Comprehensive assessment of germline pathogenic variant detection in tumor-only sequencing.

BACKGROUND: Tumor-only sequencing, implemented for the identification of somatic variants, is oftentimes used for the detection of actionable germline variants. We sought to determine whether tumor-only sequencing assays are suitable for detection of actionable germline variants, given their importance for the delivery of targeted therapies and risk-reducing measures. PATIENTS AND METHODS: The detection of germline variants affecting moderate- and high-penetrance cancer susceptibility genes (CSGs) by tumor-only sequencing was compared to clinical germline testing in 21 333 cancer patients who underwent tumor and germline testing using the Food and Drug Administration (FDA)-authorized Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Targets (MSK-IMPACT) assay. Seven homologous recombination deficiency (HRD), two DNA damage response (DDR) and four mismatch repair (MMR) genes, as well as NF1, RB1 and TP53 were included in the analysis. FDA-authorized and New York State Department of Health-approved sequencing methods for germline, tumor/normal and tumor-only sequencing assays and analytical pipelines were employed. RESULTS: In patients who underwent tumor and germline sequencing, as compared to clinical genetic testing, tumor-only sequencing failed to detect 10.5% of clinically actionable pathogenic germline variants in CSGs, including 18.8%, 12.8% and 7.3% of germline variants in MMR, DDR and HRD genes, respectively. The sensitivity for detection of pathogenic germline variants by tumor-only sequencing was 89.5%. Whilst the vast majority of pathogenic germline exonic single-nucleotide variants (SNVs) and small indels were detected by tumor-only sequencing, large percentages of germline copy number variants, intronic variants and repetitive element insertions were not detected. CONCLUSIONS: Tumor-only sequencing is adequate for the detection of clinically actionable germline variants, particularly for SNVs and small indels; however, a small subset of alterations affecting HRD, DDR and MMR genes may not be detected optimally. Therefore, for high-risk patients with negative tumor-only sequencing results, clinical genetic testing could be considered given the impact of these variants on therapy and genetic counseling.

A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.

BACKGROUND: HER2-positive (+) breast cancers, defined by HER2 overexpression and/or amplification, are often addicted to HER2 to maintain their malignant phenotype. Yet, some HER2+ tumors do not benefit from anti-HER2 therapy. We hypothesize that HER2 amplification levels and PI3K pathway activation are key determinants of response to HER2-targeted treatments without chemotherapy. PATIENTS AND METHODS: Baseline HER2+ tumors from patients treated with neoadjuvant lapatinib plus trastuzumab [with endocrine therapy for estrogen receptor (ER)+ tumors] in TBCRC006 (NCT00548184) were evaluated in a central laboratory for HER2 amplification by fluorescence in situ hybridization (FISH) (n = 56). HER2 copy number (CN) and FISH ratios, and PI3K pathway status, defined by PIK3CA mutations or PTEN levels by immunohistochemistry were available for 41 tumors. Results were correlated with pathologic complete response (pCR; no residual invasive tumor in breast). RESULTS: Thirteen of the 56 patients (23%) achieved pCR. None of the 11 patients with HER2 ratio <4 and/or CN <10 achieved pCR, whereas 13/45 patients (29%) with HER2 ratio ≥4 and/or CN ≥10 attained pCR (P = 0.0513). Of the 18 patients with tumors expressing high PTEN or wild-type (WT) PIK3CA (intact PI3K pathway), 7 (39%) achieved pCR, compared with 1/23 (4%) with PI3K pathway alterations (P = 0.0133). Seven of the 16 patients (44%) with HER2 ratio ≥4 and intact PI3K pathway achieved pCR, whereas only 1/25 (4%) patients not meeting these criteria achieved pCR (P = 0.0031). CONCLUSIONS: Our findings suggest that there is a clinical subtype in breast cancer with high HER2 amplification and intact PI3K pathway that is especially sensitive to HER2-targeted therapies without chemotherapy. A combination of HER2 FISH ratio and PI3K pathway status warrants validation to identify patients who may be treated with HER2-targeted therapy without chemotherapy.

Unravelling tumour heterogeneity using next-generation imaging: radiomics, radiogenomics, and habitat imaging.

Tumour heterogeneity in cancers has been observed at the histological and genetic levels, and increased levels of intra-tumour genetic heterogeneity have been reported to be associated with adverse clinical outcomes. This review provides an overview of radiomics, radiogenomics, and habitat imaging, and examines the use of these newly emergent fields in assessing tumour heterogeneity and its implications. It reviews the potential value of radiomics and radiogenomics in assisting in the diagnosis of cancer disease and determining cancer aggressiveness. This review discusses how radiogenomic analysis can be further used to guide treatment therapy for individual tumours by predicting drug response and potential therapy resistance and examines its role in developing radiomics as biomarkers of oncological outcomes. Lastly, it provides an overview of the obstacles in these emergent fields today including reproducibility, need for validation, imaging analysis standardisation, data sharing and clinical translatability and offers potential solutions to these challenges towards the realisation of precision oncology.

Response to dual HER2 blockade in a patient with HER3-mutant metastatic breast cancer.

BACKGROUND: HER3 activating mutations have been shown in preclinical models to be oncogenic and ligand-independent, but to depend on kinase-active HER2. PATIENTS AND METHODS: Whole-exome sequencing of the primary HER2-negative breast cancer and its HER2-negative synchronous liver metastasis from a 46-year-old female revealed the presence of an activating and clonal HER3 G284R mutation. RESULTS: HER2 dual blockade with trastuzumab and lapatinib as third-line therapy led to complete metabolic response in 2 weeks and confirmed radiological partial response after 8 weeks. Following the resection of the liver metastasis, the patient remains disease-free 40 weeks after initiation of the HER2 dual blockade therapy. Immunohistochemical analysis demonstrated a substantial reduction of phospho-rpS6 and phospho-AKT in the post-therapy biopsy of the liver metastasis. DISCUSSION: This is the first-in-man evidence that anti-HER2 therapies are likely effective in breast cancers harboring HER3 activating mutations.

Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle.

BACKGROUND: Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy. PATIENT AND METHODS: A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor. RESULTS: Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography. CONCLUSIONS: This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity. REGISTERED CLINICAL TRIAL: www.clinicaltrials.gov, NCT01090960.

Genomic analyses to select patients for adjuvant chemotherapy: trials and tribulations.

Despite improvements in adjuvant chemotherapy regimens in breast cancer, personalised use of specific cytotoxic regimens remains a clinical challenge. Defining the correct therapeutic strategy for individual patients with breast cancer based on the genomic and transcriptomic characteristics of the tumour and the patient remains an area of unmet clinical need. Despite the promise of microarray-based predictors of response to chemotherapy, clinical decisions are still guided by a limited constellation of biomarkers. In this review we will address current genomic and transcriptomic approaches to the stratification of adjuvant therapies in breast cancer, the reasons for the limited success in the incorporation of novel multi-gene predictors of response to chemotherapy in clinical practice and focus on new approaches that aim to understand the clonal evolution of the disease. The polygenic nature of drug resistance, and inter- and intra-tumour heterogeneity are considered as important research areas, given that they may constitute important challenges for the development of chemotherapy-specific response predictors.

The 70-gene prognosis signature predicts early metastasis in breast cancer patients between 55 and 70 years of age.

BACKGROUND: The majority of breast cancer patients are postmenopausal women who are increasingly being offered adjuvant chemotherapy. Since the beneficial effect of chemotherapy in postmenopausal patients predominantly occurs in the first 5 years after diagnosis, a prognostic marker for early events can be of use for adjuvant treatment decision making. The aim of this study was to evaluate the prognostic value of the 70-gene prognosis signature for early events in postmenopausal patients. METHODS: Frozen tumor samples from 148 patients aged 55-70 years were selected (T1-2, N0) and classified by the 70-gene prognosis signature (MammaPrint) into good or poor prognosis. Eighteen percent received hormonal therapy. RESULTS: Breast cancer-specific survival (BCSS) at 5 years was 99% for the good-prognosis signature versus 80% for the poor-prognosis signature group (P = 0.036). The 70-gene prognosis signature was a significant and independent predictor of BCCS during the first 5 years of follow-up with an adjusted hazard ratio of 14.4 (95% confidence interval 1.7-122.2; P = 0.01) at 5 years. CONCLUSION: The 70-gene prognosis signature can accurately select postmenopausal patients at low risk of breast cancer-related death within 5 years of diagnosis and can be of clinical use in selecting postmenopausal women for adjuvant chemotherapy.

A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks.

Gene expression microarrays allow for the high throughput analysis of huge numbers of gene transcripts and this technology has been widely applied to the molecular and biological classification of cancer patients and in predicting clinical outcome. A potential handicap of such data intensive molecular technologies is the translation to clinical application in routine practice. In using an artificial neural network bioinformatic approach, we have reduced a 70 gene signature to just 9 genes capable of accurately predicting distant metastases in the original dataset. Upon validation in a follow-up cohort, this signature was an independent predictor of metastases free and overall survival in the presence of the 70 gene signature and other factors. Interestingly, the ANN signature and CA9 expression also split the groups defined by the 70 gene signature into prognostically distinct groups. Subsequently, the presence of protein for the principal prognosticator gene was categorically assessed in breast cancer tissue of an experimental and independent validation patient cohort, using immunohistochemistry. Importantly our principal prognosticator, CA9, showed that it is capable of selecting an aggressive subgroup of patients who are known to have poor prognosis.

Refinement of breast cancer classification by molecular characterization of histological special types.

Most invasive breast cancers are classified as invasive ductal carcinoma not otherwise specified (IDC NOS), whereas about 25% are defined as histological 'special types'. These special-type breast cancers are categorized into at least 17 discrete pathological entities; however, whether these also constitute discrete molecular entities remains to be determined. Current therapy decision-making is increasingly governed by the molecular classification of breast cancer (luminal, basal-like, HER2+). The molecular classification is derived from mainly IDC NOS and it is unknown whether this classification applies to all histological subtypes. We aimed to refine the breast cancer classification systems by analysing a series of 11 histological special types [invasive lobular carcinoma (ILC), tubular, mucinous A, mucinous B, neuroendocrine, apocrine, IDC with osteoclastic giant cells, micropapillary, adenoid cystic, metaplastic, and medullary carcinoma] using immunohistochemistry and genome-wide gene expression profiling. Hierarchical clustering analysis confirmed that some histological special types constitute discrete entities, such as micropapillary carcinoma, but also revealed that others, including tubular and lobular carcinoma, are very similar at the transcriptome level. When classified by expression profiling, IDC NOS and ILC contain all molecular breast cancer types (ie luminal, basal-like, HER2+), whereas histological special-type cancers, apart from apocrine carcinoma, are homogeneous and only belong to one molecular subtype. Our analysis also revealed that some special types associated with a good prognosis, such as medullary and adenoid cystic carcinomas, display a poor prognosis basal-like transcriptome, providing strong circumstantial evidence that basal-like cancers constitute a heterogeneous group. Taken together, our results imply that the correct classification of breast cancers of special histological type will allow a more accurate prognostication of breast cancer patients and facilitate the identification of optimal therapeutic strategies.

Compromised BRCA1-PALB2 interaction is associated with breast cancer risk.

The major breast cancer suppressor proteins BRCA1 and BRCA2 play essential roles in homologous recombination (HR)-mediated DNA repair, which is thought to be critical for tumor suppression. The two BRCA proteins are linked by a third tumor suppressor, PALB2, in the HR pathway. While truncating mutations in these genes are generally pathogenic, interpretation of missense variants remains a challenge. To date, patient-derived missense variants that disrupt PALB2 binding have been identified in BRCA1 and BRCA2; however, there has not been sufficient evidence to prove their pathogenicity in humans, and no variants in PALB2 that disrupt either its BRCA1 or BRCA2 binding have been reported. Here we report on the identification of a novel PALB2 variant, c.104T>C (p.L35P), that segregates in a family with a strong history of breast cancer. Functional analyses showed that L35P abrogates the PALB2-BRCA1 interaction and completely disables its abilities to promote HR and confer resistance to platinum salts and PARP inhibitors. Whole-exome sequencing of a breast cancer from a c.104T>C carrier revealed a second, somatic, truncating mutation affecting PALB2, and the tumor displays hallmark genomic features of tumors with BRCA mutations and HR defects, cementing the pathogenicity of L35P. Parallel analyses of other germline variants in the PALB2 N-terminal BRCA1-binding domain identified multiple variants that affect HR function to varying degrees, suggesting their possible contribution to cancer development. Our findings establish L35P as the first pathogenic missense mutation in PALB2 and directly demonstrate the requirement of the PALB2-BRCA1 interaction for breast cancer suppression.

Mechanism of action of a WWTR1(TAZ)-CAMTA1 fusion oncoprotein.

The WWTR1 (protein is known as TAZ)-CAMTA1 (WC) fusion gene defines epithelioid hemangioendothelioma, a malignant vascular cancer. TAZ (transcriptional coactivator with PDZ binding motif) is a transcriptional coactivator and end effector of the Hippo tumor suppressor pathway. It is inhibited by phosphorylation by the Hippo kinases LATS1 and LATS2. Such phosphorylation causes cytoplasmic localization, 14-3-3 protein binding and the phorphorylation of a terminal phosphodegron promotes ubiquitin-dependent degradation (the phosphorylation of the different motifs has several effects). CAMTA1 is a putative tumor suppressive transcription factor. Here we demonstrate that TAZ-CAMTA1 (TC) fusion results in its nuclear localization and constitutive activation. Consequently, cells expressing TC display a TAZ-like transcriptional program that causes resistance to anoikis and oncogenic transformation. Our findings elucidate the mechanistic basis of TC oncogenic properties, highlight that TC is an important model to understand how the Hippo pathway can be inhibited in cancer, and provide approaches for targeting this chimeric protein.

Systematic screening for mutations in the 5'-regulatory region of the human dopamine D1 receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder.

A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5'-flanking region of the dopamine D1 receptor (DRD1) gene. This region has previously been shown to contain a functional promoter [Minowa et al., 1992: Proc Natl Acad Sci 89:3045-3049; Minowa et al., 1993: J Biol Chem 268:23544-23551]. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5'-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder.

Systematic screening for mutations in the human serotonin 1F receptor gene in patients with bipolar affective disorder and schizophrenia.

Using single strand conformational analysis we screened the complete coding sequence of the serotonin 1F (5-HT1F) receptor gene for the presence of DNA sequence variation in a sample of 137 unrelated individuals including 45 schizophrenic patients, 46 bipolar patients, as well as 46 healthy controls. We detected only three rare sequence variants which are characterized by single base pair substitutions, namely a silent T-->A transversion in the third position of codon 261 (encoding isoleucine), a silent C-->T transition in the third position of codon 176 (encoding histidine), and an C-->T transition in position -78 upstream from the start codon. The lack of significant mutations in patients suffering from schizophrenia and bipolar affective disorder indicates that the 5-HT1F receptor is not commonly involved in the etiology of these diseases.

Adenosine A1 receptor and bipolar affective disorder: systematic screening of the gene and association studies.

In the present study we sought to identify genetic variation in the adenosine A1 receptor (A1AR) gene on chromosome 1q31-32.1, which through alteration of protein function or level of expression might contribute to the genetic predisposition to bipolar affective disorder. We performed a systematic mutation scan of the whole coding sequence as well as 5' and 3' untranslated regions by means of single-strand conformation analysis. The region upstream to the coding sequence we investigated contains two functional promoters. Screening 42 patients with bipolar affective disorder, we detected 11 DNA sequence variants (48T/A, 267 + 275C/T, 805T/G, 1777C/A, 1827C/T, 1904C/T, 2126G/T, 2294insT, 2776C/T, 2777del36, 2819T/G). Determining the frequency of these variants in 42 anonymous blood donors, we observed a non-significant (P < 0.06) trend towards an underrepresentation of the 2126T variant in patients when compared to controls. On the other hand, the 2777del36 and the 2819G variant were not found among the controls. These findings were followed up in a large independent replication sample. However, we were not able to confirm the initial findings in the second sample. Our data suggest that genetically determined variation of the A1AR and its two promoters do not play a major role in the development of bipolar affective disorder.

Expression of a novel lacrimal gland gene lacritin in human breast tissues.

PURPOSE: The purpose of this study was to examine breast tumors and normal breast tissues for the expression of the glycoprotein lacritin. The mRNA and protein expression of lacritin has been reported to be restricted to the lacrimal gland. METHODS: We investigated 37 human primary invasive breast tumors, seven breast cancer cell lines and 16 normal breast tissues by quantitative real-time PCR for lacritin expression. RESULTS: We detected lacritin transcripts in 51% (19/37) of the primary invasive breast tumors, in 71% (5/7) of the breast cancer cell lines, and also in 56% (9/16) of the normal breast tissues. No lacritin mRNA was detectable in peripheral blood of healthy individuals. CONCLUSIONS: Here we show by quantitative real-time PCR that lacritin is expressed in human breast tumors, breast cancer cell lines, and normal breast. The previously reported restricted expression pattern of lacritin is therefore incorrect. Lacritin transcripts were not detected in peripheral blood which makes lacritin a potential candidate as a breast cancer marker gene.

5-HT2A receptor and bipolar affective disorder: association studies in affected patients.

The aim of this study was to investigate the possible involvement of genetic variation in serotonin receptors in the aetiology of bipolar affective disorder. The 5-HT2A receptor gene was systematically screened for genetic variants by single strand conformation polymorphism (SSCP) methods in subjects with bipolar affective disorder. Four polymorphisms (two structural changes, Thr25Asn and His4 M52Tyr, and two silent polymorphisms, 102-T/C and 516-C/T) which had previously been found in patients with schizophrenia and control subjects were detected. No novel polymorphisms were found in patients with bipolar affective disorder. These polymorphisms were genotyped in a sample of 129 patients and 252 controls of German origin and 176 patients and 182 controls of British origin. No strong associations were found between any of these polymorphisms and bipolar affective disorder. Genetic variation at the 5-HT2A receptor gene does not play a major role in the pathogenesis of the disorder.

PIK3CA mutation, but not PTEN loss of function, determines the sensitivity of breast cancer cells to mTOR inhibitory drugs.

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in breast cancers due to frequent mutations in PIK3CA, loss of expression of PTEN or over-expression of receptor tyrosine kinases. PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase mammalian target of rapamycin (mTOR), which can be inhibited by rapamycin analogues and by kinase inhibitors; the effectiveness of these drugs in breast cancer treatment is currently being tested in clinical trials. To identify the molecular determinants of response to inhibitors that target mTOR via different mechanisms in breast cancer cells, we investigated the effects of pharmacological inhibition of mTOR using the allosteric mTORC1 inhibitor everolimus and the active-site mTORC1/mTORC2 kinase inhibitor PP242 on a panel of 31 breast cancer cell lines. We demonstrate here that breast cancer cells harbouring PIK3CA mutations are selectively sensitive to mTOR allosteric and kinase inhibitors. However, cells with PTEN loss of function are not sensitive to these drugs, suggesting that the functional consequences of these two mechanisms of activation of the mTOR pathway are quite distinct. In addition, a subset of HER2-amplified cell lines showed increased sensitivity to PP242, but not to everolimus, irrespective of the PIK3CA/PTEN status. These selective sensitivities were confirmed in more physiologically relevant three-dimensional cell culture models. Our findings provide a rationale to guide selection of breast cancer patients who may benefit from mTOR inhibitor therapy and highlight the importance of accurately assessing the expression of PTEN protein and not just its mutational status.

No common denominator for breast cancer lymph node metastasis.

The axillary lymph node status is the most powerful prognostic factor for breast cancer patients to date. The molecular mechanisms that control lymph node metastasis, however, remain poorly understood. To define patterns of genes or gene regulatory pathways that drive breast cancer lymph node metastasis, we compared the gene expression profiles of 15 primary breast carcinomas and their matching lymph node metastases using microarrays. In general, primary breast carcinomas and lymph node metastases do not differ at the transcriptional level by a common subset of genes. No classifier or single gene discriminating the group of primary tumours from those of the lymph node metastases could be identified. Also, in a series of 295 breast tumours, no classifier predicting lymph node metastasis could be developed. However, subtle differences in the expression of genes involved in extracellular-matrix organisation and growth factor signalling are detected in individual pairs of matching primary and metastatic tumours. Surprisingly, however, different sets of these genes are either up- or downregulated in lymph node metastases. Our data suggest that breast carcinomas do not use a shared gene set to accomplish lymph node metastasis.