induce CD34+ cells from AB (increased), CB (decreased), or PV and PMF (unaffected) into cycle and erythroblasts in proliferation (increased for AB and PV and unaffected for CB and PMF). Differences in expansion of erythroblasts from AB, CB, and PV were associated with differences in activation of TGF-ß signaling (SHCY317, SMAD2S245/250/255, and SMAD1S/S/SMAD5S/S/SMAD8S/S) detectable in these cells by phosphoproteomic profiling. In conclusion, treatment with TGF-ß receptor-1 kinase inhibitors may reactivate normal hematopoiesis in PMF patients, providing a proliferative advantage over the unresponsive malignant clone.

Comparison of diffusion-weighted imaging and gadoxetic acid-enhanced MR images in the evaluation of hepatocellular carcinoma and hypovascular hepatocellular nodules.

PURPOSE: To compare diffusion-weighted imaging (DWI) and gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) magnetic resonance imaging (MRI) in the evaluation of hepatocellular carcinoma (HCC) and nodules at high risk of HCC transformation. MATERIALS AND METHODS: We evaluated nodules' size, vascular pattern, and signal intensity on hepatobiliary phase images and on DWI of 105 nodules (41 cirrhotic patients). RESULTS: A total of 35/66 HCCs identified on Gd-EOB-DTPA MRI showed hyperintensity on DWI. A total of 25/39 nodules (hypovascular and hypointense nodule on hepatobiliary phase images) progressed to HCC (higher risk for nodules ≥10mm in size and hyperintense on DWI, P<.05). CONCLUSION: Gd-EOB-DTPA MRI demonstrated a significant role in the identification of nodule at higher risk of HCC transformation, and hyperintensity on DWI was associated with progression to HCC.

Preparation and use of reverse protein microarrays.

Reverse-phase protein array (RPPA) is a multiplex, high-throughput proteomic technique for profiling the activation status of signal transduction pathways involved in cancer survival and progression, potentially allowing for identification of new biomarkers and drug targets. On RPPA, the entire patient proteome is immobilized on a spot and single proteins can be quantified across a set of samples, spotted on the same array, with high specificity and sensitivity. Array immunostaining and signal amplification systems are used to generate a signal proportional to the concentration of the analyte. Dedicated scanners and software are used to detect spots, measure intensity, subtract background, normalize signal, and generate a numeric value as output. The generated output file is then analyzed using several different bioinformatic and biostatistical tools. In this unit, the RPPA procedure is described in depth, from sample handling and preparation to data analysis, with particular emphasis on tissue sample analysis.