Phillip Stahl

Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer

Using an Affymetrix 10K SNP array to screen for gene copy number changes in breast cancer, we detected a single-gene amplification of the ESR1 gene, which encodes estrogen receptor alpha, at 6q25. A subsequent tissue microarray analysis of more than 2,000 clinical breast cancer samples showed ESR1 amplification in 20.6% of breast cancers. Ninety-nine percent of tumors with ESR1 amplification showed estrogen receptor protein overexpression, compared with 66.6% cancers without ESR1 amplification (P < 0.0001). In 175 women who had received adjuvant tamoxifen monotherapy, survival was significantly longer for women with cancer with ESR1 amplification than for women with estrogen receptor–expressing cancers without ESR1 amplification (P = 0.023). Notably, we also found ESR1 amplification in benign and precancerous breast diseases, suggesting that ESR1 amplification may be a common mechanism in proliferative breast disease and a very early genetic alteration in a large subset of breast cancers.

A Mechanism for Cancer-Associated Membranous Nephropathy

The cause of paraneoplastic membranous nephropathy is unclear. In the current case, new expression of THSD7A in a gallbladder carcinoma and the development of membranous nephropathy may indicate a potential mechanism for the association between cancer and this nephropathy.

An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target antigen identified in adult membranous nephropathy (MN) along with the major antigen phospholipase A2 receptor 1 (PLA2R1). The prevalence of THSD7A-Ab-positive patients is unknown, and it is unclear whether the clinical presentation differs between patients positive for PLA2R1-Ab or THSD7A-Ab. We screened serum samples of 1276 patients with MN from three different cohorts for the presence of THSD7A-Ab by Western blot analysis and a newly developed indirect immunofluorescence test (IFT). Compared with Western blot analysis, the IFT had a 92% sensitivity and a 100% specificity. The prevalence of THSD7A-associated MN in a prospective cohort of 345 patients with MN was 2.6%, and most were women. In this cohort, the percentage of patients with THSD7A-associated MN and malignant disease significantly exceeded that of patients with PLA2R1-associated MN and malignant disease. In all cohorts, we identified 40 patients with THSD7A-associated MN, eight of whom developed a malignancy within a median time of 3 months from diagnosis of MN. In one patient with THSD7A-associated MN and metastases of an endometrial carcinoma, immunohistochemistry showed THSD7A expression on the metastatic cells and within follicular dendritic cells of the metastasis-infiltrated lymph node. We conclude that the IFT allows sensitive and specific measurement of circulating THSD7A-Ab in patients with MN. Patients with THSD7A-associated MN differ in their clinical characteristics from patients with PLA2R1-associated MN, and more intensive screening for the presence of malignancies may be warranted in those with THSD7A-associated MN.

MALDI imaging–based identification of prognostically relevant signals in bladder cancer using large-scale tissue microarrays

OBJECTIVE Although most patients with urinary bladder cancer present with noninvasive and low-malignant stages of the disease, about 20% eventually develop life-threatening metastatic tumors. This study was designed to evaluate the potential of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify molecular markers predicting the clinical course of bladder cancer. MATERIALS AND METHODS We employed MALDI-MSI to a bladder cancer tissue microarray including paraffin-embedded tissue samples from 697 patients with clinical follow-up data to search for prognostically relevant associations. RESULTS Analysis of our MALDI imaging data revealed 40 signals in the mass spectra (m/z signals) associated with epithelial structures. The presence of numerous m/z signals was statistically related to one or several phenotypical findings including tumor aggressiveness (stage, grade, or nodal status; 30 signals), solid (5 signals) or papillary (3 signals) growth patterns, and increased (6 signals) or decreased (12 signals) cell proliferation, as determined by Ki-67 immunohistochemistry. Two signals were linked with tumor recurrence in noninvasive (pTa category) tumors, of which one was also related to progression from pTa-category to pT1-category disease. The absence of one m/z signal was linked with decreased survival in the subset of 102 muscle-invasive cancers. CONCLUSION Our data demonstrate the suitability of combining MSI and large-scale tissue microarrays to simultaneously identify and validate clinically useful molecular markers in urinary bladder cancer.

MALDI imaging on large-scale tissue microarrays identifies molecular features associated with tumour phenotype in oesophageal cancer

Matrix‐assisted laser desorption/ionisation mass spectrometry imaging (MALDI‐MSI) and tissue microarray (TMA) technologies were jointly utilized to search for molecular features associated with clinicopathological parameters in oesophageal cancer.

Y chromosome loss is a frequent early event in urothelial bladder cancer

Aims: Y chromosome losses have been described in 10–40% of bladder cancers and were suggested to be age‐related. The clinical significance of chromosome Y losses is largely unknown, since only small sets of male bladder cancer patients have been evaluated in previous studies. The aim of this study was to further clarify the potential relevance of Y chromosome losses in bladder cancer with respect to clinical outcome and patient age. Methods: A pre‐existing bladder cancer tissue microarray (TMA) with clinical follow‐up data including 516 urothelial bladder cancers from male patients was utilised in this study. Y chromosome losses were analysed by multicolour fluorescence in situ hybridisation (FISH) using a centromere Y probe and a centromere X probe. p53 immunostaining data were available for all patients from a previous study. Results: Y chromosome losses were seen in 23% of 477 interpretable cancers from male patients. There was no significant difference in patient age in tumours with (67.4 ± 4.3 years) or without (67.3 ± 2.3 years) Y chromosome losses (p = 0.9068). Y chromosome losses were equally frequent in tumours of all grades (p = 0.7831) and stages (p = 0.6140). There was also no association with p53 immunostaining (p = 0.4092). Y chromosome losses were not associated with survival in 224 invasive urothelial cancers (pT2–4; p = 0.2324), an increased risk for recurrences in 197 pTa tumours (p = 0.7649) or increased progression risk in 76 pT1 tumours (p = 0.4582). Conclusion: The data of this study show that Y chromosome losses are frequent in urothelial bladder cancer of all grades and stages, which could imply that loss of the Y chromosome is an early event in bladder cancer development. p53 mediated genomic instability is evidently not required for the development of Y chromosome losses. Since there was no correlation between Y chromosome losses and clinical outcome, detection of Y losses has no clinical relevance in urothelial bladder cancer.

PTEN deletion is rare but often homogeneous in gastric cancer

Background and aim Gastric carcinoma is the second most frequent cause of cancer-related death worldwide. As PTEN is a potential modifier of tumour response to trastuzumab, a recently approved therapy in metastatic HER2 positive gastric cancer, the existence of PTEN deletions in primary gastric cancer was investigated. Methods 230 primary gastric cancers were analysed in a tissue microarray format by dual labelling fluorescence in situ hybridisation for PTEN deletion. HER2 analysis was also performed. To study PTEN deletion heterogeneity, all available large tissue sections from primary cancer and corresponding metastases were analysed in seven patients. Results Eight of 180 interpretable primary gastric cancer spots showed PTEN deletions (4.4%), including seven hemizygous and one homozygous deletion. PTEN deletion was correlated with nodal (8 of 122 cases (6.6%); p=0.041) and distant metastases (4 of 19 (21.1%); p<0.001). Large section validation showed a homogeneous distribution of PTEN deletion. HER2 positivity was seen in one PTEN deleted case. Conclusion Genomic PTEN deletion is a rare event in gastric adenocarcinoma but correlates with metastatic disease. The homogeneous distribution pattern indicates that this alteration occurs early in tumour development.

Altered PTEN function caused by deletion or gene disruption is associated with poor prognosis in rectal but not in colon cancer

Colorectal cancer is the third most common malignancy worldwide. Anti-epidermal growth factor receptor (EGFR)-targeted therapy shows clinical evidence in this malignancy and improves outcome. The tumor suppressor gene phosphatase and tensin homologue (PTEN) is considered a potential predictor of nonresponse to anti-EGFR agents. The purpose of this study was to assess whether associations between PTEN alterations (PTEN gene deletion or PTEN gene disruption) and clinical outcome could be caused by a prognostic (and not predictive) effect of PTEN inactivation. Therefore, we analyzed 404 colorectal cancers not previously treated with anti-EGFR drugs in a tissue microarray format. PTEN deletion and PTEN gene rearrangements were analyzed by fluorescence in situ hybridization. Heterogeneity analysis of all available large tissue sections was performed in 6 cases with genomic PTEN alteration. Twenty-seven (8.8%) of 307 analyzable colorectal cancer spots showed genomic PTEN alterations including 24 hemizygous and 1 homozygous deletion as well as 2 PTEN gene disruptions. Genomic PTEN alterations were associated with reduced patient survival in rectal cancer in univariate and multivariate analyses (P = .012; hazard ratio, 2.675; 95% confidence interval, 1.242-5.759) but not in colon cancer. Large-section evaluation revealed a homogeneous distribution pattern in all 4 analyzed cases with PTEN deletion and in both cases with a PTEN gene disruption. In conclusion, genomic PTEN gene alterations caused by deletion or gene disruption characterize a fraction of rectal cancers with particularly poor outcome.

MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large-scale tissue microarrays

For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer, we applied Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to a tissue micro array containing formalin-fixed and paraffin-embedded tissue samples from 349 patients. Analysis of our MALDI-IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki-67 labeling index. Fifteen out of 27 IMS signals revealed a significant association with survival. For seven signals (m/z 654, 776, 788, 904, 944, 975 and 1013) the absence and for eight signals (m/z 643, 678, 836, 886, 898, 1095, 1459 and 1477) the presence were associated with decreased life expectancy, including five masses (m/z 788, 836, 904, 944 and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these five masses resulted in a three-step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis and cell proliferation. Our data demonstrate the suitability of combining IMS and large-scale tissue micro arrays to simultaneously identify and validate clinically useful molecular marker. Copyright

THSD7A Expression in Human Cancer

We recently described a case of a Thrombospondin Type‐1 Domain containing 7A (THSD7A) associated membranous nephropathy in a female patient who was synchronously suffering from a THSD7A‐positive malignancy. We here investigated the role of THSD7A as a new potential tumor antigen by evaluating over 20 000 tissue spots in more than 70 different tumor entities by immunohistochemistry using tissue microarrays. THSD7A expression was highly variable in different neoplasias with differing staining patterns. Both gain and loss of THSD7A expression compared to expression status in non‐tumor tissue were linked to tumor‐specific markers in the different tumor entities and were of prognostic value. The potential role of THSD7A in tumor development and therapy needs further investigation.