EGFR alterations and EML4-ALK rearrangement in primary adenocarcinoma of the urinary bladder

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Abstract

The identification of mutations in epidermal growth factor receptor (EGFR) and translocations involving anaplastic lymphoma kinase (ALK) in lung adenocarcinoma has drastically changed understanding of the disease and led to the development of targeted therapies. Adenocarcinoma of the urinary bladder is rare and poorly understood at the molecular level. We undertook this study to determine whether EGFR mutations, increases in EGFR copy number, or ALK translocations are present in these tumors. Twenty-eight cases of primary bladder adenocarcinoma were analyzed. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro Kits. All cases were analyzed via fluorescence in situ hybridization (FISH) using Vysis ALK Break Apart FISH Probes for detection of ALK chromosomal translocation and Vysis Dual Color Probes to assess for increased gene copy number of EGFR. None of the 28 cases examined showed mutational events in EGFR or ALK rearrangements. EGFR polysomy was seen in 10 out of 28 (36%) cases. No correlation with EGFR polysomy was seen in the tumors with respect to age, histologic subtypes, pathologic stage, or lymph node metastasis. In summary, EGFR mutations and ALK rearrangements do not appear to be involved in the development of primary adenocarcinoma of the urinary bladder. A subgroup of cases (36%), however, demonstrated increased gene copy number of EGFR by FISH.

Main

Approximately 356 000 cases of urinary bladder cancer are diagnosed per year worldwide; however, primary adenocarcinoma accounts for less than 2% of these tumors.1, 2, 3 In some cases, primary adenocarcinoma has been associated with bladder exstrophy.4, 5, 6 Patients with primary urinary bladder adenocarcinoma tend to present at a higher stage than those with urothelial carcinoma, although in some circumstances survival appears similar to or better than that of urothelial carcinoma.6, 7 Histologically, tumors tend to demonstrate enteric morphology, closely resembling colonic adenocarcinoma. Therefore, care must be taken to exclude the possibility of secondary involvement of the urinary bladder in a primary colorectal tumor.8 The presence of signet-ring cell morphology is reported to portend a worse outcome.9, 10 Radical surgery is the definitive treatment, although postoperative radiotherapy has been reported to enhance survival.4, 5, 11 Nonetheless, there is a need for better understanding of the pathogenesis of urinary bladder adenocarcinoma to guide more effective therapy.

Mutations in the epidermal growth factor receptor (EGFR) have been shown to be a driver mutation in tumorigenesis and cancer progression in many organs.12, 13, 14, 15 Therapies targeting the mutant EGFR have allowed for new and more personalized treatment options in these cancers.16, 17, 18 Rearrangements involving anaplastic lymphoma kinase (ALK), most notably the EML4-ALK rearrangement, have shown considerable clinical utility in the treatment of certain lung cancers.13, 17, 19, 20, 21 Although currently less specific and less predictive of therapeutic results, the detection of EGFR polysomy has been seen in many tumors to correlate with tumor activity and infers a role of EGFR in the propagation of tumors.22, 23, 24, 25 The goal of this study was to determine whether EGFR mutations, EGFR polysomy, or ALK rearrangements are present in primary adenocarcinoma of the urinary bladder as they may provide therapeutic targets in this disease.

Materials and methods

Cases

Twenty-eight patients with adenocarcinoma of the urinary tract (urinary bladder and urachus) were identified from the surgical pathology case files of the participating institutions (Figures 1a–d). The hematoxylin and eosin (H&E) slides of each case, along with clinical histories, were reviewed to confirm diagnosis and primary tumor origin within the urinary bladder. Clinicopathologic information was also gathered via medical chart review.

Figure 1
figure1

Morphology and interphase FISH for EGFR copy number and ALK gene rearrangement in primary adenocarcinoma of the urinary bladder. (a) H&E-stained section of primary adenocarcinoma of the urinary bladder. The tumor infiltrates the lamina propria with extravasation of mucin. (b) Adenocarcinoma composed of signet-ring cells infiltrating the bladder wall. (c) Enteric-type adenocarcinoma forming irregularly shaped cribriform glandular structures. (d) Higher magnification demonstrates a close resemblance to colorectal adenocarcinoma. (e) EGFR polysomy as detected by interphase FISH using LSI EGFR/CEP7 Dual Color Probe containing centromeric chromosome 7 (spectrum green) and EGFR probes (EGFR, spectrum orange). The cells showed EGFR polysomy as revealed by >2 copies per cell of signal pairs with red/green (EGFR/CEP7) signal ratio <2. (f) ALK alteration testing with ALK break-apart probes. The ALK gene showed a wild-type pattern as evidenced by fused red (3′ of ALK) and green (5′ of ALK) signals.

EGFR Mutational Analysis

DNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissue samples was performed using the Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, USA). DNA concentration was determined using the NanoDrop Spectrophotometer and adjusted to approximately 10 ng/μl in dH2O. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro Kits (Qiagen, Valencia, CA, USA). The pyrosequencing kit tests for mutations in the exon 18 codon 719 region, deletions in exon 19, mutations in the exon 20 codon 768 and codon 790 regions, and mutations in exon 21 codon regions 858–861. Resulting amplicons were purified, denatured, and sequenced using mutation-adjacent primers. Pyrograms were generated by software and interpreted for the presence of mutations in the corresponding codons.

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) studies were performed on 4-μm-thick paraffin sections. Briefly, slides were deparaffinized with two 15-min washes in xylene and subsequently washed twice with 100% ethanol for 10 min each and air dried. The sections were heated at 95 °C in 0.1 mM citric acid (pH 6) solution (Invitrogen, Carlsbad, CA, USA) for 10 min, rinsed with distilled water for 3 min, and washed with 2 × saline–sodium citrate (SSC) for 5 min. Tissue digestion was performed by applying 0.4 ml of pepsin (Sigma, St Louis, MO, USA) solution (4 mg/ml in 0.9% NaCl in 0.01 N HCl) to each slide and incubating the slides in a humidified box for 40 min at 37 °C. The slides were rinsed with distilled water for 5 min, washed with 2 × SSC for 5 min, and then air dried.

EGFR alterations were detected by using LSI EGFR/CEP7 Dual Color Probe containing a centromeric probe for chromosome 7 (CEP7, spectrum green) and an EGFR probe located at 7p12 (EGFR, spectrum orange) (Vysis, Downers Grove, IL, USA).24, 25, 26 ALK gene rearrangement was detected using dual-color Vysis LSI ALK Break Apart Rearrangement Probe Kit (Vysis, Downers Grove, IL, USA). Five microliters of diluted probe was applied to each slide; coverslips were placed over the slides and sealed with rubber cement. The slides were denatured at 80 °C for 10 min and hybridized at 37 °C overnight. The coverslips were removed, and the slides were extensively washed with two 0.1 × SSC/1.5 M urea solutions at 45 °C for 20 min, in 2 × SSC at 45 °C for 10 min, and then in 2 × SSC/0.1% NP40 at 45 °C for 10 min. Finally, the slides were washed with 2 × SSC at room temperature for 5 min, air dried, counterstained with 10 μl DAPI/Antifade (DAPI in Fluorguard, 0.5 g/ml, Insitus), and sealed with nail polish.

The hybridized slides were observed and documented using a MetaSystem system (Belmont, MA, USA) under a × 100 oil objective. The images were acquired with a CCD camera and analyzed with MetaSystem Isis software (Belmont, MA, USA). The following filters were used: SP-100 for DAPI, FITC MF-101 for spectrum green, and Gold 31003 for spectrum orange signals. Signals from each color channel (probe) were counted under pseudo color, with computerized translation of each color channel into blue, green, and red. Four sequential focus stacks with 0.3 μm intervals were acquired and integrated into a single image to reduce thickness-related artifacts. A minimum of 100 nonoverlapping cancer cells were evaluated for each case, totaling 100–200 cells per case. Only if ≥90% of cells demonstrated sufficient signal was the slide considered to be qualified for counting.

The criteria for the EGFR FISH enumeration were adapted from Varella-Garcia et al27 and recommend criteria of FDA-approved PathVysion HER-2 DNA Probe Kit (package insert; Abbott Molecular, Des Plaines, IL, USA; January 2010). The criteria for the ALK FISH enumeration were according to the recommended criteria of FDA-approved Vysis ALK Break Apart FISH Probe Kit (package insert; Abbott Molecular, Des Plaines, IL, USA; August 2011) and Camidge et al.28 Briefly, EGFR amplification was considered to be present if ≥10% of the nuclei contained multiple EGFR signals and the EGFR/CEP7 ratio was ≥2. Polysomy for an individual tumor was defined as follows: ≥10% of the nuclei contained >2 signals of CEP7 or EGFR and the EGFR/CEP7 ratio was <2. For ALK, two fused or closely approximated red and green signals were considered the wild-type pattern. FISH was considered to be negative for ALK rearrangement if <5 cells out of 50 or <10% of cells demonstrated split red and green signals. ALK fusion was interpreted as present if red and green signals were separated by a distance >1.5 signal diameters or single red signals were identified in >25 cells out of 50 or >50% of cells show split signals. If the average percentage of positive cells from two separate readers was ≥15% (≥15/100), the sample was considered to be positive. These parameters are consistent with methods used in studies examining both EGFR and ALK in lung adenocarcinoma.13, 27, 28, 29

Statistical Analysis

Statistical analyses were performed using SPSS (Chicago, IL, USA). Fisher exact test was used to analyze association between genetic alterations. Statistical significance was defined as P<0.05, and all P-values were two sided.

Results

The average age of the patients in the series was 62 years, with a range of 32–87 years. The male-to-female ratio was 1.6:1. Of the studied cases, 9 out of the 28 (32%) cases were from either transurethral resection or biopsy specimens. Of these cases, the majority (six out of nine, 67%) were pT1 at the time of diagnosis. Of the 19 cystectomy or partial cystectomy specimens, 13 (68%) cases were either pT3 or pT4 at the time of resection. Seven patients had lymph node metastasis, and two had distant metastases. The enteric-type pattern was the most common histology, present in 20 (71%) of cases as either the sole or a constituent histologic pattern. Other patterns included mucinous, signet-ring cells and adenocarcinoma with neuroendocrine features.

In all 28 cases, EGFR mutational events were not identified by PCR methods. Using FISH to detect EGFR gains, polysomy was seen in 10 out of 28 (36%) cases (Figure 1e), although EGFR amplification was not observed. Of the cases with EGFR polysomies, no distinct statistically significant correlation was seen with regard to age, sex, pathologic stage, or lymph node metastasis (all P-values were >0.05). It was observed that 3 out of the 10 cases with EGFR polysomy were found to be pT4 at the time of diagnosis (representing half of all pT4 cases). Seven cases had lymph node metastases, one of which had EGFR polysomy. In cases with increased EGFR copy number, enteric-type histology was present in 9 out of the 10 cases but was not statistically significant (P=0.19). ALK translocations were not identified in any of the 28 cases (Figure 1f).

Discussion

The detection of driving mutations within tumors that allow for prediction of targeted therapeutic benefits has radically changed the outlook and future goals of cancer treatment. As targeted therapies to these mutational events already exist, we sought to determine whether similar mutations were present in adenocarcinoma of the urinary bladder. Our results show that no identifiable mutations were seen in the analyzed domains of EGFR, nor were rearrangements involving ALK present by FISH. We did, however, find EGFR polysomies in 10 tumors (36%), indicating an increase in EGFR copy number. Polysomy status did not correlate with age, histologic subtype, pathologic stage, or lymph node metastasis. Enteric-type histology closely resembling colonic adenocarcinoma was the most common manifestation of primary urinary bladder adenocarcinoma (71%). Most presented at a high pathologic stage (pT3 or pT4), particularly when definitive resection was performed and accurate pathologic staging could be determined. Ninety percent of cases with identified EGFR polysomy were enteric type.

The detection of EGFR polysomy (increased gene copy number) has been reported in a wide range of tumors, including lung adenocarcinoma, colonic adenocarcinoma, squamous-cell carcinoma of the vulva and head and neck, clear-cell renal-cell carcinoma, sinonasal adenocarcinoma, and glioblastoma.3, 6, 12, 15, 22, 23, 30, 31, 32, 33 In both the lung and the colon, some authors have reported a correlation with the presence of increased EGFR copy number and response to EGFR tyrosine kinase inhibitors and anti-EGFR monoclonal antibodies, respectively. Others have found conflicting results and, therefore, current recommendations for lung adenocarcinoma favor mutational testing over EGFR polysomy.15, 23 This conflicting data and limited evidence showing a predictive nature to EGFR polysomy have limited its clinical utilization in these tumors. Further investigation is warranted.

Although this study analyzes the presence of molecular alterations in EGFR or ALK translocations, it does not serve to fully evaluate the entire spectrum of possible molecular alterations contributing to the tumorigenesis of primary bladder adenocarcinoma. Despite analyzing EGFR mutational events via PCR and EGFR polysomy by FISH, other alterations in EGFR have been reported that are not detected by these methods. The most widely reported of these alterations is the EGFRvIII mutation.13, 17 However, the mutational events tested in this study are those most strongly associated with prediction to targeted therapies in lung adenocarcinoma and, as such, were the only ones examined.13, 34 In addition, we chose to not evaluate EGFR expression by immunohistochemistry. This has been reported to be a poor predictor of response to therapy in lung adenocarcinoma and is seen to lack correlation with EGFR mutations.35, 36 Prior studies performed at our institution have also shown that a poor correlation between EGFR polysomy and overexpression of EGFR is present in the prostate and germ cell tumors.24, 25, 26

As these results indicate that EGFR and ALK do not play a significant role in the development of primary bladder adenocarcinoma, other mutational events must be occurring to drive the development and propagation of these tumors. Little, however, has been done to explore the molecular landscape of this tumor. A previous study by our group demonstrated that KRAS mutations were present in 12% of cases examined.37 Additional genetic studies on the tumor are limited, but Kunze and Schlott38 have reported a high frequency of epigenetic alterations within primary urinary bladder adenocarcinoma. In particular, they described these tumors having a high degree of promoter methylation of the 14-3-3 sigma (SFN) and CAGE1 genes.

Significantly more studies have been performed exploring the genetic landscape of the much more commonly occurring urothelial carcinoma.39 Investigations of BRAF mutations in urothelial carcinoma have mostly shown these to be a rare event, although occurring in up to 7% of cases.40, 41, 42 In addition, mutations in TSC2, PIK3R1, PIK3CA, and FGFR3 have been reported in urothelial carcinoma.42, 43 Mutations in these genes lead to effects on well-studied downstream pathways such as AKT, RAS, and mTOR. These mutations, however, have not been reported or studied in primary bladder adenocarcinoma. Skeldon et al44 have shown an increased risk of urothelial cancer in patients with Lynch syndrome. We are unaware of any studies examining a correlation with Lynch syndrome or defects in mismatch repair genes when relating to primary bladder adenocarcinoma. These mutations occurring in urothelial carcinoma provide initial targets for further investigation as similar genetic events may be a possibility. The use of high-throughput sequencing techniques, such as next-generation sequencing, may expedite the discovery of additional genetic alterations and possible therapeutic targets.

Our findings indicate that primary urinary bladder adenocarcinoma lacks mutations in EGFR and ALK gene rearrangements. A significant minority of cases (36%), however, demonstrated EGFR polysomy. With increasing evidence indicating that EGFR overexpression may be an indicator of treatment response, further studies are required to determine the possible clinical utility of EGFR as a therapeutic target in the setting of primary adenocarcinoma of the urinary bladder.

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Correspondence to Liang Cheng.

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The authors declare no conflict of interest.

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Keywords

  • adenocarcinoma
  • EML4-ALK rearrangement
  • epidermal growth factor receptor (EGFR)
  • gene copy number
  • polysomy
  • targeted therapy
  • urinary bladder

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