Profiling molecular regulators of recurrence in chemorefractory triple-negative breast cancers.

BACKGROUND: Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development. METHODS: We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes. RESULTS: Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2. CONCLUSIONS: We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation.

Evaluation of conventional training in Clinical Breast Examination (CBE).

BACKGROUND: Clinical Breast Examination (CBE) is the examination of a women's breasts by a healthcare professional, such as a breast surgeon, family physician or breast-care nurse who is trained to recognise many different types of abnormalities and warning signs in the breast [1]. CBE is particularly important in rural areas and developing countries who have limited access to technology such as mammography. CBE needs to be taught to health professionals like any other clinical skill used by medical professionals in the workplace. CBE in part involves palpation of the breast, that is, determining by touch which breast lumps are normal and which are suspicious in feeling. The gold standard for assessing tactile skills in CBE is seeing whether students can accurately identify and discriminate between different breast lumps also known as masses (IDBM) on actual patients in a clinical setting. However, this is not practical in a medical education setting. Usually the testing methods 'go through the motions' of feeling the breast as part of CBE. So the students' technique is examined either using unrealistic simulation models or using an intimate examination associate (IEA), an actor/volunteer who permits students to examine their intimate body parts such as breast or genitals for teaching purposes. These volunteers do not have any abnormalities so this teaching does not include the actual detection of suspicious lumps. We undertook a study of clinical skill with 10 medical students to examine different methods of assessing novice student clinical skills after a brief training in CBE. OBJECTIVES: This study aims to evaluate the effectiveness of current training and assessment of novice students in CBE and their capacity to identify and discriminate breast masses (IDBM) on actual patients. METHODS: We assessed each student's IDBM ability in an actual clinical situation, a breast clinic with a mixture of eight IEAs and one real patient with a large, easily palpable, putative breast cancer. We recruited 10 clinically inexperienced medical students, who were trained for 30 minutes by two breast surgeons using an IEA. Students were tested in a simulated clinical setting, a breast clinic where each examined 4 IEAs and one patient. The students were blind to who was the real patient and who was an IEA. Patients were examined by a breast surgeon in private prior to the commencement in the study. The breast surgeon recorded any clinical finding on the patients during the initial examination. The surgeon coached each patient on how to mark the students and showed the patient their results so the patients had a benchmark. After each examination was finished the students had four different assessments: 1) patients marked each student, 2) students were independently proctored - that is, marked by an expert, 3) students recorded their clinical findings and 4) students recorded how confident they were that they had the correct findings. Results from different kinds of student assessments were compared.RESULTSA chi-square test for independence between true positive or negative masses versus student-assessed positive or negative masses was not significant at alpha = .05. This means that there was no statistical association in the indication of positive or negative presence of masses versus whether such masses were actually present or absent. By comparison, experts (breast surgeons) were able to detect normal and abnormal breast masses by palpation alone 100% of the time and rate their confidence level as 'certain'. Unlike the experts, student self-reported confidence was unrelated to their competence score (CS). Proctoring was inversely related to the students' CS.CONCLUSIONSThe main conclusion is that novice students do not seem to be able to accurately detect breast masses in a clinical setting even after training. On the basis of these results, we believe that a comprehension component in the current CBE testing is needed in addition to the current methods of testing.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array.

BACKGROUND: Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. RESULTS: We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. CONCLUSION: Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.

Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.

OBJECTIVE: Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs). DESIGN: Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility. RESULTS: RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale). CONCLUSIONS: Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.

Integrated detection and population-genetic analysis of SNPs and copy number variation.

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.