Advances in Anatomic Pathology:
Morphologic Features of Carcinomas With Recurrent Gene Fusions
Qi, Mei MD*; Li, Yanjiang MD, PhD†; Liu, Jun MD†; Yang, Xiaoqing MD*; Wang, Lin MSc*; Zhou, Zhiqiang BSc‡; Han, Bo MD, PhD*,‡
*Department of Pathology, Shandong University Medical School
‡Department of Pathology, Shandong University Qilu Hospital, Jinan
†Department of Urology, The Affiliated Hospital of Qingdao University Medical College, Qingdao, China
Supported by the National Natural Science Foundation of China (Grant No. 81072110), Independent Innovation Foundation of Shandong University (Grant No. 2010TB012).
The authors have no conflicts of interest to disclose.
Reprints: Bo Han, MD, PhD, Department of Pathology, Shandong University Medical School, #44, Wenhua Xi Rd, Jinan 250012, P.R. China (e-mail: firstname.lastname@example.org). All figures can be viewed online in color at http://www.anatomicpathology.com.
Recurrent gene fusions have been thought to play a central role in leukemias, lymphomas, and sarcomas, but they have been neglected in carcinomas, largely because of technical limitations of cytogenetics. In the past few years, an increasing number of recurrent gene fusions have been recognized in epithelial cancers. The majority of prostate cancers, for example, have an androgen-regulated fusion of one of the ETS transcription factor gene family. Notably, the fusion genes can often serve as specific diagnostic markers, criteria of molecular classification and therefore potential therapeutic targets. Recent studies have focused on investigations of morphologic features (phenotype) of recurrent gene fusions (genotype) in malignancies. In this review, we will summarize the histologic features of known recurrent genomic rearrangements in carcinomas, especially focusing on TMPRSS2-ERG fusion in prostate cancer, EML4-ALK in lung cancer, ETV6-NTRK3 in secretory breast cancer, RET/PTC and PAX8/PPARγ1 rearrangements in thyroid cancer. In addition, we will describe how these features could potentially be used to alert the pathologists of the diagnosis of fusion-positive tumor. A combination of histologic validation with other screening strategies (eg, immunohistochemistry) for recognition of recurrent gene fusions is also highlighted.
Histologic recognition of a tumor that is associated with specific genetic aberrations provides a link between pathology and molecular characterization. It may help us understand specific biological pathways associated with special phenotype. In addition, depending on the clinical significance, it may become relevant for patient management.
The common genetic aberrations described in cancer include base substitutions, insertions, deletions, translocations, and chromosomal gains and losses. First hypothesized in the early 1900s, there is now compelling evidence for a causal role for chromosomal rearrangements in cancer.1 Recurrent chromosomal rearrangements have been thought to be primarily characteristic of leukemias, lymphomas, and sarcomas. The prototypic example of this translocation is the BCR-ABL gene fusion in chronic myeloid leukemia.1 Carcinomas, which are much more common, until recently, comprised <1% of the known, disease-specific chromosomal rearrangements. However, in 2005, Tomlins et al2 reported that over half of all prostate cancer patients harbored a particular gene fusion: TMPRSS2-ERG. Since then, a growing body of evidence suggested that gene fusions play an important role in the common epithelial cancers, such as prostate and lung cancers.3–5
The idea that gene rearrangements are associated with specific phenotype in carcinomas is an emerging concept in the field of gene fusion biology. Through careful characterization of tumors, it is now clear that the morphologic phenotype (histology) may suggest an underlying genotype (genomic rearrangement). For example, Mosquera et al6 identified that blue-tinged mucin, cribriform growth pattern, macronucleoli, intraductal tumor spread, and signet-ring cell features are significantly associated with TMPRSS2-ERG fusion in prostate cancer. Likewise, some researchers observed that EML4-ALK-positive lung cancers in Asian countries are more likely to exhibit acinar-predominant structure, less-differentiated grade, intracytoplasmic and/or extracytoplasmic mucin, and cribriform pattern.7–9 In breast cancer, ETV6-NTRK3 has been reported to be exclusively present in the majority of secretory breast carcinomas (SBCs) , a rare type of invasive breast cancer.10
In this review, we will summarize the histologic features of known recurrent genomic rearrangements in carcinomas including prostate, breast, lung, and thyroid cancer and describe how these features could potentially be used to alert the pathologist in the diagnosis of a fusion-positive cancer. A combination of histologic validation with other screening strategies (eg, immunohistochemistry) for recognition of gene fusion tumors will also be highlighted.
Prostate cancer is the most commonly diagnosed nonskin cancer in western countries. Recently, recurrent gene fusions involving oncogenic ETS transcription factors ERG, ETV1, ETV4, ETV5, and ELK4, which are fused to TMPRSS2 (a prostate-specific, androgen-responsive gene) or other upstream partners, have been identified in a majority of prostate cancers.2,3 Among ETS gene fusions, the TMPRSS2-ERG fusion is the most prevalent, occurring in approximately 50% of prostate cancer in the western countries.3 Although some controversy exists, emerging data have suggested that TMPRSS2-ERG fusion prostate cancer is associated with a more aggressive phenotype.3,11 As TMPRSS2 and ERG are located ∼3 Mb apart on chromosome 21, the TMPRSS2-ERG fusions can occur through interchromosomal insertion or through interstitial deletion (EDel).12
To date, a series of studies have investigated morphologic features of the TMPRSS2-ERG gene fusion in prostate cancer. Tu et al13 first reported that mucin-positive prostate cancers harbored TMPRSS2-ERG gene fusions more often when compared to mucin-negative tumors. In another study, Mosquera et al6 comprehensively assessed common histologic features in >100 prostate cancer cases with TMPRSS2-ERG fusions. Five morphologic features identified as blue-tinged mucin, cribriform growth pattern, macronucleoli, intraductal tumor spread, and signet-ring cell morphology are significantly associated with the presence of the TMPRSS2-ERG fusion. Cases found to harbor ≥3 of these features were almost always fusion-positive, and only 24% of ETS fusion-positive cases did not display any of these histologic features. Recently, in an attempt to characterize the prevalence of TMPRSS2-ERG fusion in prostate cancer cases with needle biopsy, the same group identified a total of 4 features, including cribriform growth, blue-tinged mucin, macronucleoli, and collagenous micronodules significantly associated with TMPRSS2-ERG fusion–positive prostate cancer biopsies.14
Histologic variants of prostate cancer account for 5% to 10% of the disease and often differ from the conventional acinar type in clinical, genetic, and biological potential. For example, small cell carcinoma (SCC) and ductal adenocarcinoma are known to exhibit an aggressive clinical behavior with poor prognosis. Most recently, we reported that 55% (27/49) of variant cancers harbor ETS aberrations by fluorescence in situ hybridization (FISH), most of which demonstrate TMPRSS2-ERG fusion.15 For the first time, our data suggested that mucinous prostate cancer was significantly associated with the presence of TMPRSS2-ERG fusion, whereas foamy gland variant was not. Interestingly, TMPRSS2-ERG fusion through EDel was observed exclusively in prostatic SCC. Later, Guo et al16 evaluated ERG genetic aberration in SCC of different organs. Notably, ERG gene rearrangement was identified in the majority (67%) of prostatic SCCs, but absent in any SCC of the urinary bladder or lungs. This finding was further supported by Lottan et al.17 Their studies collectively demonstrated the specific presence of TMPRSS2-ERG gene fusion in prostatic SCC, which may be helpful in distinguishing SCC of prostatic origin from nonprostatic origins. In another FISH-based study, we comprehensively assessed ERG aberrations in intraductal carcinoma of the prostate, a distinct histopathologic entity in prostate cancer.18 Intraductal carcinoma of the prostate has been described as a lesion associated with poor prognostic features in prostate cancer.19 In a series of 48 cases, ERG rearrangement was identified in 75% (36/48) of the cases. Of these 36 cases, 65% were through EDel and 35% were through insertion.18
Representative morphology and corresponding FISH images of prostate cancers associated with ERG rearrangement are shown in Figure 1. As mentioned above, these studies suggested that the biological effects of TMPRSS2-ERG overexpression may drive pathways that favor these common morphologic features that pathologists observe daily. Additional larger follow-up studies may help further establish the phenotypic associations of the gene fusions, and these histologic features may also be helpful in diagnosing TMPRSS2-ERG fusion prostate cancer, which may have both prognostic and therapeutic implications. However, all these histologic features lack sufficient specificity to suggest the presence of ERG rearrangement in prostate cancer.
Given the high yield and feasibility of performing immunohistochemistry versus FISH, immunohistochemistry has been employed to evaluate the ERG rearrangement in situ. Most recently, using a novel anti-ERG monoclonal antibody, several groups have reported high sensitivity and specificity of ERG rearrangement status assessed by immunohistochemistry.20–22 Therefore, through histologic analysis and immunohistochemistry, researchers are able to determine whether the patients are required to further validate TMPRSS2-ERG fusion. Another significant clinical implication for this finding is the potential utility of assessing the TMPRSS2-ERG fusion status in problematic prostate needle core biopsies and adjacent small atypical glands.
Recent advances in next-generation transcriptome sequencing facilitated the discovery of RAF kinase gene fusions, SLC45A3-BRAF, ESRP1-RAF1, and RAF1-ESRP1 in advanced prostate cancers.23 Although they are only detected in about 1% of prostate cancers, RAF kinase fusions represent the first “driver” fusion in prostate cancers that do not involve an ETS family member. So far, histologic analysis is lacking in this distinct molecular subtype.
Lung cancer remains the leading cause of cancer death in both men and women with an estimated ∼1.3 million deaths worldwide annually.24 Recurrent gene fusions, such as activating fusions of the ALK and ROS tyrosine kinases, have also been reported in non–small cell lung cancer (NSCLC).25–27 Among them, EML4-ALK fusion was most prevalent, which was firstly identified by Soda et al25 in 2007, occurring in 6.7% (5/75) Japanese NSCLC patients. Additional studies that usually involve east Asian patients, have reported that approximately 5% of lung cancers harbor EML4-ALK fusions, equivalent to over 70,000 patients diagnosed annually worldwide.4,27 Recent studies tend to recognize EML4-ALK fusion as an intriguing potential target for lung cancer diagnosis and treatment.
EML4-ALK appears to be specific to lung cancer, but absent in gastrointestinal and breast cancers.28 The vast majority of ALK-rearranged NSCLC cases are adenocarcinomas. Occasionally, EML4-ALK fusion has also been reported in adenosquamous cell carcinomas as well.29 Of note, ALK rearrangements occur almost exclusively in adenocarcinoma but not in squamous cell carcinoma.30 Overall, a variety of histologic features are associated with the presence of ALK rearrangement. Signet-ring cell morphology was reported mostly in the western patients,31,32 which was similar to those more frequently seen in gastric cancer. The majority of western patients showed tumor cells with a solid or sheet-like pattern, easily distinguishable from the acinar, papillary, or bronchioloalveolar patterns, which contrasts the acinar pattern commonly observed in Asian populations (Fig. 2A). Cytologically, abundant intracellular mucin and small, marginalized nuclei are often identified in the majority of ALK-rearranged tumors. Jokoji et al9 reported that EML4-ALK-positive lung adenocarcinomas showed significant associations with intracytoplasmic and/or extracytoplasmic mucin, and cribriform pattern with excessive extracytoplasmic mucin. In another study, Inamura et al7 found an interesting histotype-genotype relationship in a subset of Japanese EML4-ALK lung cancers, that is, all 3 EML4-ALK variant 2 cases were acinar adenocarcinomas, and the other 2 ALK-rearranged cases were mixed-type adenocarcinomas, showing papillary with bronchioloalveolar components. This finding was further supported by Wong et al.8 In their cohort, 4.9% (13/266) of Chinese prostate cancer patients harbored EML4-ALK, most of which were adenocarcinomas. Of the ALK-rearranged adenocarcinomas, 82% (9/11) had the mixed tumor subtype, which displayed papillary, micropapillary, and cribriform growth.
A most recent comprehensively histologic analysis of ALK-rearranged lung cancers was performed by Yoshida et al30 in 2011. They noted that solid or acinar growth pattern, cribriform structure, presence of mucous cells (signet-ring cells or goblet cells), abundant extracellular mucus, lack of lepidic growth, and significant nuclear pleomorphism were more common in ALK-rearranged lung cancers. Of note, the majority (78%) of ALK-positive tumors harbored solid signet-ring cell and mucinous cribriform patterns, whereas only 1% of ALK-negative tumors had the 2 features.
EML4-ALK NSCLC represents a unique subset of lung cancer patients for whom ALK inhibitors may represent a very effective therapeutic strategy.33 The challenge is to incorporate and disseminate widespread use of diagnostic testing for EML4-ALK to identify this patient subset. So far, FISH analysis remains the classic method to detect ALK rearrangements although it is time-consuming and labor intensive. Most recently, a combination of histologic evaluation with immunohistochemical analysis has been successfully applied to detect ALK fusion protein in lung cancers.9,34 Immunohistochemistry can be a useful test for screening ALK FISH-positive cases in advanced lung cancers.
Taken together, the above studies revealed the association of EML4-ALK fusion and acinar-predominant morphology in lung cancer. Although additional larger scale studies are needed, a combination of histologic and immunohistochemical analysis may be helpful in diagnosing ALK fusion–positive lung cancer.
Breast cancer is a heterogeneous disease, encompassing multiple entities associated with distinctive histologic features, clinical presentations, and biological behaviors. Two recent reviews assessing >30 studies revealed the direct genotypic-phenotypic correlation in breast cancer.35,36 So far, recurrent gene fusions that have been reported in breast cancer mainly involve ETV6-NTRK310,37 and MYB-NFIB,38 which only occur in rare histologic types. Other gene fusions such as RPS6KB1-VMP1 and ESR1-C6ORF97 were also identified in breast cancer,39,40 but seem to be extremely rare.
SBC accounts for <1% of all breast cancers. Previously, Tognon et al10 reported the great majority of SBCs (12/13, 92%) were shown to harbor ETV6-NTRK3 gene fusion. Using conditional node mice models, they provided direct evidence for the causative role of ETV6-NTRK3 in the carcinogenesis of SBC of breast cancer. This finding was further supported by Makretsov et al.37 Notably, none of the ETV6-NTRK3 fusion–negative breast cancers in their cohort revealed SBC histology. Therefore, ETV6-NTRK3 fusion seems to be restricted to SBC as indicated. Most recently, Lae et al41 showed that SBCs with ETV6-NTRK3 fusion gene belonged to the phenotypic basal-like spectrum of breast carcinomas. Their data further suggested the concept that ETV6-NTRK3 fusion is specific for SBC in the context of breast malignancies. Morphologic image of SBC associated with ETV6 translocation is shown in Figure 2C.
Another recurrent gene fusion that occurs in breast cancer is MYB-NFIB fusion. In 2009, Persson et al38 reported MYB-NFIB fusion in adenoid cystic carcinomas (ACCs). Most recently, Wetterskog et al42 revealed that 12 out of the 13 breast ACCs displayed the MYB-NFIB fusion gene using FISH. They identified that MYB-NFIB fusion, as a candidate therapeutic target, was a hallmark of breast ACCs. Taken together, their results demonstrate that the vast majority, but not all of breast ACCs harbor the MYB-NFIB fusion gene. Of note, strong MYB immunostaining is very specific for ACCs and can be utilized as a useful diagnostic marker for a subset of ACCs.38,43–45
In the past few years, with development of new technologies, such as massively parallel RNA sequencing, many novel gene fusions have been identified in breast cancer, for example, ARHGEF2-SULF2, AHCYL1-RAD51C, and RAD51C-ATXN7 in MCF-7 cell line.46 In 2011, Robison et al47 identified 2 classes of recurrent gene rearrangements which are genes encoding microtubule-associated serine-threonine kinase and members of the Notch family. These findings suggest that recurrent gene rearrangements have key roles in subsets of breast carcinomas. Of note, most of the current data are from individual cell lines, so we do not know how recurrent the fusions are in patient cohort. In addition, although some gene fusions in breast cancer are potentially “targetable,” no morphology analysis has been performed because of its rare nature.
Collectively, these data provided specific gene fusions associated with certain types of breast cancer such as ETV6-NTRK3 in SBC and MYB-NFIB in ACC. Understanding the molecular basis of histologic features is a crucial step for unraveling the complexity and heterogeneity of breast cancer that will pave the way for better classification systems and effectively tailoring the therapies of breast cancer patients.
So far, RET/PTC and PAX8/PPARγ1 rearrangements, point mutations of BRAF and RAS represent the most common genetic aberrations in thyroid cancers. Histologically, thyroid cancers are mainly divided into papillary, follicular, medullary, and anaplastic carcinomas. Among them, papillary thyroid carcinoma (PTC) is the most prevalent, and comprises of ∼80% of all thyroid cancers. To date, at least 15 different types of RET/PTC chimeric oncogene have been identified in this entity.48 Although with some controversy, it is generally believed that RET/PTC is the major molecular marker for PTC. In 1997, Nikiforov et al49 identified that RET/PTC1 is associated with classic papillary histologic features, whereas RET/PTC3 is more common in solid variants and radiation-induced sporadic thyroid papillary carcinomas. Interestingly, this correlation was proven in transgenic mice models. Santoro et al50 reported that thyroid-specific expression of the RET/PTC1 rearrangement under the control of thyroglobulin promoter can lead to the development of thyroid tumors with micropapillary architecture, mixture of solid/cribriform growth with papillary infolding, and cytologic features of papillary carcinoma, including irregularly shaped nuclei, intranuclear inclusions, and grooves. Recently, Adeniran et al51 comprehensively validated common histologic features of 97 papillary carcinoma cases with RET/PTC fusions. They noted that RET/PTC-positive papillary carcinomas frequently metastasize to lymph nodes. A total of 6 diagnostically relevant nuclear features were significantly associated with the presence of RET/PTC fusion, that is, nuclear enlargement; nuclear contour irregularity, chromatin clearing, nuclear crowding/overlapping, nuclear grooves, and pseudoinclusions. Three other common microscopic features, such as lymphocytic inflammationl psammoma bodies and tumor fibrosis can also be observed. Although RET/PTC genetic rearrangement is highly correlated with PTC, it has been reported that other thyroid diseases, such as Hashimoto thyroiditis, oncocytic adenomas, may also harbor this genetic aberration. It should be noted that gene fusions involving the NTRK1 gene, may act as an alternative to RET fusions in papillary thyroid carcinogenesis. Recently, Ciampi et al52 also reported AKAP9-BRAF fusion in 11% (3/28) early radiation-associated PTCs.
In contrast, ∼50% human follicular thyroid carcinomas (FTCs) presented PAX8-PPARγ1 fusion, which is highly specific to FTC. Kroll et al53 firstly reported that PAX8-PPARγ1 were identified in thyroid FTCs (5/8, 62.5%) at mRNA and protein expression level, but not in PTCs, follicular adenomas, or multinodular hyperplasia. Interestingly, emerging data revealed that PAX8-PPARγ fusion exhibit significant differences in prevalence among different ethnic groups. For example, Chia et al54 detected PAX8-PPARγ translocation in a small subset (2/18, 11.1%) of Malaysian FTC patients using FISH analysis, which was significantly lower than that of western countries (26% to 78%). In 2004, Hibi et al55 detected no PAX8-PPARγ translocation in advanced FTC or in follicular adenoma in Japanese patients.
Other gene fusions involved in the PPARγ1 gene are much rarer in FTC. For example, Lui et al56 identified that <3% FTCs harbored the CREB3L2-PPARγ1. In summary, RET/PTC rearrangement is more relevant to PTC, and PAX8-PPARγ1 seems to be associated with FTC in thyroid cancers. All these observations may help design better prevention and treatment strategies for thyroid cancers.
OTHER EPITHELIAL CANCERS
Although rare, recurrent gene fusions have also been identified in other epithelial malignancies. Translocation renal cell carcinoma (RCC), also known as juvenile RCC, represents 15% of RCC in patients younger than 45 years.57 The best characterized subtype of translocation RCC is the TFE3 family translocation RCC. These fusions include PRCC-TFE3, ASPL-TFE3, and CLTC-TFE3.57 Argani et al58 reported that ASPL-TFE3 fusion–positive renal tumors were characterized by nested and pseudopapillary patterns of growth, psammomatous calcifications, epithelioid cells with abundant clear cytoplasm, and well-defined cell borders (Fig. 2B). The same group further identified that irregular nuclei with vesicular chromatin, small nucleoli, and cytoplasm from clear to densely granular were more common in PRCC-TFE3 RCCs.59 All variants of TFE3 translocation RCC result in overexpression of TFE3 protein, which can be detected by immunohistochemistry.60 Positive immunostain for TFE3 is essential in confirming the diagnosis of Xp11 translocation RCC. Another less common translocation subtype, t (6; 11) (p21; q22), involving a fusion α/TFEB, induces overexpression of transcription factor EB (TFEB), which can be detected by immunohistochemistry.60
MECT1-MAML2 has been recognized in up to 70% of mucoepidermoid carcinomas.61 Clinically, MECT1-MAML2-positive patients had a better prognosis compared to fusion-negative patients. Therefore, molecular classification of mucoepidermoid carcinomas on the basis of the MECT1-MAML2 fusion oncogene is histopathologically and clinically relevant.61 Mammary analogue secretory carcinoma is a recently described salivary gland tumor with ETV6 translocation.62 So far, the largest series of mammary analogue secretory carcinoma (n=16) is that described in the report by Skalova et al.63
In 2011, Gu and colleagues identified the presence of ROS kinase fusions in 8.7% (2/23) of cholangiocarcinoma patients, and ROS kinase has been a promising candidate for a diagnostic molecular marker and therapeutic target in cholangiocarcinoma.64 Most recently, ESRRA-C11orf20 fusion was identified in serous ovarian cancer65 and CD44-SLC1A2 gene fusion was recognized in 1% to 2% of gastric cancer.66 With rapid development of new technologies, more and more novel gene fusion have been identified, and morphologic assessment of these gene fusions would provide helpful information in the diagnosis and molecular characterization in research community.
CONCLUSIONS AND FUTURE PERSPECTIVE
This is the first review to systematically summarize morphologic feature of gene fusions in carcinomas. Table 1 summarized morphologic features or associated histologic subtypes of recurrent gene fusions in carcinomas. Notably, some histologic features could be helpful to the pathologist to diagnose a fusion-positive cancer. Depending on the clinical significance, histologic recognition of a tumor associated with specific gene rearrangement may become relevant for patient management. However, it is notable that the histologic features lack sufficient specificity to suggest the presence of the translocation in the majority of carcinomas, such as TMPRSS2-ERG fusion in prostate cancer, EML4-ALk fusion in lung adenocarcinomas. Association of morphology and gene fusions seems to be tighter in a subset of rare carcinomas or histologic variants of malignancies.
Here, several interesting questions arise, for example, why some recurrent gene fusions seem to be restricted to some specific tumor types, such as ETV6-NTRK3 in SCCs of breast cancer? What are the underlying molecular mechanisms of this phenotype-genotype association? Integration of morphologic analysis and molecular biology may help to address these issues.
In the last 7 years, the discoveries of recurrent gene fusions, such as TMPRSS2-ETS in prostate cancer, have opened up new avenues for cancer research and treatment. The data summarized in this review indicate that the pathogenetic mechanisms involved in epithelial cancer may be similar to those known to operate in hematological and soft-tissue malignancies; these functional gene fusions may therefore serve as ideal diagnostic markers, provide insight into tumor biology, and most importantly, serve as specific therapeutic targets. In addition, pathologists should strive for developing a standardized morphologic validation and molecular classification for these fascinating entities with gene fusions, which may lead to a better understanding of their biology and their clinical behavior and management.
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gene fusion; carcinoma; morphology; histology
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