Deborah A. Dillon

Kirsten ras mutations in patients with colorectal cancer: The 'RASCAL II' study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Expression of c-met is a strong independent prognostic factor in breast carcinoma†

The c‐met protooncogene encodes the met protein, the receptor for scatter factor/hepatocyte growth factor, a growth factor that modulates the motility and stable interaction of the epithelial cells. This study assesses the expression of met receptor in breast carcinoma and its prognostic value with respect to survival.

Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma : A Search for the Early Precursors of Papillary Cancer

Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer.

Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements

The ability to conveniently and rapidly profile a diverse set of proteins has valuable applications. In a step toward further enabling such a capability, we developed the use of rolling-circle amplification (RCA) to measure the relative levels of proteins from two serum samples, labeled with biotin and digoxigenin, respectively, that have been captured on antibody microarrays. Two-color RCA produced fluorescence up to 30-fold higher than direct-labeling and indirect-detection methods using antibody microarrays prepared on both polyacrylamide-based hydrogels and nitrocellulose. Replicate RCA measurements of multiple proteins from sets of 24 serum samples were highly reproducible and accurate. In addition, RCA enabled reproducible measurements of distinct expression profiles from lower-abundance proteins that were not measurable using the other detection methods. Two-color RCA on antibody microarrays should allow the convenient acquisition of expression profiles from a great diversity of proteins for a variety of applications.

Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.

Optimized Normalization for Antibody Microarrays and Application to Serum-Protein Profiling

The measurements of coordinated patterns of protein abundance using antibody microarrays could be used to gain insight into disease biology and to probe the use of combinations of proteins for disease classification. The correct use and interpretation of antibody microarray data requires proper normalization of the data, which has not yet been systematically studied. Therefore we undertook a study to determine the optimal normalization of data from antibody microarray profiling of proteins in human serum specimens. Forty-three serum samples collected from patients with pancreatic cancer and from control subjects were probed in triplicate on microarrays containing 48 different antibodies, using a direct labeling, two-color comparative fluorescence detection format. Seven different normalization methods representing major classes of normalization for antibody microarray data were compared by their effects on reproducibility, accuracy, and trends in the data set. Normalization with ELISA-determined concentrations of IgM resulted in the most accurate, reproducible, and reliable data. The other normalization methods were deficient in at least one of the criteria. Multiparametric classification of the samples based on the combined measurement of seven of the proteins demonstrated the potential for increased classification accuracy compared with the use of individual measurements. This study establishes reliable normalization for antibody microarray data, criteria for assessing normalization performance, and the capability of antibody microarrays for serum-protein profiling and multiparametric sample classification.

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Significance This study is the first demonstration, to our knowledge, of the application of desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for discrimination of breast cancer and delineation of tumor margins. Using DESI-MSI, it is possible to discriminate between cancerous and adjacent normal tissue on the basis of the detection and specific spatial distributions of different lipid species. This study proves the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. It will allow the surgeon to access to this information in real time so as to make accurate intraoperative decisions quickly. It will result in improved cosmesis and decrease the need for multiple operations for margin reexcision. Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.

PIK3CA Mutations in In situ and Invasive Breast Carcinomas

The PIK3 signaling pathway has been identified as one of the most important and most frequently mutated pathways in breast cancer. Somatic mutations in the catalytic subunit of PIK3CA have been found in a significant fraction of breast carcinomas, and it has been proposed that mutant PIK3CA plays a role in tumor initiation. However, the majority of primary human tumors analyzed for genetic alterations in PIK3CA have been invasive breast carcinomas and the frequency of PIK3CA mutations in preinvasive lesions has not been explored. To investigate this, we sequenced exons 9 and 20 of PIK3CA in pure ductal carcinoma in situ (DCIS), DCIS adjacent to invasive carcinoma, and invasive ductal breast carcinomas. In a subset of cases, both in situ and invasive areas were analyzed from the same tumor. We found that the frequency of PIK3CA mutations was essentially the same ( approximately 30%) in all three histologic groups. In some cases, in situ and invasive areas of the same tumor were discordant for PIK3CA status, and in two cases in which multiple invasive and adjacent in situ areas within the same tumor were analyzed independently, we detected intratumor heterogeneity for PIK3CA mutations. Our results suggest that mutation of PIK3CA is an early event in breast cancer that is more likely to play a role in breast tumor initiation than in invasive progression, although a potential role for exon 9 mutations in the progression of a subset of DCIS cases cannot be excluded.

Trastuzumab for the Treatment of Salivary Duct Carcinoma

OBJECTIVE Salivary duct carcinoma (SDC) is a rare and aggressive malignancy with high mortality and poor response to treatment. A significant fraction of SDCs are HER2 positive. This retrospective review examines HER2 testing in SDC and the outcome of trastuzumab-based therapy in adjuvant and palliative settings. METHODS A total of 13 patients with SDC and HER2/neu expression by immunohistochemistry of 1-3+ were treated with trastuzumab in adjuvant (n = 8) or palliative (n = 5) setting. Adjuvant therapy consisted of concurrent radiation and chemotherapy with weekly paclitaxel, carboplatin, and trastuzumab (TCH) for 6 weeks followed by TCH for 12 weeks and trastuzumab alone for 1 year. Palliative treatment for metastatic disease consisted of TCH every 3 weeks for 6 cycles followed by trastuzumab for variable time periods with or without second-line chemotherapy for progression. All patients had fluorescence in situ hybridization testing for HER2/neu gene amplification. RESULTS The median duration of follow-up was 27 months (range: 8-48 months). In all, 62% of adjuvant patients (5/8) had no evidence of disease more than 2 years from completion of therapy. All patients with metastatic disease (5/5 patients) responded to treatment with TCH. One patient achieved a complete response and remains with no evidence of disease 52 months after initiation of TCH. The median duration of response was 18 months (range: 8-52 months). CONCLUSION HER2/neu positivity and treatment with trastuzumab correlated well with long-term survival and response in our patients. Based on this data, we propose that HER2/neu status be examined routinely in all patients with SDCs and the treatment be directed accordingly.

Overcoming Therapeutic Resistance in HER2-Positive Breast Cancers with CDK4/6 Inhibitors

Using transgenic mouse models, cell line-based functional studies, and clinical specimens, we show that cyclin D1/CDK4 mediate resistance to targeted therapy for HER2-positive breast cancer. This is overcome using CDK4/6 inhibitors. Inhibition of CDK4/6 not only suppresses Rb phosphorylation, but also reduces TSC2 phosphorylation and thus partially attenuates mTORC1 activity. This relieves feedback inhibition of upstream EGFR family kinases, resensitizing tumors to EGFR/HER2 blockade. Consequently, dual inhibition of EGFR/HER2 and CDK4/6 invokes a more potent suppression of TSC2 phosphorylation and hence mTORC1/S6K/S6RP activity. The suppression of both Rb and S6RP enhances G1 arrest and a phenotype resembling cellular senescence. In vivo, CDK4/6 inhibitors sensitize patient-derived xenograft tumors to HER2-targeted therapies and delay tumor recurrence in a transgenic model of HER2-positive breast cancer.