Caveolin Gene Expression Predicts Clinical Outcomes for Early-Stage HER2-Negative Breast Cancer Treated with Paclitaxel-Based Chemotherapy in the GeparSepto Trial.

PURPOSE: Caveolin-1 and -2 (CAV1/2) dysregulation are implicated in driving cancer progression and may predict response to nab-paclitaxel. We explored the prognostic and predictive potential of CAV1/2 expression for patients with early-stage HER2-negative breast cancer receiving neoadjuvant paclitaxel-based chemotherapy regimens, followed by epirubicin and cyclophosphamide. EXPERIMENTAL DESIGN: We correlated tumor CAV1/2 RNA expression with pathologic complete response (pCR), disease-free survival (DFS), and overall survival (OS) in the GeparSepto trial, which randomized patients to neoadjuvant paclitaxel- versus nab-paclitaxel-based chemotherapy. RESULTS: RNA sequencing data were available for 279 patients, of which 74 (26.5%) were hormone receptor (HR)-negative, thus triple-negative breast cancer (TNBC). Patients treated with nab-paclitaxel with high CAV1/2 had higher probability of obtaining a pCR [CAV1 OR, 4.92; 95% confidence interval (CI), 1.70-14.22; P = 0.003; CAV2 OR, 5.39; 95% CI, 1.76-16.47; P = 0.003] as compared with patients with high CAV1/2 treated with solvent-based paclitaxel (CAV1 OR, 0.33; 95% CI, 0.11-0.95; P = 0.040; CAV2 OR, 0.37; 95% CI, 0.12-1.13; P = 0.082). High CAV1 expression was significantly associated with worse DFS and OS in paclitaxel-treated patients (DFS HR, 2.29; 95% CI, 1.08-4.87; P = 0.030; OS HR, 4.97; 95% CI, 1.73-14.31; P = 0.003). High CAV2 was associated with worse DFS and OS in all patients (DFS HR, 2.12; 95% CI, 1.23-3.63; P = 0.006; OS HR, 2.51; 95% CI, 1.22-5.17; P = 0.013), in paclitaxel-treated patients (DFS HR, 2.47; 95% CI, 1.12-5.43; P = 0.025; OS HR, 4.24; 95% CI, 1.48-12.09; P = 0.007) and in patients with TNBC (DFS HR, 4.68; 95% CI, 1.48-14.85; P = 0.009; OS HR, 10.43; 95% CI, 1.22-89.28; P = 0.032). CONCLUSIONS: Our findings indicate high CAV1/2 expression is associated with worse DFS and OS in paclitaxel-treated patients. Conversely, in nab-paclitaxel-treated patients, high CAV1/2 expression is associated with increased pCR and no significant detriment to DFS or OS compared with low CAV1/2 expression.

Inferred Immune-Cell Activity Is an Independent Predictor of HER2-Negative Breast Cancer Prognosis and Response to Paclitaxel-Based Therapy in the GeparSepto Trial.

PURPOSE: Tumor microenvironment (TME) immune markers have been correlated with both response to neoadjuvant therapy and prognosis in patients with breast cancer. Here, immune-cell activity of breast cancer tumors was inferred by expression-based analysis to determine if it is prognostic and/or predictive of response to neoadjuvant paclitaxel-based therapy in the GeparSepto (G7) trial (NCT01583426). EXPERIMENTAL DESIGN: Pre-study biopsies from 279 patients with HER2-negative breast cancer in the G7 trial underwent RNA-seq-based profiling of 104 immune-cell-specific genes to assess inferred Immune Cell Activity (iICA) of 23 immune-cell types. Hierarchical clustering was used to classify tumors as iICA "hot," "warm," or "cold" by comparison of iICA in the G7 cohort relative to that of 1,467 samples from a tumor database established by Nantomics LLC. Correlations between iICA cluster, pathology-assessed tumor-infiltrating lymphocytes (TIL), and hormone receptor (HR) status for pathologic complete response (pCR), disease-free survival (DFS), and overall survival (OS) were determined. RESULTS: iICA cluster correlated with TIL levels. The highest pCR rates were observed in hot cluster tumors, and those with relatively higher TILs. Greater inferred activity of several T-cell types was significantly associated with pCR and survival. DFS and OS were prolonged in patients with hot or warm cluster tumors, the latter particularly for HR negative tumors, even if TILs were relatively low. CONCLUSIONS: Overall, TIL level better predicted pCR, but iICA cluster better predicted survival. Differences in associations between TILs, cluster, pCR, and survival were observed for HR-positive tumors versus HR-negative tumors, suggesting expanded study of the implication of these findings is warranted.

Spindle cell embryonal rhabdomyosarcoma of the prostate in an adult patient - case report and review of clinicopathological features.

BACKGROUND: Embryonal rhabdomyosarcoma of the prostate in an adult is a very rare event with only a few cases published. Diagnosis usually occurs with advanced disease frequently already with metastatic spread. In adults prognosis is very poor, therefore early diagnosis is crucial. To date, only three cases of spindle cell subtype of embryonal rhabdomyosarcoma of the prostate in an adult have been published. CASE PRESENTATION: We report an additional case of prostatic spindle cell embryonal rhabdomyosarcoma subtype in an adult. CONCLUSIONS: We discuss relevant clinicopathological features of spindle cell embryonal rhabdomyosarcoma of the prostate in adult patients in the context of the literature.

Plasma membrane calcium ATPase expression in human colon multistep carcinogenesis.

The expression of the plasma membrane Ca(2+) ATPase (PMCA) was analyzed in a series of 84 formalin-fixed and paraffin embedded colon samples including normal mucosa (n = 32), adenoma (n = 19), adenocarcinoma (n = 27), and lymph node metastasis (n = 6) using (i) immunohistochemistry, (ii) mRNA in situ hybridization, and (iii) quantitative reverse-transcriptase PCR. A marked reduction of PMCA4 protein was observed in high-grade adenoma, colon cancer as well as lymph node metastasis, pointing to its potential role in the progression of cancer. However, PMCA4 RNA transcripts were unchanged or even increased in colon carcinomas, suggesting posttranscriptional regulation of PMCA4 during carcinogenesis.

Microarray analysis reveals differential expression of benign and malignant pheochromocytoma.

The diagnosis of a malignant pheochromocytoma (PC) can only be established by the presence of distant metastases, but a subset of apparently benign PCs develop metastases. We have employed a microarray analysis to identify a typical gene expression profile which distinguishes malignant from benign PC. Total RNA was isolated from fresh-frozen tissue of five benign and five malignant PCs. The reference consisted of laser microdissected tissue from normal adrenal medulla. After generating Cy3- and Cy5-fluorescently labeled cDNAs, F-chips containing 11 540 spots were hybridized. Data were analyzed with the IMAGENE 3.0 software. Gene expression levels were validated by real-time (RT)-PCR and immunohistochemistry (IHC). The analysis revealed a more than twofold difference in expression between benign and malignant PCs in 132 genes: 19 were up-regulated and 113 were down-regulated. Expression differences of six genes (calsequestrin, NNAT, neurogranin, secreted protein acidic and rich in cysteine (SPARC), EGR2, and MAOB) were confirmed by RT-PCR in 25 PCs. IHC for calsequestrin revealed an overexpression in malignant PCs (7/10 vs 1/10, P=0.03). Comparative analysis by microarray of all ten PCs (benign/malignant) versus normal adrenal medulla revealed a more than twofold expression difference in 455/539 and 491/671 genes respectively. Several of these genes are known to participate on adrenal tumorigenesis, potential tumor suppressor genes, and oncogenes. Comprehensive gene expression analysis of malignant and benign PCs revealed different gene profiles, which could be used to discriminate between malignant and benign PCs. Based on these findings, the strategy for further follow-up and treatment could be modified accordingly.

Genomic instability at both the base pair level and the chromosomal level is detectable in earliest PanIN lesions in tissues of chronic pancreatitis.

OBJECTIVE: Chronic pancreatitis (CP) is a predisposing disease for pancreatic carcinoma (PC), however, precise molecular mechanisms of cancer development in the background of CP are ill defined. METHODS: A total of 443 laser-microdissected pancreatic intraepithelial neoplasias (PanINs), acinar-ductal metaplasia (ADM), and normal ducts from 21 patients with CP were analyzed for loss of heterozygosity (LOH) and immunohistochemical protein expression of p53, p16, and DPC4. Pancreatic intraepithelial neoplasias were analyzed for mutations in p53, p16, and Ki-ras genes by ABI sequencing. Aneuploidy was determined by fluorescence in situ hybridization with probes for chromosomes 3, 7, 8, and 17. RESULTS: Loss of heterozygosity rate in PanIN-1 and ADM was between 1.7% (p53) and 5.8% (p16). In PanIN-3, p53 protein overexpression and loss of expression for p16 and DPC4 protein were seen. Heterozygous mutations of p53 and p16 without LOH were found in PanIN-1A and ADM, whereas homozygous mutations were found in PanIN-3. Aneuploidy increased from PanIN-1A to PanIN-3. Ki-ras mutations were discovered first in PanIN-1. CONCLUSIONS: Heterozygous mutations of p53- and p16 genes together with chromosomal instability occur early in CP and are clonally expanded, but final inactivation mostly by LOH happens later in pancreatic carcinogenesis. Determination of aneuploidy in pancreatic juice may be of value for early detection and risk assessment in patients with long-standing CP.

Molecular genetic analysis of surveillance biopsy samples from Barrett's mucosa--significance of sampling.

Our aim was to determine the spectrum and accumulation of mutations in surveillance biopsies from Barrett's mucosa of individual patients during follow-up. We performed loss of heterozygosity (LOH) analysis of six recently described tumor suppressor genes relevant for the carcinogenesis of Barrett's adenocarcinoma from laser-microdissected, paraffin-embedded biopsy samples of Barrett's mucosa without or with low-grade dysplastic change. 118 biopsy samples from 26 patients were taken during surveillance programs in time intervals ranging between 6 and 51 months. We found no significant increase in LOH events at least in a 51-month interval. In two patients, Barrett's adenocarcinoma was diagnosed 6 months after the first diagnosis of Barrett's mucosa. Six of 26 patients did not show LOH. The remaining patients exhibited a striking variation of LOH patterns and accumulations in biopsy samples during follow-up. From our microsatellite marker panel, we were not able to define a single surrogate marker that could serve as a potential biomarker, indicating an increased risk of progression to Barrett's adenocarcinoma. However, LOH combinations, especially APC/p16(INK4) or APC/p53, deserve attention as putative biomarkers in future studies. Our results raise important questions regarding the biological dynamics of mutations in Barrett's mucosa in addition to the influence of sampling, especially with regard to the number of biopsies taken from Barrett's mucosa.

[Intraepithelial neoplasms (PanIN) and intraductal papillary-mucinous neoplasms (IPMN) of the pancreas as precursor lesions of pancreatic carcinoma]. / Intraepitheliale Neoplasien (PanIN) und intraduktale papillär-muzinöse Neoplasien (IPMN) des Pankreas als Vorläufer des Pankreaskarzinoms.

Due to the fatal prognosis of pancreatic carcinoma, great efforts have been made to investigate precursor lesions of invasive neoplasia during the last few years. Pancreatic intraepithelial neoplasias (PanIN) have been recognized as precursor lesions of ductal adenocarcinoma, and are classified into different grades from PanIN-1A, -1B, -2, to -3. Molecular analyses have helped to define a progression model for pancreatic neoplasia. The most important step seems to be the occurrence of a PanIN-3 lesion defining a high risk of malignant transformation. As in PanINs, different types of intraductal papillary-mucinous neoplasms (IPMN) can be discriminated ranging from benign to invasive lesions. Becoming invasive, some of these tumors appear as ductal adenocarcinoma, others as colloid carcinoma with a much better prognosis. In this review, the characteristics of these two precursor lesions and their genetic alterations are summarized.

Alterations of the tissue inhibitor of metalloproteinase-3 (TIMP3) gene in pancreatic adenocarcinomas.

OBJECTIVES: Tissue inhibitor of metalloproteinase-3 (TIMP3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastases. In the present study, the involvement of TIMP3 in the tumorigenesis of 34 pancreatic adenocarcinomas was evaluated. METHODS: Immunohistochemistry, methylation-specific PCR, and RNA expression analysis (RT-PCR) of TIMP3 were performed in 34 resected and microdissected primary pancreatic adenocarcinomas. RESULTS: Immunohistochemistry showed loss or strongly reduced protein expression in 17 of 34 pancreatic adenocarcinomas (50%) that corresponded to loss of TIMP3-RNA-expression. Promoter hypermethylation was identified in 2 of 34 tumors (6%). It was tumor specific and corresponded to a loss of TIMP3 protein expression. TIMP3 alterations did not correlate with any clinical feature such as tumor size or survival. CONCLUSION: TIMP3 seems to play an important role in the tumorigenesis of primary pancreatic adenocarcinomas. In contrast to other tumors, hypermethylation seems not to be the key mechanism for the inactivation of TIMP3. Other methods of gene inactivation need to be identified.

Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry.

Hereditary non-polyposis colorectal cancer (HNPCC) accounts for approximately 2 to 4% of the total colorectal cancer burden. For economic reasons a diagnostic "stepladder" is recommended. After evaluation of the family history, diagnostic microsatellite instability (MSI) analysis has found its place as a valuable screening tool for HNPCC. Immunohistochemical analysis can help to pinpoint the affected gene. The detection of a mutation in one of the responsible mismatch repair gene confirmed the diagnosis HNPCC. Here we demonstrate our experience of some important pitfalls that will be discussed in this study. In MSI testing, one potential source for false-negative results is intralesional heterogeneity. We demonstrate examples of a flat adenoma and a carcinoma, which required laser microdissection to correctly determine the microsatellite status. In these lesions manual microdissection, the most frequently applied method, was not sufficient. However, the number of cells obtained by using laser microdisssection can fall below a necessary minimum, which can also cause false-negative results of MSI analysis, as shown here in a mucinous carcinoma. In addition, evaluation of immunohistochemically stained tissue slides requires experience to avoid false-positive or false-negative interpretation. A case with two synchronous colorectal cancers revealed loss of MSH2 expression in one carcinoma, whereas the second carcinoma stained positively leading to a false-negative interpretation. In some cases, false-positive results can be obtained, if a perinuclear-staining pattern is interpreted as positive. In summary, there are several potential pitfalls in the molecular screening for HNPCC. Therefore the importance of correct interpretation of clinical data, immunohistochemistry, and microsatellite analysis in combination, performed by a pathologist with experience in molecular genetics is essential. In addition, laser microdissection of tumor areas that have been chosen by a pathologist is highly recommended in cases that cannot be resolved with manual microdissection.

Single-cell immunohistochemical mutation load assay (SCIMLA) using human paraffin-embedded tissues.

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species.

Laser microdissection of small tissue samples--application to chronic pancreatitis tissues.

Laser microdissection is considered to be the gold standard of tissue sampling, especially if a defined small tissue area consisting of single or few cells within a heterogeneous tissue compartment is of interest. This sophisticated technique offers the opportunity of rapid and contamination-free tissue sampling for RNA- or DNA-based molecular genetic studies. We have applied laser microdissection to a molecular genetic study of pancreatic intraductal lesions (PanINs) in tissues of chronic pancreatitis, where an exact microdissection of small ducts within a dense fibrous tissue is of paramount importance for following analysis. From nine patients suffering from chronic pancreatitis, formalin-fixed, paraffin-embedded tissue specimens were laser microdissected, and a total of 202 normal ducts and PanINs of grade PanIN-1A to grade PanIN-2 were harvested. After whole genome amplification by improved primer extension and preamplification PCR (I-PEP-PCR), microsatellite-PCR based loss of heterozygosity analysis (LOH) of the tumor suppressor gene loci TP53, p16INK4, and DPC4 was performed. One of 85 informative duct lesions (1.2%) had LOH of TP53, 1 of 76 duct lesions (1.3%) had LOH of DPC4, and 2/29 duct lesions (6.9%) showed LOH of p16INK4. Microsatellite instability (MSI) was seen in 2 of 178 duct lesions (1.1%). Immunohistochemical staining of p53 protein and DPC4 protein revealed no aberrant expression. These preliminary data indicate that LOH of tumor suppressor genes, important in pancreatic cancer genesis or MSI, can be found in chronic pancreatitis tissues, but their incidence is low.

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.