Molecular Profiling Assays Research Articles

Abstract B094: Targeted expression and molecular profiling assay for tumor microenvironment

Abstract New rapid and robust transcriptome-based methods for cellular characterization of the tumor microenvironment and biomarker discovery are required to improve cancer prognosis and treatment. However, challenges with current approaches for the above applications include high sample requirements, poor sensitivity, low dynamic range, and limited throughput. To address these limitations, we have developed a unique approach for targeted transcriptome profiling using validated targeted primers that leverages the sensitivity of multiplex RT-PCR with the throughput of Next-Generation Sequencing (NGS) technology. By combining these methods, just 10-100 ng of total RNA is sufficient to quantify over 5 orders of magnitude variation in gene expression levels. Further, the use of targeted primers enables direct analysis of total RNA isolate and obviates the need for globin depletion from whole blood samples. Finally, using a defined set of amplicons to assess expression levels of all protein-coding genes facilitates and simplifies data analysis and allows more precise sample-to-sample normalization. We will present profiling results that demonstrate how this assay can be used to analyze the level of immune cell infiltration, assess intact and deficient immune mechanisms, and generally elucidate the tumor microenvironment of breast cancer samples. Citation Format: Alex Chenchik, Andrey Komarov, Michael Makhanov, Sunitha Sastry, Costa Frangou. Targeted expression and molecular profiling assay for tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B094.

TumorNext: A comprehensive tumor profiling assay that incorporates high resolution copy number analysis and germline status to improve testing accuracy.

The development of targeted therapies for both germline and somatic DNA mutations has increased the need for molecular profiling assays to determine the mutational status of specific genes. Moreover, the potential of off-label prescription of targeted therapies favors classifying tumors based on DNA alterations rather than traditional tissue pathology. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext, which can detect single nucleotide variants, small insertions and deletions in 142 genes that are frequently mutated in somatic and/or germline cancers. TumorNext also detects gene fusions and structural variants, such as tandem duplications and inversions, in 15 frequently disrupted oncogenes and tumor suppressors. The assay uses a matched control and custom bioinformatics pipeline to differentiate between somatic and germline mutations, allowing precise variant classification. We tested 170 previously characterized samples, of which > 95% were formalin-fixed paraffin embedded tissue from 8 different cancer types, and highlight examples where lack of germline status may have led to the inappropriate prescription of therapy. We also describe the validation of the Affymetrix OncoScan platform, an array technology for high resolution copy number variant detection for use in parallel with the NGS panel that can detect single copy amplifications and hemizygous deletions. We analyzed 80 previously characterized formalin-fixed paraffin-embedded specimens and provide examples of hemizygous deletion detection in samples with known pathogenic germline mutations. Thus, the TumorNext combined approach of NGS and OncoScan potentially allows for the identification of the “second hit” in hereditary cancer patients.

Abstract 3166: Querying, viewing and analyzing colorectal cancer translational research studies in tranSMART

Abstract All translational research projects basically share the same design, which is correlating variation in disease phenotype to variation in underlying biology. Typical questions to be addressed are: ‘Which (out of thousands) biomarkers predict good/bad outcome?’ and ‘Which (out of thousands) biomarkers predict whether a patient will benefit from a particular therapy?’. In line with this concept, many research teams have generated large amounts of experimental molecular data from patient samples, yet generally this information is inaccessible for examination due to local storage of both (meta)data and the data processing workflows used. Alternatively, data stored in central databases may only be available for exploration and interpretation by data specialists, provided that the processing workflow has been published and is available. Thus, if data is not recorded and easily retrievable, validation of obtained results (e.g. promising biomarkers) may be virtually impossible. Additionally, it will be difficult to query existing data sets to answer new questions, which may lead to experiments being unnecessarily repeated and biological materials being wasted. In the Netherlands, the Translational Research IT (TraIT) project initiated by the Center for Translational Molecular Medicine (CTMM, www.ctmm-trait.nl) aims to provide IT solutions to support translational research from start to end, including sustainable management and analysis of data. These data types involve the clinical, biomedical imaging, biobanking, (molecular) experimental data domains and their associated workflows. In addition to domain-specific solutions to manage these data types, the processed or ‘final’ data of these different domains will become available in the data-integration platform tranSMART for querying, visualization and analysis. To ensure sustainable data stewardship and provide easy access to existing data for the CTMM colorectal cancer project ‘Decrease in Colorectal Cancer Death (DeCoDe)’, data has been made available in tranSMART and more datasets are being added. The data encompasses both clinical and (molecular) experimental data from non-high-throughput molecular profiling (NHTMP) assays and array-based profiling techniques. It is now possible with tranSMART to explore the respective DeCoDe datasets and perform various analyses (survival, Fisher-exact tests, ANOVA, aCGH tests etc.), without needing deep bioinformatics expertise. Through metadata tags it is possible to trace back raw or pre-processed data in other tools (e.g. clinical data in OpenClinica, array-data in GEO, or histological images of tissue microarrays). Data can be examined in more detail by domain experts using tools they are familiar with or other tools provided by CTMM-TraIT. Thus, existing rich data are made findable, accessible, interoperable and reusable (FAIR) and source data can be traced back for customized processing. Citation Format: Mariska Bierkens, Wim van der Linden, Ward Weistra, Kees van Bochove, Jeroen A.M. Beliën, Remond J.A. Fijneman, Rita Azevedo, Jan-Willem Boiten, Gerrit A. Meijer. Querying, viewing and analyzing colorectal cancer translational research studies in tranSMART. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3166.

Proteoform profiling of peripheral blood serum proteins from pregnant women provides a molecular IUGR signature

Intrauterine growth restriction (IUGR) is an important cause of perinatal morbidity and mortality and contributes substantially to medically indicated preterm birth; preventing fetal death. Molecular profiling of the mothers' peripheral blood was desired to monitor the health conditions of the fetuses. To develop such a minimally invasive assay, we applied a protein affinity fractionation method to peripheral blood serum samples from pregnant women belonging to either the IUGR or to the control group. Proof-of-principle was shown by relative quantitation analysis of mixtures of intact proteoforms using MALDI-ToF mass spectrometry. The two best differentiating proteins and proteoforms, respectively, were apolipoprotein C-II and apolipoprotein C-III0. Together with three robustly expressed protein proteoforms proapolipoprotein C-II, apolipoprotein C-III1, and apolipoprotein C-III2, which served as landmarks for relative quantitation analysis, they constituted the maternal IUGR proteome signature. Separation confidence of our IUGR proteoform signature reached a sensitivity of 0.73 and a specificity of 0.87 with an area under curve of 0.86 in receiver operator characteristics. SignificanceIdentification of IUGR newborns in the case room is required as children are severely diseased and need specialized care during infancy. Yet, at time of birth there is no readily applicable clinical test available. Hence, a molecular profiling assay is highly desired.It needs to be mentioned that current clinical definitions and recommendations for IUGR are unfortunately misleading and are not universally applicable. The most commonly adopted definition is an abdominal circumference (AC) or estimated fetal weight measurement <10th percentile. Although both, the American College of Obstetricians and Gynecologists (ACOG) and the Royal College of Obstetricians and Gynecologists (RCOG) agree that at this cut-off the risk of perinatal morbidity and mortality increases, this definition does not take into account the individualized growth potential of each fetus. In particular its sole use fails to identify larger fetuses that have not achieved their growth potential and may be at risk of adverse outcomes. Also, this definition, when solely applied, will result in the misdiagnosis of IUGR for some constitutionally small fetuses. It needs to be pointed out that the above mentioned criteria can only be determined during pregnancy in case mothers report from early on during pregnancy.We have developed a test that relies on mass spectrometric analysis of the mother's serum protein composition (IUGR signature) which can be determined just ahead of delivery and at date of delivery, respectively using a minimal invasive blood sampling approach. With this manuscript we describe the use of a mass spectrometric profiling method of 30 peripheral blood samples from pregnant women prior to giving birth of either unsuspicious newborns or IUGR-affected infants.We report for the first time that maternal blood sample analysis via affinity mass spectrometry differentiates IUGR infants from controls with high confidence.

Expansion platform type II: testing a treatment strategy

Expansion platform type II: testing a treatment strategy

Back to Basics: Traditional Nottingham Grade Mitotic Counts Alone are Significant in Predicting Survival in Invasive Breast Carcinoma.

Newer multigene molecular profiling assays for breast carcinoma rely heavily on the quantification of genes of proliferation, whereas traditional histological grading reports the mitotic count. The mitotic activity of invasive breast carcinomas may be undervalued; therefore, an evaluation of the prognostic significance of mitotic score in predicting prognosis was performed. Retrospective analysis of a single institutional cohort of newly diagnosed estrogen receptor positive (ER+), HER2 negative (HER2-) unilateral invasive breast carcinomas was performed. Mitotic scores from the 3-part Nottingham combined histological grade were compared with clinical parameters. Mitoses were counted on Olympus BX50 microscopes and assigned scores of 1-3 based on observed mitoses. A total of 1292 ER+, HER2- invasive breast carcinoma patients were identified, with a median follow-up time of 2.6 years (range 0-14 years). Higher mitotic score was significantly associated with younger age, larger tumor size, angiolymphatic invasion, node-positive disease, higher stage, and the use of hormonal and cytotoxic chemotherapy. Mitotic score was significant in modeling time to local/regional recurrence (p = 0.02), recurrence-free survival/RFS (p < 0.001), and overall survival/OS (p = 0.01) with higher mitotic scores associated with worse outcomes. Higher mitotic score correlated significantly with intermediate/high risk Oncotype Dx recurrence scores (p = 0.009). First-generation molecular profiling assays for estrogen receptor positive invasive breast carcinomas derive much of their predictive power from quantifying genes of proliferation into a single score. Sometimes overlooked in the profusion of molecular data, the time-tested, mitotic count in the Nottingham combined histological grade is a good single-parameter predictor of survival.

Fighting a smarter war against cancer

Molecular Profiling in Cancer: Research or Practice, Georgetown University Hotel and Conference Center, Washington, DC, USA, 6–7 December 2013 On the 6th and 7th of December 2013, the Ruesch Center for the Cure of Gastrointestinal Cancers (DC, USA) hosted its fourth annual symposium entitled Molecular Profiling in Cancer: Research or Practice. This topic was selected for the 2013 symposium because of the explosion of molecular profiling assays that are available to us as practicing oncologists, which are believed, by most, to be a significant advancement in cancer care innovation. The Ruesch Center has been hosting topical symposia for the last 4 years. We have focused on: biomarker use in cancer medicine (2010); defining value in cancer care (2011); how to engage the ‘other 97%’ of patients in clinical trial enrollment, when currently we capture only 3% of patients on average (2012); and, of course, molecular profiling in cancer care (2013).

Molecular profiling and commercial predication assays in ovarian cancer: still not ready for prime time?

Short of early detection to allow curative primary intervention, the other major barrier to further success in treatment of ovarian cancers is matching the best treatment to the proper ovarian cancer type and to the individual patient. There are several decades of experience applying in vitro chemoresponse testing for solid tumors including ovarian cancer. This concept, first described in 1979, has yet to receive level one evidence supporting its application, despite the testing of numerous assays commercially as well as in academic centers and its use for tens of thousands of patients at a significant cost. The approach-rather than undergoing rigorous scientific examination-is now being muddied by the development of commercial molecular profiling assays from which treatment suggestions are provided. Molecular profiling as a research tool has added value to our understanding and treatment of patients with ovarian cancer. Morphologic and histochemical characterizations coupled now with increasing knowledge of ovarian cancer type-specific molecular patterns is improving our ability to properly diagnosis ovarian cancer type and thus guide therapy. With the exception of the role of germ-line and possibly somatic BRCA1 and BRCA2 mutations and their true predictiveness for probable response to poly(ADP-ribose) polymerase inhibition, molecular typing and profiling has yet to identify druggable molecular targets in ovarian cancer. Its use should be continued as a research and learning tool, and its results should be subjected to clinical trial validation. For very different reasons, neither chemoresponse assays nor molecular profiling are ready for prime time, yet.

Overview of Various Techniques/Platforms With Critical Evaluation of Each

Patients with cancer of unknown primary are common and can present in a variety of ways with different histological features. Despite best clinical effort in pretreatment diagnostic workup, many patients classified as having cancer of unknown primary (CUP) often are still left with no definitive diagnosis of the primary organ or tissue of origin to account for the metastatic disease. Whereas advances in immunohistochemical techniques have improved the diagnostic yield to some extent, the challenges remain substantial for most patients with CUP in whom initial therapy is typically chosen empirically. In recent years, development of molecular gene profiling of tumor offers new possibilities to better characterize, diagnose, and classify the tissue of origin of various metastatic CUP to better inform optimal therapy. The premise behind the development of improved diagnostic tools to better diagnose the organ or tissue of origin for metastatic disease of unknown primary is that an organ/tissue-specific tailored therapy of choice would favorably impact the treatment outcome. There are now three commercially available molecular profiling platforms for the purpose of diagnosing the tissue of origin in the otherwise CUP patients: 1) bioTheranostics: Cancer TYPE ID® (qRT-PCR for mRNA); 2) Pathworks®: Tissue of origin test (microarray for mRNA expression); and 3) Rosetta Genomics-Prometheus: miRview™ mets (ProOnc Tumor SourceDxT) (qRT-PCR for microRNA). Whereas these are new technologic platforms that offer new promise for better diagnostics and perhaps better therapeutic strategies in cancer therapy, each of the platforms has its own strengths and limitations due to their test of choice and assay source materials and technical platform itself. However, a fundamental question that needs be further addressed regarding the utility of these novel molecular profiling assays is whether they represent more superior approaches than genomic profiling assays using rapidly emerging cancer genomics next-generation sequencing (NSG) platforms. Because cancer is nowadays understood as genomic disease, the genomic alterations (e.g., mutations, copy number variations, chromosomal translocations, splicing variants) may offer more important insights into the cancer pathogenesis. More importantly, these genomic information may be more relevant in guiding personalized/precision cancer therapy than merely empiric chemotherapy based on tissue/organ-of-origin information. Ideally, further comparative studies and demonstration of utilities would be needed and eagerly anticipated to determine which diagnostic approach ultimately could impact the clinical outcome of patients with CUP.

The molecular landscape of diffuse glioma and prospects for biomarker development

High-throughput molecular profiling is transforming long-standing conceptions of diffuse gliomas, the most common primary brain tumors. Indeed, comprehensive genomic, transcriptomic and epigenomic analyses have not only provided striking mechanistic insights into the pathogenesis of diffuse gliomas but also greatly enriched the pool of potential biomarkers for prognostic and predictive patient stratification. This article summarizes significant recent developments in the molecular characterization of diffuse gliomas, focusing on implications for biomarker development and application. In doing so, we will also address relevant high-throughput molecular profiling technologies and both the opportunities and challenges implicit in their widespread incorporation into disease management workflows. Although the number of validated biomarkers guiding diffuse glioma management is currently quite small, rapidly progressing molecular annotation continues to provide a steady stream of clinically relevant candidates, many of which show promise for predictive capabilities in the context of specific targeted therapeutics. Such potential now requires rigorous validation in well-designed clinical trials supported by robust molecular profiling assays operative from standard clinical material.

Cancer of Unknown Primary Site: Improved Patient Management with Molecular and Immunohistochemical Diagnosis

Cancer of unknown primary site (CUP) is a common heterogeneous clinicopathologic syndrome, but investigations and publications regarding these patients are rare. For the last 20 years, empiric "broad-spectrum" chemotherapy has been the standard therapy for the majority of these patients. More recently, improved immunocytochemistry and advent of gene-expression profiling have provided the diagnostic tools necessary to accurately define the tissue of origin in most patients. Molecular profiling assays complement standard pathologic diagnosis, and a recently reported large prospective study demonstrated an improvement in outcome for patients treated with site-specific therapy directed by the molecular assay diagnoses compared with empiric chemotherapy. Survival in molecularly diagnosed patients was as expected for those particular tumor types. The evaluation of patients has become more standardized. The empiric-chemotherapy era is ending and customized therapies based on accurate tissue of origin diagnoses have arrived. Eventually the recognition of the molecular aberrations responsible for the growth and metastasis of solid tumors, regardless of the tissue of origin, will lead to more precise and effective therapy for patients with advanced cancers.

Cancer of Unknown Primary Site: Improved Patient Management with Molecular and Immunohistochemical Diagnosis

Cancer of unknown primary site (CUP) is a common heterogeneous clinicopathologic syndrome, but investigations and publications regarding these patients are rare. For the last 20 years, empiric “broad-spectrum” chemotherapy has been the standard therapy for the majority of these patients. More recently, improved immunocytochemistry and advent of gene-expression profiling have provided the diagnostic tools necessary to accurately define the tissue of origin in most patients. Molecular profiling assays complement standard pathologic diagnosis, and a recently reported large prospective study demonstrated an improvement in outcome for patients treated with site-specific therapy directed by the molecular assay diagnoses compared with empiric chemotherapy. Survival in molecularly diagnosed patients was as expected for those particular tumor types. The evaluation of patients has become more standardized. The empiric-chemotherapy era is ending and customized therapies based on accurate tissue of origin diagnoses have arrived. Eventually the recognition of the molecular aberrations responsible for the growth and metastasis of solid tumors, regardless of the tissue of origin, will lead to more precise and effective therapy for patients with advanced cancers.

StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health

Much of the excitement generated by induced pluripotent stem cell technology is concerned with the possibility of disease modeling as well as the potential for personalized cell therapy. However, to pursue this it is important to understand the ‘normal’ pluripotent state including its inherent variability. We have performed various molecular profiling assays for 21 hESC lines and 8 hiPSC lines to generate a comprehensive snapshot of the undifferentiated state of pluripotent stem cells. Analysis of the gene expression data revealed no iPSC-specific gene expression pattern in accordance with previous reports. We further compared cells, differentiated as embryoid bodies in 2 media proposed to initiate differentiation towards separate cell fates, as well as 20 adult tissues. From this analysis we have generated a gene list which defines pluripotency and establishes a baseline for the pluripotent state. Finally, we provide lists of genes enriched under both differentiation conditions which show the proposed bias toward independent cell fates.

Development of a molecular profiling assay for circulating tumor cells (CTCs) utilizing automated multiplexed in situ hybridization for metastatic castrate-resistant prostate cancer (mCRPC).

39 Background: ERG gene rearrangements lead to overexpression of the ERG oncogene in 50% of localized and metastatic prostate cancers. Recent studies have indicated that this gene fusion correlates with androgen sensitivity in prostate cancer patients and an association with increased DNA Repair activity (PARP1). The PTEN tumor suppressor has also been shown to regulate the AKT pathway, and related to increased PARP1 activity. We hypothesized that future PARP1 inhibitors and other targeted therapies for mCRPC will likely require the development of a companion diagnostic assay for measuring ERG rearrangement and PTEN deletion status in CTCs. Methods: We isolated CTCs utilizing the CellSearch system from mCRPC patients for enumeration. Following enumeration, the cells were transferred to glass slides and interrogated utilizing the automated Benchmark staining platform with multiplexed Quantum Dot FISH inclusive of probes for 5’ERG, 3’ERG, PTEN, and Cen 10. FISH signals were visualized utilizing spectral imaging. Results: Successful transfer of the CTCs from the CellSearch to a glass slide were achieved In model cell line systems, in cultured cells (LnCaP and VCaP) either directly or after spiking to PBL and five out of 6 times we were able to successfully demonstrate simultaneous single copy 5’ and 3’ ERG and PTEN/Cen 10 labeled probes. Studies are in progress to extend the clinical utility of this assay to isolated CTC from cases with metastatic prostate cancer. Conclusions: CTCs are likely to be utilized clinically for monitoring disease progression mCRPC patients as measures for therapeutic efficacy. As more therapeutics are approved for use in mCRPC patients, the ability for oncologists to molecularly characterize the type of CTCs driving tumor burden is likely to play a significant role in optimizing patient management by determining the type and the sequence of future targeted therapies. We have for the first time demonstrated a platform to molecularly characterize patient CTCs on an automated platform that is amenable to standard clinical use.

A Retrospective Study of Treatment Outcomes in Patients With Carcinoma of Unknown Primary Site and a Colorectal Cancer Molecular Profile

Molecular tumor profiling is a new method of identifying the tissue of origin in patients with carcinoma of unknown primary (CUP) site. However the value of this information in improving treatment outcomes is undefined. We evaluated results of site-specific treatment in a group of patients with CUP in whom molecular profiling predicted a colorectal site of origin. Tissue of origin predictions by a 92-gene real-time polymerase chain reaction (RT-PCR) molecular profiling assay (CancerTYPE ID; bioTheranostics, Inc, San Diego, CA) from March 2008 to August 2009 were reviewed. One hundred twenty-five of 1544 patients (8%) assayed were predicted to have a colorectal tissue of origin with > 80% probability. Surveys were sent to the physicians of these 125 patients requesting deidentified patient information. Information was provided for 42 of 125 patients (34%). Thirty-two patients received either first- or second-line therapy with colorectal cancer regimens; the overall response rate was 50%. Patients who received first-line empirical therapy for CUP had an overall response rate of 17%. The median survival of patients who received site-specific therapy for colorectal cancer was 27 months. Patients predicted to have a colorectal site of origin by molecular tumor profiling had median survival when treated with site-specific regimens that was similar to survival in patients with known metastatic colon cancer. The median survival in this group was substantially better than the historical median survival for patients with CUP (range 8-11 months) when treated with empirical CUP regimens. Molecular tumor profiling seems to improve survival by allowing specific therapy in this patient subgroup; prospective trials are ongoing to confirm these observations.

Gene expression profiling from formalin-fixed, paraffin-embedded tissue for tumor diagnosis

BackgroundMolecular profiling assays have emerged as a promising tool in tumor diagnosis. Recent advances have allowed the use of formalin-fixed, paraffin-embedded (FFPE) tissues in such assays involving the use of microarrays. The Pathwork Tissue of Origin (TOO) Test was developed for use with FFPE tissue to aid tumor diagnosis. We sought to determine the performance of the TOO test on routine specimens from an independent laboratory. MethodsForty-five blinded, archived clinical specimens from the UCSF Department of Pathology were tested. Total RNA was processed to prepare labeled cDNA for hybridization to Pathchip microarrays. Hybridization data was analyzed with a 2000-gene classification model to quantify similarity between RNA expression of the study specimens and the 15 tissues on the test panel. Results44/45 (98%) specimens were successfully processed. 37 cases met study inclusion criteria. Of these, 35 (95%) gave results which were in agreement with the reference diagnosis. In no case was the reference diagnosis ruled out. ConclusionsThe Tissue of Origin Test gave a high agreement with the reference diagnosis when archived clinical specimens from UCSF were assessed. Molecular profiling assays are highly accurate, and can be a useful tool in cancer diagnosis.

Molecular tumor classification using a 92-gene assay in the differential diagnosis of squamous cell lung cancer

Recent progress in targeted cancer therapies for advanced non-small cell lung cancer (NSCLC) has expanded the selection of optimal treatment beyond platinum-based chemotherapy and identifi ed differential response and safety profi les based on histology. Given that accurate pathologic classifi cation is of increasing clinical interest, molecular diagnostics presents a standardized approach to determining the tissue of origin and the histologic subtype in conjunction with routine evaluation. The objective of this retrospective case study was to characterize the clinical utility of molecular profi ling in diagnoses and treatment of squamous cell lung cancer by (1) comparing molecular results with pathologic correlates in lung classifi cation and subclassifi cation, (2) characterizing case features to better understand the clinical indications for molecular testing, and (3) evaluating the potential clinical impact based on how test results were utilized in rendering clinical diagnoses and treatment selections. Sixty-two cases predicted to be squamous cell lung cancer by a 92-gene molecular profi ling assay (CancerTYPE ID) were evaluated. Specimen characteristics and abstracted pathology information revealed that the clinical indications for CancerTYPE ID were high-grade tumors with equivocal histopathology and/or immunohistochemistry and were utilized to potentially resolve a differential diagnosis (47%), to identify an unknown/uncertain primary tumor origin (32%), or to confi rm a primary tissue origin (21%). A physician survey of clinical strategy pre and post assay showed that CancerTYPE ID guided, confi rmed, or changed the primary tumor site for treatment purposes in 85% of the 20 evaluable cases and the tumor subtype in 60% of the tumors tested. Results of this case series present preliminary evidence that molecular profi ling provides additional information on tumor site and subtype in conjunction with standard clinical evaluation.

Diagnosis of Metastatic Breast Cancer—A Case Report Using Molecular Cancer Classification

An accurate cancer diagnosis is critical as it can direct the use of site-directed, and potentially more effective, treatment options for specific types of cancer. A differential or uncertain diagnosis could prevent cancer patients from receiving optimal treatment, thus affecting their overall prognosis. Advances in molecular technology have led to the development of molecular cancer classifiers that can direct or confirm the diagnosis of metastatic cancers which would otherwise be considered uncertain or unknown. This case report describes the role of molecular diagnostics in the evaluation of a patient with a large pancreatic mass and a history of breast cancer. Results from a 92-gene molecular profiling assay (CancerTYPE ID®) predicted that this new mass was breast cancer. This diagnosis allowed for effective treatment and complete response in this patient.

Molecular Classification of Cancers of Unknown Primary Site

Cancer of unknown primary site is a common metastatic cancer, diagnosed in about 50 000 patients per year in the US. The diagnosis, classification, and management of patients with carcinoma of unknown primary site has been difficult and frustrating. The therapy has usually been empiric for the majority of patients, and their prognosis has been poor. Molecular classification of metastatic cancers with known primary sites has been accurate (76-89%), as reported from several studies. Molecular profiling of initial biopsy specimens has tremendous potential as a test to diagnose the site of tumor origin in patients with unknown primary cancer. Several retrospective studies of molecular profiling assays have provided indirect validation of the accuracy of primary site prediction, based on correlations with clinicopathologic features. One additional study of initial diagnostic biopsies in unknown primary cancer patients, where primary tumor sites were identified months to years later, has provided more direct validation of the accuracy of molecular classification (the primary tumor sites of 15 of 20 patients were correctly predicted). The ability to diagnose and classify unknown primary cancer more precisely would allow for more site-specific or targeted therapy, and likely improve patient outcomes. Several clinical studies are in progress or planned to test this concept.

Histopathology of primary breast cancer 2003

The histologic and immunophenotypic characterisation of primary breast carcinoma admittedly is a finite and imperfect source of information for an ideal prognostic and predictive evaluation, and for tailoring the most appropriate adjuvant intervention for each individual patient. Complementary data coming from molecular profiling assays hopefully will significantly add to our current skills in predicting the future. Undoubtedly, however, the traditional morphological and immunohistochemical features are the faithful mirror of the differential expression of hundreds of genes, which may therefore be safely and consistently evaluated even by simple histologic examination and immunohistochemical assays. What is nowadays especially needed is to improve our skills and technical means for assuring accuracy and reproducibility of the morphological and immunohistochemical findings.