Advances in Anatomic Pathology:
Clinical Applications of Recent Molecular Advances in Urologic Malignancies: No Longer Chasing a “Mirage”?
Netto, George J. MD
Departments of Pathology, Urology and Oncology, Johns Hopkins University, Baltimore, MD
The author has no funding or conflicts of interest to disclose.
All figures can be viewed online in color at http://http://www.anatomicpathology.com.
Reprints: George J. Netto, MD, Departments of Pathology, Urology and Oncology, Johns Hopkins University, 401N. Broadway, Weinberg 2242, Baltimore, MD 21231 (e-mail: firstname.lastname@example.org).
As our understanding of the molecular events leading to the development and progression of genitourologic malignancies, new markers of detection, prognostication, and therapy prediction can be exploited in the management of these prevalent tumors. The current review discusses the recent advances in prostate, bladder, renal, and testicular neoplasms that are pertinent to the anatomic pathologist.
Recent advances in cancer genetics and genomics are reshaping management paradigms in solid tumor patients. Molecular diagnostics have become an integral part of clinical management algorithms of patients with lung, colon, and brain cancers. Till recently, implementation of molecular biomarkers in the management of urologic malignancies has been an illusive target.
In prostate cancer (PCa), the continuous debate on whether current serum prostate-specific antigen (PSA)-based screening strategies are potentially leading to “overtreatment” of a subset of PCa patients has further fueled the interest in pursuing clinicopathologic and molecular parameters that may help identify patients with biologically “significant” PCas.1,2 More recently, applying sophisticated bioinformatics tools to increasing number of datasets generated from genomic, transcriptomic, and proteomic studies of PCa has led to the discovery and validation of a growing list of molecular biomarkers with potential role in predicting disease progression, response to therapy, and survival in PCa patients.3–62 Identifying molecular profiles that can accurately predict “insignificant” PCa tumors would be valuable for alternative PCa management approaches such as “proactive surveillance” that are increasingly offered.
For bladder cancer (BC) patients, the need for new treatment options that can improve upon the modest outcomes currently associated with muscle invasive bladder cancer (MI-BC) is evident. Validated prognostic molecular biomarkers that can help clinicians identify patients in need of early aggressive management are lacking. Robust predictive biomarkers that can stratify responses to emerging targeted therapies are also needed.
Similarly, the increasingly momentous adoption of targeted systemic therapy strategies in advanced clear cell renal cell carcinoma (RCC) has incited interest in identifying predictive markers of response and in exploring new targets of therapy for other types of renal tumors.
Emerging Prognostic Factors
Firmly established parameters such as clinical stage, pathologic stage, histologic Gleason grade, and serum PSA levels are routinely used for prognostication and guidance of disease management in PCa.13–15 As the molecular events underpinning the development of PCa and the pathogenetic steps detailing the epigenetic and genetic alterations involved in the progression of PCa have been brought into focus (Fig. 1), an extensive list of molecular biomarkers have been evaluated in the last decade for their potential role in predicting disease outcome.3,7,10–12,16–20 The wide array of molecular-based PCa markers include proliferation index (ki67),21–27 microvessel density,28–33 nuclear morphometry,34–37 tumor suppression genes (eg p53, p21, p27, NKX3.1, PTEN, retinoblastoma gene “Rb”), oncogenes (eg Bcl2, c-myc, EZH2, and HER2/neu), adhesion molecules (CD44, E-cadherin), PI3K/akt/mTOR pathway,38 apoptosis regulators (eg, survivin and transforming growth factor β-1), androgen receptor status,39 neuroendocrine differentiation markers,40–45 and prostate tissue lineage-specific markers expression (PSA, PSAP, and prostate-specific membrane antigen “PSMA”).
Epigenetic Changes in PCa
Changes in DNA methylation marks, accompanied by epigenetic gene silencing, seem to be the earliest somatic genome changes in PCa18 (Table 1). New generation of assay strategies for detection of specific DNA sequences carrying 5-meC, offer promising opportunities for clinical tests for potential PCa screening, detection, diagnosis, staging, and risk stratification. Hypermethylation of glutathione S-transferase-π (GSTP1) transcriptional regulatory sequences has been consistently detected in >90% of PCas. GSTP1, encodes an enzyme responsible for detoxifying electrophiles and oxidants thus shielding cell from genome damage. Loss of GSTP1 expression seems to be an early event in the initiation of prostatic carcinogenesis as evidenced by the presence of GSTP1 methylation in 5% to 10% of proliferative inflammatory atrophy lesions thought by some to be the earliest PCa precursors, and in >70% of high-grade prostatic intraepithelial neoplasia lesions.46,47 In addition to GSTP1, >40 other genes have been shown to be altered by epigenetic hypermethylation.48 Yegnasubramanian et al49,50 found hypermethylation at GSTP1, APC, RASSF1a, COX2, and MDR1 to be detected both in localized and in metastatic PCa, whereas hypermethylation of other genes such as ERα, hMLH1, and p14/INK4a were more likely to be found in latter stages of PCa progression suggesting “2 waves” of epigenetic alterations in PCa.
ERG-ETS Gene Fusions
In 2005, Tomlins et al51,52 identified a recurrent chromosomal rearrangement in over one half of their analyzed PCa cases. The recurrent chromosomal rearrangements lead to a fusion of the androgen-responsive promoter elements of the TMPRSS2 gene (21q22) to 1 of 3 members of the E26 transformation-specific (ETS) transcription factors family members ERG, ETV1, and ETV4 located at chromosomes 21q22, 7p21, and 17q21, respectively. Although the prognostic role of assessing TMPRSS2-ETS rearrangements in PCa tissue samples has been called into question by recent well-designed large cohort studies including ours,53,54 the discovery had great implications in terms of furthering our understanding development and pathogenesis of PCa and provide a new marker for molecular diagnosis in PCa.55–62,64,65 The potential diagnostic and prognostic role of detecting TMPRSS2-ERG in postprostate massage urine samples requires further investigation.66–68 Figure 2 depicts a commonly used FISH split apart-based approach for the evaluation of ERG gene fusion. Recently, commercial anti-ERG monoclonal antibodies became available that makes it possible to use IHC for evaluating ERG protein expression as a surrogate approach to detecting TMPRSS2-ERG fusion by FISH. We, and others, have demonstrated a strong correlation between ERG overexpression by IHC and ERG fusion status with over 86% sensitivity and specificity rates. ERG IHC may offer an accurate, simpler, and less costly alternative for evaluation of ERG fusion status in PCa on needle biopsy and radical prostatectomy samples69,70 (Fig. 3).
The PI3K/mTOR (mammalian target of rapamycin) pathway plays an important role in cell growth, proliferation, and oncogenesis in PCa.71–77 PTEN is a negative regulator of this pathway. Several recent well-designed retrospective studies have revealed that loss of PTEN tumor suppressor gene (TSG) activity and the ensuing mTOR pathway activation is associated with poor prognosis in PCa. In a recent large-nested cases control, tissue microarray-based, study from our institution, we were able to show loss of immunoexpression of PTEN to be a predictor of biochemical recurrence after radical prostatectomy independent of Gleason grade, cancer stage, and other clinicopathologic parameters.78 In a second study from our group by Lotan et al,79 the prognostic role of PTEN alteration was further linked to adverse pathologic features and decreased time to metastatic disease in a surgical cohort of high-risk PCa patients. The correlation of PTEN immuostains with genomic loss of PTEN gene was also established is the later study. The mTOR pathway is also a potential target for PCa treatment and several rapamycin analogs are currently being tested as potential therapeutic agents for PCa.76,80 We recently reported the results of a pilot study evaluating the pharmacodynamic efficacy of neoadjuvant rapamycin therapy in PCa.80 Using IHC analysis, we found a significant decrease in Phos-S6 protein, the main downstream effector of mTOR pathway, in patients receiving neoadjuvant mTOR inhibitor agent.80
Other Tumor Suppressor Genes and Oncogenes
Among TSGs, the role of p53 expression in predicting prognosis in prostate carcinoma has been extensively studied. Brewster et al81 found p53 expression and Gleason score in needle biopsy to be independent predictors of biochemical relapse after radical prostatectomy. Another study found p53 status on prostatectomy but not needle biopsies to be predictive raising the issue of sampling.82 Many studies evaluating prostatectomy specimens found p53 to be of prognostic significance independent of grade, stage, and margin status.26,33,83–88 More recent genome-wide studies seems to support the prognostic role of p53 alterations89 (see below section on Integrated Genomics). The majority of studies of another TSG p27, a cell cycle inhibitor, have also supported a correlation with progression after prostatectomy. Although less robust evidence exists for the prognostic role of p21,90 a downstream mediator of p53, and transcription factors such as NKX3.1,19,91 preponderance of evidence supports a prognostic role for Bcl222,81,83,85,87 and myc oncogenes92,93 as potential adjuncts to histologic prognostic parameters.
Gene expression profiling studies using cDNA microarrays containing 26,000 genes identified 3 subclasses of prostate tumors based on distinct patterns of gene expression.4 High-grade and advanced stage tumors, as well as tumors associated with recurrence, were disproportionately represented among 2 of the 3 subtypes, one of which also included most lymph node metastases. Furthermore, 2 surrogate genes were differentially expressed among tumor subgroups by immunohistochemistry. These included: MUC1, a gene highly expressed in the subgroups with “aggressive” clinicopathologic features and AZGP1, a gene highly expressed in the favorable subgroup. The surrogate genes were strong predictors of tumor recurrence independent of tumor grade, stage, and preoperative PSA levels. Such study suggests that prostate tumors can be usefully classified according to their gene expression patterns, and these tumor subtypes may provide a basis for improved prognostication and treatment stratification.
In another study, Tomlins et al6 used laser-capture microdissection to isolate 101 cell populations to illustrate gene expression profiles of PCa progression from benign epithelium to metastatic disease. By analyzing expression signatures in the context of over 14,000 “molecular concepts,” or sets of biologically connected genes, the authors generated an integrative model of progression. Molecular critical transitions in progression included protein biosynthesis, ETS family transcriptional targets, androgen signaling, and cell proliferation. Known prognostic markers such as grade could be ascribed to noted attenuated androgen signaling signature seen in high-grade cancer (Gleason pattern 4), similar to metastatic PCa, which may reflect dedifferentiation and explain the clinical association of grade and prognosis. Taken together, these data show that analyzing gene expression signatures in the context of a compendium of molecular concepts is useful in understanding cancer biology.
Lapointe et al94 complemented their above-mentioned gene expression findings by looking for associated copy number alterations using array-based comparative genomic hybridization (array CGH). They were able to identify recurrent copy number genetic aberrations that corresponds to 3 prognostically distinct groups of PCa: (i) deletions at 5q21 and 6q15 deletion group associated with favorable outcome group; (ii) a 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion) deletion group, and (iii) 8q24 (MYC) and 16p13 gains, and loss at 10q23 (PTEN) and 16q23 groups correlating with metastatic disease and aggressive outcome.
Finally, in a recent genome-wide analysis of PCa, Taylor et al95 elegantly illustrated how detailed annotation of PCa genomes can impact our understanding of the disease and its treatment strategy. Assessing DNA copy number, mRNA expression, and focused exon resequencing in 218 PCa tumors, the authors identified the role of nuclear receptor coactivator NCOA2 as a novel oncogene in 11% of PCa cases. TMPRSS2-ERG fusion was associated with novel prostate-specific deletion at chromosome 3p14 that may implicate FOXP1, RYBP, and SHQ1 as potential cooperative tumor suppressors. Most intriguing was their ability to define clusters of low-risk and high-risk disease beyond that achieved by Gleason score using DNA copy number data. Six clusters of PCa tumors are identified by unsupervised hierarchical clustering with distinct risk for biochemical recurrence.
In summary, genomic studies suggest that PCas develop through a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating PCa biology and explain in part the clinical heterogeneity of the disease.
Markert et al89 also illustrated the potential utility of molecular signatures as prognosticator in PCa. The authors assessed microarray dataset characterizing 281 PCa patients from a Swedish watchful-waiting cohort. mRNA microarray signature profiles for gene signatures reflecting embryonic stem cell, induced pleuripotent stem cell, and polycomb repressive complex-2 phenotypes, in addition to inactivation of the tumor suppressors p53 and PTEN loss and the TMPRSS2-ERG fusion were assessed. Unsupervised clustering identified PCa subset with “stem-like signatures” combined with p53 and PTEN inactivation to be associated with very poor survival outcome. PCa tumors characterized by TMPRSS2-ERG fusion had intermediate survival outcome, whereas remaining groups demonstrated more favorable outcome (Fig. 4). The exciting findings were further validated in an independent clinical cohort at Memorial Sloan-Kettering Cancer Center. This classification was independent of Gleason score and therefore can provide additional added value in prognostication in patients with lower Gleason Grade PCa.
Emerging Early Detection Markers and Targets of Therapy
Markers of PCa detection that can be applied to blood, urine, or prostatic secretion fluid (ejaculate or prostate massage fluids) have been the focus of active recent research. Markers that have been investigated in the urine or prostatic secretions include gene promoter hypermethylation profile assays48,96–98 and DD3 (differential display code 3), also known as PCA3. DD3 is a noncoding RNA that was initially identified by Bussemakers et al99 as one of the most specific markers of PCa. PCA3 gene is located on chromosome 9q21.2 (Fig. 5). Quantitative real-time reverse transcriptase PCR (RT-PCR) assay detecting PCA3 can be applied to blood, urine, or prostatic fluid.100
Evaluation of PCA3 in postattentive prostate massage urine samples using transcription-mediated amplification technology has shown to be superior to serum PSA in prediction biopsy outcome with sensitivity and specificity approximating 70% and 80%, respectively, and a negative predictive value of 90%101–104; it is currently under evaluation for Food and Drug Administration (FDA) approval in the United States. Encouraging data from the REDUCE trial support a role for evaluation of PCA3 in postattentive prostate massage urine sample in predicting positive prostate needle biopsy in immediately subsequent as well as future biopsies after initial negative biopsy. PCA3 may also have a role in predicting the risk for higher Gleason score and larger tumor volume on radical retropubic prostatectomy. If confirmed, the latter could be of great value in treatment options algorithm and delineation of candidates for active surveillance.105–108 Multiplex urine assays to include PCA3, TMPRSS2-ERG, SPINK1, and GOLPH2 are also under evaluation with recent data suggesting an improved performance of such assays compared to PCA3 alone.109
Finally, several markers are being investigated as potential targets of therapy for PCa. The list includes: tyrosine kinase receptors (eg, EGFR), angiogenesis targets [(eg, vascular endothelial growth factor (VEGF)],110 fatty acid synthase,111 PI3K/akt/mTOR mammalian target of rapamycin,76,80,112 endothelin receptors,113,114 and PSMA115–118 to name a few.
In summary, a wide array of molecular markers discussed in this chapter may be utilized in the near future as adjuncts to currently established prognostic parameters and early detection markers. Loss of PTEN and PCA3 are 2 such markers that are most likely to soon gain widespread utilization. Current research efforts in prostate are also focused on biological markers that can serve as target of therapy.
In 2012, over 73,510 new cases of urothelial carcinoma (URCa) were diagnosed in the United States leading to over 14,880 deaths.119 BC is the fourth most common tumor in males and 11th most common in females. Because of the high rate of tumor recurrence and the need for frequent cystoscopy, URCa is the cancer with the highest cost per patient with an annual burden of over 4 billion dollars to our health care system. Nevertheless, URCa presents us with unique challenges and opportunities given urine samples amenable to the application of noninvasive molecular detection methods and the relative ease of delivery of molecular targeted therapy to a topographically accessible tumor.120
Clinically, URCa present 2 distinct phenotypes. The first phenotype is superficial (nonmuscle invasive) URCa representing three fourth of cases. Fifty percent of these superficial tumors will recur as nonmuscle invasive tumors and only 5% to 10% of progressing into muscle invasive disease.
The mainstay of therapy in superficial tumors is TURB with or without intravesical chemotherapy and immune therapy (BCG). The second phenotype is the muscle invasive URCa representing 20% to 30% of all URCa. Only 15% of muscle invasive URCa have a prior history of superficial URCa and represent a progression from the superficial phenotype, whereas the majority (80% to 90%) are “primary” de novo muscle invasive URCa. Currently, patients suffering from muscle invasive high-grade tumors are destined to a disappointing 50% to 60% overall survival despite aggressive combined treatment modalities including cystectomy and chemotherapy.
Accumulating molecular genetic evidence supports 2 distinct broad pathogenetic pathways for BC development that seem to parallel the contrasting biological and clinical phenotypes of nonmuscle invasive (superficial) and muscle invasive URCa (Fig. 6). Although the majority of invasive URCas are thought to originate through progression from dysplasia to flat CIS and high-grade noninvasive lesions, superficial urothelial lesions are thought to originate from benign urothelium through a process of urothelial hyperplasia. Progression from nonmuscle invasive to muscle invasive disease accounts for only a small percentage (10% to 15%) of the entire pool of noninvasive lesions. Genetic instability is key to the accumulation of genetic alterations required for progression to MI-BC.122–125
Clinically, a significant proportion of nonmuscle invasive bladder cancer (NMI-BC) (pTa and pT1) are deemed to recur after transurethral resection (TURB) with only a minority of cases enduring progression to high-grade carcinoma that will ultimately progress to MI-BC.
Three primary genetic alterations have consistently been associated with the pathogenesis pathway of NMI-BC. These include tyrosine kinase receptor FGFR-3, H-RAS,126 and PIK3CA.124,127,128 Alterations in the RAS-MAPK and PI3K-Akt pathways are in large part responsible for promoting cell growth in urothelial neoplasia. Activating mutations in RAS lead to activation of mitogen-activated protein kinases (MAPK) and PI3K pathways. Not surprisingly, activating mutations in upstream tyrosine kinase receptor FGFR3 seems to be mutually exclusive with RAS mutations given that both signal through a common downstream pathway in urothelial oncogenesis. PIK3CA and FGFR3 mutations generally co-occur suggesting a potential synergistic additive oncogenic effect for PIK3CA mutations.
The pathogenic pathway for MI-BC primarily involves alterations in TSGs involved in cell cycle control including p53, p16, and Rb122,125,129 (Fig. 7). As illustrated in Figure 6, progression of the subset of NMI-BC into higher grade muscle invasive disease is similarly based on alterations in p53 and Rb TSGs.
Established clinicopathologic prognostic parameters for NMI-BC include: pT stage, WHO/ISUP grade, tumor size, tumor multifocality, presence of CIS, and frequency and rate of prior recurrences.130 Prognostic parameters that can accurately predict progression in patients with NMI-BC tumors are actively sought to further facilitate identification of those in need of vigilant surveillance and aggressive treatment plan. The latter is especially pertinent in a disease where the financial burden and quality of life for patients under surveillance is significant. Per patient, BC is the most expensive single solid tumor in the United States with a staggering 3 billion US Dollars estimated annual cost to our health care system.131 Furthermore, given the current poor outcome of muscle invasive disease (≤60% overall survival rate), markers that can improve prognostication in this group of patients are equally needed.132–134
As our understanding of molecular pathways involved in urothelial oncogenesis increasingly come into focus, the translational field of molecular prognostication, theranostics, and targeted therapy in BC has sharply gained momentum.135–153 Evidently, a rigorous validation process ought to precede the incorporation of such molecular biomarkers in clinical management. Initial retrospective discovery studies need to be confirmed and validated in large independent cohorts. The subsequent crucial step is validating the robustness of the proposed biomarker in well-controlled multi-institutional randomized prospective study. Such prospective study should support an additive role for the inclusion of the new biomarker over existing management algorithm(s).154,155 It is the lack of the latter crucial steps in biomarkers development that had hindered the streamlining of clinical utilization of several promising markers in BC patient management.63,121,156,157
Emerging Molecular Diagnostic Applications
Chromosomal Numerical Alteration
Chromosome 9 alterations are the earliest genetic alterations in both of the above-described divergent pathways of BC development. They are responsible for providing the necessary milieu of genetic instability that in turn allows for the accumulation of subsequent genetic defects.125 Several additional structural/numerical somatic chromosomal alterations are also a common occurrence in BC. Among these, gains of chromosomes 3q, 7p, and 17q and 9p21 deletions (p16 locus) are of special interest given their potential diagnostic and prognostic value.158,159 A multitarget interphase FISH-based urine cytogenetic assay was developed160 based on the above numerical chromosomal alterations and is now commercially available and commonly used in clinical management (Fig. 8). Initially FDA approved for surveillance of recurrence in previously diagnosed BC patients, the test subsequently gained approval for screening in high-risk (smoking exposure) patients with hematuria. The multicolor FISH assay seems to enhance the sensitivity of routine urine cytology analysis and can be used in combination with routine cytology as a reflex testing in cases with atypical cytology. A sensitivity range of 69% to 87% and a specificity range of 89% to 96% have been reported with the multitarget interphase FISH assay.162 With the exception of 1 study,161 the multitarget FISH urine assay has been shown to be more sensitive than routine cytology. An additional advantage of urine-based FISH testing could be the anticipatory positive category of patients identified by such assay. This refers to patients where FISH assay detects molecular alteration of BC in urine cells several months before cancer detection by cystoscopy or routine cytology. In the study by Yoder et al,163 two thirds of the 27% of patients categorized as “anticipatory positive” developed BC that was detected by cystoscopy up to 29 months later. Such encouraging results point to the great potential of molecular testing in early detection and allocation of vigorous frequent follow-up cystoscopy in at-risk patients.164–167
Finally, several recent studies have pointed to the potential prognostic role for multitarget FISH analysis.158,159,168–170 Maffezzini et al were able to demonstrate that low-risk FISH-positive patients, defined as 9p21 loss/Ch3 abnormalities, had a higher rate of recurrence as compared with FISH-negative patients.169 The recurrence rate was even greater in patients with a high-risk positive FISH (Ch7/Ch17 abnormality). Both Kawauchi and colleagues, using bladder washings, and Kruger and colleagues, using formalin-fixed paraffin-embedded (FFPE) transurethral biopsy samples, independently found loss of 9p21 to predict recurrence but not progression in NMI-BC. Furthermore, both Savic et al168 and Whitson et al170 found urine cytology and FISH in post-BCG bladder washings to be predictive of failure to BCG therapy in patients with nonmuscle invasive disease. Such promising prognostic role for multitarget FISH awaits prospective randomized trial before clinical integration into practice algorithm. Clear guidelines for interpretation and test performance parameters in terms of interobserver reproducibility is also needed.171
Receptor Tyrosine Kinases
Recent studies have pointed to the potential prognostic value of evaluating the expression of receptor tyrosine kinases (RTK) such as FGFR-3, EGFR, and other ERB family members (HER2/neu and ERBB3)125,142,172–182 in NMI-BC and MI-BC disease.
FGFR3 mutations are a common occurrence in NMI-BC and can theoretically be used alone or combined with RAS and PIK3CA oncogenes as markers of early recurrence during surveillance. Both Zuiverloon et al183 and Miyake et al184 independently developed sensitive PCR assays for detecting FGFR3 mutations in voided urine. A positive urine sample by the assay developed by Zuiverloon group was associated with concomitant or future recurrence in 81% of NMI-BC cases. An even higher positive predictive value of 90% was achieved in patients with consecutive FGFR3-positive urine samples. Similarly, Miyake and colleagues were able to detect FGFR3 mutations in 53% of their 45 patients and found their assay to be superior to cytology (78% vs. 0%) in detecting post-TURB recurrence in NMI-BC harboring FGFR3 mutations in primary tumors.
Kompier et al128 were recently able to develop multiplex PCR assay for mutational analysis detecting the most frequent mutations hot spots of HRAS, KRAS, NRAS, FGFR3, and PIK3CA in FFPE TURB samples. They demonstrated evidence of at least 1 mutation in up to 88% of low-grade NMI-BC samples. Hernandez et al185 revealed that FGFR3 mutations were more common among low malignant potential neoplasms (77%) and TaG1/TaG2 tumors (61%/58%) than among TaG3 tumors (34%) and T1G3 tumors (17%). On multivariable analysis, mutations were associated with increased risk of recurrence in NMI-BC.
Van Rhijn et al186 previously proposed a molecular grade parameter (mG) based on a combination of FGFR3 gene mutation status and MIB-1 index as an alternative to pathologic grade in NMI-BC. Recently,172 the same group elegantly validated their previously proposed mG parameter and compared it to the European Organization for Research and Treatment of Cancer (EORTC) NMI-BC risk calculator187 (weighted score of 6 variables including WHO 1973 grade, stage, presence of CIS, multiplicity, size, and prior recurrence rate). The mG was more reproducible than the pathologic grade (89% vs. 41% to 74%). FGFR3 mutations significantly correlated with favorable disease parameters, whereas increased MIB-1 was frequently seen with pT1, high grade, and high EORTC risk scores. EORTC risk score remained significant in multivariable analyses for recurrence and progression. Importantly, mG also maintained independent significance for progression and disease-specific survival (DSS) and the addition of mG to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7%.
Several studies have suggested a negative prognostic role for HER2/neu amplification/overexpression in MI-BC.154,188–190 Most recently, Bolenz et al180 found HER2/neu-positive MI-BC patients to be at twice increased risk for recurrence and cancer-specific mortality on multivariable analyses adjusted for pathologic stage, grade, lymphovascular invasion, lymph node metastasis, and adjuvant chemotherapy.
P53, Cell Cycle Regulators, and Proliferation Index Markers
Early studies by Sarkis et al191–193 revealed p53 alterations to be a strong independent predictor of disease progression in BC (NMI-BC, MI-BC, as well as CIS). P53 has also been shown to be predictive of increased sensitivity to chemotherapeutic agents that lead to DNA damage.194–196 Recent studies have further supported the prognostic role of p53197 in pT1-pT2 patients after cystectomy showing an independent role for p53 alteration in predicting disease-free survival (DFS) and DSS.
Among other G1-S phase cell cycle regulators, cyclin D3 and cyclin D1, p16, p21, and p27 have also been evaluated as prognosticators in NMI-BC.153,196,198–202 Lopez-Beltran et al198 confirmed their initial finding196 of the independent prognostic role of cyclin D3 and cyclin D1 overexpression in predicting progression in pTa and pT1 tumors. Their findings, however, are in contrast to subsequent findings by Shariat et al153 emphasizing the need for further validation in multi-institutional large cohorts of patients.
A synergistic prognostic role for combining p53 evaluation with other cell cycle control elements such as pRb, cyclin E1, p21, and p27 is emerging both in NMI-BC and MI-BC.145,147,194,203,204 In a study by Shariat et al,147 NMI-BC patients with TURB demonstrating synchronous immunohistochemical alterations in all 4 tested markers (p53, p21, pRb, and p27) were at significantly lower likelihood of sustaining DFS compared with patients with only 3 markers. The negative predictive effect was decreased with decreasing number of altered markers (3 vs. 2 vs. 1).
Similarly, the same group later found that combining p53, p27, and Ki67 assessment in pT1 radical cystectomy specimens improved the prediction of DFS and DSS.204
A similar synergistic prognostic role for the assessment of immunoexpression of multiple molecular markers (p53, pRb, and p21) was demonstrated by Chatterjee et al145 in patients undergoing cystectomy for MI-BC (Fig. 9). The superiority of multimarker approach compared with prior single marker approach certainly merits further assessment. Such multimarker approach of prognostication could soon be integrated in the standard of care in BC management once additional multi-institutional prospective trials confirm the above promising findings.
Tumor proliferation index measured immunohistochemically by either ki67 or MIB-1 has been consistently shown to be a prognosticator in BC.172,186,196,201,205–208 As mentioned above, tumor proliferation index (MIB1) in NMI-BC plays a prognostic role as one of the elements of the mG parameter forwarded by van Rihjn et al.186 The independent prognostic role of proliferation index measured by Ki67 has also been shown. In the study by Quintero et al,205 Ki67 index in NMI-BC TURB biopsy was predictive of progression-free survival (PFS) and DSS.
A similar role for proliferation index assessment as prognosticator is established in MI-BC. Building on initial findings of significance in an organ-confined subset of MI-BC by Margulis et al,206 a recent report of the bladder consortium multi-institutional trial (7 institutions; 713 patients) again confirmed the role of proliferation index, measured in cystectomy specimens.207 In the later study, Ki 67 improved prediction of both PFS and DSS when added to standard prediction models supporting a role for proliferation index assessment in stratifying patients for perioperative systemic chemotherapy. This has certainly taken Ki67 assessment a step closer to clinical applicability in MI-BC.
Gene Expression and Genomic Analysis
Several recent gene expression studies have highlighted sets of differentially expressed genes that may play a role in diagnosis and in predicting recurrence and progression in BC.122,136–138,150,152,209–217
In a landmark study by Sanchez-Carbayo et al,136 oligonucleotide arrays were used to analyze transcript profiles of β5 cases of NMI-BC and MI-BC. Hierarchical clustering and supervised algorithms were used to stratify bladder tumors based on stage, nodal metastases, and overall survival. Predictive algorithms were 89% accurate for tumor staging using genes differentially expressed in nonmuscle invasive versus muscle invasive tumors. Accuracies of 82% (entire cohort) and 90% (MI-BC) were also obtained for predicting overall survival. A genetic profile consisting of 174 probes was able to identify patients with positive lymph nodes and poor survival. Furthermore, 2 independent Global Test runs confirmed the robust association of the suggested profile with lymph node metastases and overall survival simultaneously. As another layer of validation, one of the top-ranked genes coding for a soluble protein, synuclein, was selected from the gene expression profile to evaluate associations in its prognostic significance.
Immunohistochemical analyses on a tissue arrays confirmed the significant association of synuclein with tumor staging and clinical outcome independent of clinicopathologic parameters.
Recently, Birkhahn et al150 attempted to identify genes predictive for recurrence and progression in Ta using a quantitative pathway-specific approach in a set of 24 key genes by real-time PCR in tumor biopsies at initial presentation. They found CCND3 expression to be highly sensitive and specific for recurrence (97% and 63%, respectively). Although HRAS, E2F1, BIRC5/Survivin, and VEGFR2 were predictive for progression by univariate analysis, on multivariable analysis the combination of HRAS, VEGFR2, and VEGF expression status predicted progression with an impressive 81% sensitivity and 94% specificity.
In a recent study, Lindgren et al215 suggested that a combined molecular and histopathologic classification of BC may prove more powerful in predicting outcome and stratifying treatment. The authors combined gene expression analysis, whole-genome array-CGH analysis, and mutational analysis of FGFR3, PIK3CA, KRAS, HRAS, NRAS, TP53, CDKN2A, and TSC1 to identify 2 intrinsic molecular signatures (MS1 and MS2). Genomic instability was the most distinguishing genomic feature of MS2 signature, independent of TP53/MDM2 alterations. Their genetic signatures were validated in 2 independent datasets that successfully classified URCas into low-grade and high-grade tumors as well NMI-BC and MI-BC with high precision and sensitivity. Furthermore, a gene expression signature that independently predicts metastasis and DSF was also defined. This clearly supports the role of molecular grading as a complement to standard pathologic grading.
Mengual et al152 performed gene expression analysis in 341 urine samples from NMI-BC and MI-BC patients and 235 controls by TaqMan Arrays. A 12+2 gene expression signature demonstrated a staggering 98% sensitivity and 99% specificity in discriminating between BC and control and 79% sensitivity and 92% specificity in predicting tumor aggressiveness (NMI-BC vs. MI-BC). The signature was then validated in voided urine samples and maintained accuracy. In an integrated genetic/epigenetic approach, Serizawa et al214 prospectively performed mutational screen of a set of 6 genes (FGFR3, PIK3CA, TP53, HRAS, NRAS, and KRAS) and quantitatively assessed promoter methylation status of 11 additional genes (APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5, and WIF1) in NMI-BC tumor biopsies and corresponding urine samples from 118 patients and 33 controls. A total of 95 oncogenic mutations and 189 hypermethylation events were detected. The total panel of markers provided a sensitivity of 93% and 70% in biopsies and urine samples, respectively. FGFR3 mutations in combination with 3 methylation markers (APC, RASSF1A, and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity.
Selecting patients for neoadjuvant chemotherapy on the basis of risk of node-positive disease has the potential to benefit high-risk patients, whereas sparing other patients toxic effects and delay to cystectomy. Smith and colleagues recently reported a 20 gene expression model to predict the pathologic node status in primary tumor tissue from 3 independent cohorts that clinically lacked evidence of nodal metastasis. The model was developed in 2 separate training cohorts (90 and 66 patients). But most importantly was then assessed for its ability to predict node-positive disease in tissues from a separate phase III trial cohort (185 patients). The model was able to predict nodal status independent of standard clinicopathologic prognostic criteria.
The validation step in an independent cohort is vital given recent reports that gene expression tumor signatures are not portable across cohorts.218
With the impending cost and turn-around time advantages of next generation sequencing technology, the power of genomic approach in providing a noninvasive diagnostic and predictive tool should be actively pursued in a prospective large cohort.
Epigenetic analysis is also gaining momentum in BC as a noninvasive diagnostic tool for screening and surveillance. As a prognostic tool, epigenetic analysis has similarly shown promising potential in BC patients.214,219–231
In an early study by Catto et al,226 hypermethylation analysis at 11 CpG promoters islands was performed by MSP-PCR in 116 bladder and 164 upper urinary tract tumors. Promoter methylation was found in 86% of all tumors and the incidence was relatively higher in upper tract tumors compared with BC. Methylation was associated with advanced tumor stage and higher tumor progression and mortality rates. Most importantly, on multivariate analysis, methylation at the RASSF1A and DAPK genes promoters was associated with disease progression independent of tumor stage and grade.
The same group231 using quantitative methylation-specific PCR (MSP) at 17 candidate gene promoters found 5 loci to be associated with progression (RASSF1a, E-cadherin, TNFSR25, EDNRB, and APC). Multivariate analysis revealed that the overall degree of methylation was more significantly associated with subsequent progression and death than tumor stage. An epigenetic predictive model developed using artificial intelligence techniques identified likelihood and timing of progression with 97% specificity and 75% sensitivity.
Among the studies evaluating the diagnostic role of promoter hypermethylation, the study by Lin et al220 used MSP assay in 4 genes (E-cadherin, p16, p14, and RASSF1A) in primary tumor DNA and urine sediment DNA from 57 BC patients. MSP detected hypermethylation in the urine of 80% of tested patients. Hypermethylation analysis of E-cadherin, p14, or RASSF1A in urine sediment DNA detected 85% of superficial and low-grade BC and 79% of high grade and 75% of invasive BCs. The study highlighted the great potential of such test in detecting NMI-BC. Similar diagnostic role was also found by Cabello et al222 using a novel technology, methylation-specific multiplex ligation-dependent probe amplification assay, to analyze 25 TSGs that have been thought to play a role in BC oncogenesis. The TSG included PTEN, CD44, WT1, GSTP1, BRCA2, RB1, TP53, BRCA1, TP73, RARB, VHL, ESR1, PAX5A, CDKN2A, and PAX6. The authors found BRCA1, WT1, and RARB to be the most frequently methylated TSGs with receiver operating characteristic curve analyses revealing significant diagnostic accuracies in 2 additional validation sets.
Finally, assessment of promoter hypermethylation is giving additional insights on BC oncogenesis. Promoter hypermethylation of CpG islands and “shores” controlling miRNA expression is 1 such example.224
Ploidy and Morphometric Analysis
Several studies have pointed to the independent prognostic role of ploidy and S phase analysis in NMI-BC.208,232–238 Ploidy analysis can be performed by flow cytometry or automated image cytometry (ICM) and is applicable to urine cytology specimens as well as biopsy supernatant233 and disaggregated TURB FFPE specimens.234
In one of the largest studies assessing DNA ploidy in NMI-BC (377 test set; 156 validation set), Ali-El-Dein et al232 found stage, DNA ploidy, tumor multiplicity, history of recurrence, tumor configuration, and type of adjuvant therapy to independently predict recurrence. Recurrence at 3 months, grade, and DNA ploidy were the only predictors of progression to muscle invasion. The constructed “Predictive-Index” model successfully stratified patients in a second validation set into 3 risk groups. Likewise, Baak et al234 were able to show ploidy status and S phase measured by ICM to be strong independent predictors of recurrence and progression in pTa and pT1 patients.
Despite all the above encouraging data, ploidy analysis still await prospective randomized trials to bring ICM or flow cytometry technique into current standard management algorithms for NMI-BC.
Other biomarkers with encouraging but less robust data on their potential prognostic role in BC include tumor microenvironment markers, such as cell adhesion markers, such as E-cadherin and N-cadherin141,239 and angiogenesis modulators such as HIF 1a, HIF 2, VEGF, CA IX, and Thrombospondin-1139,140,240–244; in addition, our group and others have demonstrated a potential prognostic role for mTOR pathway markers.141,239,245–247 Other markers such as Aurora-A have also been investigated in this setting.248,249 Finally, miRNA profile alterations will certainly be a new area of heavy investigation as a noninvasive diagnostic and as a prognostic tool in BC patients.224,250–252
Targeted Therapy and Predictive Markers in Bladder Cancer
RTK-HRAS-MAPK, mTOR, as well as angiogenesis pathway of the tumor microenvironment offer promising opportunities for new targeted treatments of BC.128,146,245,247,253–266 Among RTK, HER2 has been targeted in a multicenter phase II trial reported in 2007 by Hussain et al.267 Forty-four advanced BC patients with metastatic disease and evidence of tumor HER2 positivity by either immunohistochemical, FISH, or elevated serum extracellular HER2 domain levels, were treated with a combination of carboplatin, paclitaxel, and gemcitabine with the humanized monoclonal anti-HER2 antibody trastuzumab. Approximately 70% of treated patients demonstrated partial (59%) or complete (11%) response with a median overall survival of 14.1 months. A higher response rate was associated in patients with 3+ HER2 expression by IHC or HER2 gene amplification by FISH compared with those with 2+ HER2 expression and FISH-negative tumors. Interestingly, in contrast with the strongly correlated HER2 gene amplification and 3+ IHC HER2 overexpression usually seen in breast cancer, the majority of HER2 overexpression in bladder UC are not associated with HER2 gene amplification.268
A second ongoing randomized phase II trial is evaluating the role of anti-EGFR recombinant humanized murine monoclonal antibody cetuximab. Patients with metastatic, locally recurrent, or nonresectable disease are treated with standard GC (gemcitabine and carboplatin) chemotherapy with or without cetuximab. By blocking EGF binding to the extracellular EGFR domain, cetuximab inhibits downstream signal transduction pathway accounting for its antiproliferative activity in solid tumors. In BC, a potential added synergistic antiangiogenic effect could be also at play.269,270 A separate phase II Cancer and Leukemia Group B trial investigated the role of a small molecule inhibitor of EGFR (gefitinib) in advanced BC patients. Gefitinib in combination with GC had no survival or time to progression advantage over GC alone.271,272
On the basis of the results of a phase II single arm trial273 suggesting a therapeutic advantage for lapatinib (a tyrosine kinase inhibitor targeting both EGFR and HER2) in EGFR or HER2-positive BC tumors, a phase II/II randomized trial is underway looking at the role of maintenance with lapatinib (vs. placebo) in patients with objective response to first-line chemotherapy and positive for either marker by IHC or FISH studies.
In an attempt to target BC dependence on angiogenesis, monoclonal antibodies and small molecule inhibitors of angiogenesis are under investigation in advanced disease. An initial phase II trials evaluating the role of bavacizumab, a recombinant humanized monoclonal anti-VEGF antibody, in combination with GC as a first-line therapy in metastatic BC, revealed objective response in two thirds of patients with 6 of 43 patients showing complete response albeit with significant treatment-related toxicity.274 A CALBG phase III randomized trial for GC with and without bevacizumab for metastatic UC is now underway as well as other phase II trials for bevacizumab in combination with other chemotherapeutic agents such as M-VAC (methotrexate, vinblastine, adriamycin, and cisplatin).275
The role of multitargets tyrosine kinase inhibitors in BC has also been investigated with mixed results. Although sorafenib (inhibits Raf kinase, PDGFR-B,VEGFR-2, and VEGFR-3) phase II trials have failed to show significant objective response, sunitinib (inhibits VEGFR-2 and PDGFR-B) has shown a more promising effect in a recent phase II trial involving 77 patients at Memorial Sloan Kettering Cancer Centre, where clinical benefit was observed in almost one third of patients. A subsequent randomized double-blind phase II trial is underway investigating the efficacy of sunitinib in delaying progression as a maintenance agent in patients with initial response to standard chemotherapy.276 Finally, given the recent evidence suggesting the presence of mTOR pathway alterations in BC, a phase II trial evaluating the potential role of everolimus, an inhibitor of mTor pathway, in advanced BC is underway.245–247
In summary, as our understanding of the complex molecular mechanisms involved in BC development has come into a sharper focus, our approaches to diagnosis and management of BC continue to evolve. In the not so distant future, the current paradigm of clinicopathologic-based prognostic approach to predicting progression in NMI-BC210,277–279 is to be supplemented by a molecular-guided approach based on some of the markers listed in Table 2.130,132,145,156,157,204,226,231,242,244,245,247,280–282 Several new targeted therapy agents are under investigation in randomized trials in combination with standard chemotherapy agents either as first-line treatment or on a maintenance base to prolong response in patient with advanced BC.
Renal carcinoma continues to be a major cause of morbidity and mortality worldwide. Last year, approximately 64,770 new renal tumor patients were diagnosed and 15,570 deaths were ascribed to renal cancer in the United States. RCC is the seventh most common neoplasm in American males and the eighth most common neoplasm in females.119 There is a 2- to 3-fold male predominance of RCC incidence but no obvious racial predilection. Recognized risk factors include: tobacco smoking, obesity (BMI>29 may double the risk of RCC), and acquired or hereditary polycystic diseases. The classic clinical presentation symptom triad of flank pain, hematuria, and palpable mass is no longer the leading form of presentation. Patient presentation as a result of RCC-associated paraneoplastic syndromes due to secreted parathyroid hormone, erythropoietin, prostaglandins, or ACTH is also unusual at present. This change in clinical presentation is mainly due to a marked increase in incidentally found smaller RCC lesions during imaging studies performed for a variety of other causes.
Anatomic Molecular Diagnostic Applications
Current established prognostic parameters in RCC include: pTNM stage, Fuhrman grade, histologic subtype, and clinical parameters such as ECOG performance status, hemoglobin level, and LDH levels among others.283–285 Continuous refinements of staging criteria and development of nomograms to integrate the above factors promise to yield a better prognostic and management discriminators.286
A large number of biomarkers are under current intense investigation for their potential utility as prognosticators and/or therapy predictors in RCC.286–293 Table 3 lists some of these markers. Kim et al288 evaluated a set of immunohistochemical markers including Ki67, CAIX, CAXII, p53, PTEN, gelsolin, EpCAM, and vimentin in combination with established parameters. Their study suggested a new combined molecular and clinicopathologic prognostic model (CAIX, vimentin, p53, pTNM, and ECOG performance status) to be superior to prior models of clinical and pathologic parameters alone including the commonly used UISS clinical model. Parker et al294 derived another biomarker-based scoring system from 634 patients with CCRCC. This weighted algorithm (BioScore) integrated dichotomized expression of B7-H1, Ki-67, and survivin. Patients with high BioScores (>4) were 5 times more likely to die from RCC than those with low scores. In addition, the sequential use (as opposed to integration into a new model) of BioScore with existing clinicopathologic scoring systems (TNM, UISS, and SSIGN) further enhanced the predictive ability compared with each of these scoring systems alone. Prospective validation of these biomarker prognostic models is required before routine clinical use can come about.
A promising prognostic role for mTOR pathway members was initially pointed out by Pantuck et al.295,296 Their study revealed an independent negative prognostic role for PTEN loss and pS6k overexpression. The same study showed increased pAKT cytoplasmic expression and loss of pAKT nuclear expression to be negative predictors of survival. We recently analyzed immunoexpression status and prognostic significance of mTOR and hypoxia-induced pathway members in 135 primary and 41 metastatic CCRCC using tissue microarrays (Fig. 10). We found significantly lower PTEN levels in primary and metastatic CCRCCs compared with benign tissues. phos-S6 and 4EBP1 levels were higher in primary CCRCC compared with benign tissues. HIF-1α levels were also significantly higher in primary and metastatic tumors. In primary CCRCC, levels of all mTOR and hypoxia-induced pathway members were significantly associated with pT stage. Tumor size, HIF-1α, and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary CCRCC.297,298 Using a similar tissue microarray and immunohistochemistry analysis approach, we recently were also able to demonstrate evidence of dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways in papillary RCC. However, the mTOR pathway and HIF-1α alterations lacked prognostic significance in our cohort for papillary RCC.297,298
A prognostic role for angiogenesis pathway is illustrated in RCC. Bui et al299 demonstrated that both low expression of CAIX and high Ki 67 proliferation index were independent negative predictors of survival in CCRCC. Interestingly, CAIX overexpression predicted response to IL2 immune therapy in metastatic RCC, a finding also documented in the study by Atkins et al.300 In another study by Jacobsen et al,301 VEGF expression seemed to correlate with tumor size and pTNM stage in RCC. The authors found high VEGF expression to be a negative survival prognosticator on univariate but not multivariate analysis. Separately, Kluger et al289 analyzed tissue microarrays containing 330 CCRCC and PRCC using a novel method of automated quantitative analysis of VEGF and VEGF-R expression by fluorescent immunohistochemistry. Unsupervised hierarchical clustering classified tumors by coordinated expression of VEGF and VEGF-Rs. The authors found high expression of VEGF and VEGF-Rs in tumor cells to be associated with poor survival. Finally, a study by Lidgren et al302 revealed high expression of HIF-1α to be an independent negative prognosticator in CCRCC.
Among cell cycle control molecules, p27 (Kip1) and cyclin D1 seem to have a promising prognostic role in CCRCC. Migita et al303 found loss of p27 expression to be an independent predictor of poor DSS. Similar p27 findings were also documented by Hedberg et al.304,305 Analysis of molecular expression profiles permits simultaneous measurement of thousands of genes to create a global picture of cellular function. Multiple groups have evaluated gene expression profiling in RCC with variable results. Kosari et al306 found significant differences in gene expression between nonaggressive and aggressive CCRCC. Thirty-four of the 35 transcripts that displayed the most significant differential expression by microarray analysis also displayed significant differential expression in independent validation studies using quantitative RT-PCR. Hierarchical clustering of the quantitative RT-PCR data using candidate markers accurately grouped 88% of aggressive and 100% of nonaggressive CCRCC samples. The group showed the expression of one of their candidate markers (survivin) to inversely predict cancer-specific survival in a separate cohort of 183 CCRCC patients treated at Mayo Clinic.
Genomic and Theranostic Applications
Renal tumors are unique among human neoplasms in terms of the tight correlation between their different morphologic phenotypes and underlying genetic alterations. Lessons learned from the relatively rare familial renal cancer syndromes have helped unlock the complex molecular oncogenic mechanisms involved in their sporadic counterparts. As a result, many potential new targets of therapy are now under investigation in the hereto unsuccessful endeavor of treating advanced RCC. The following discussion of the molecular basis of von Hippel-Lindau disease (VHL) best illustrates the great therapeutic and theranostic potentials of uncovering the molecular mechanisms of renal oncogenesis.307
VHL syndrome is a rare autosomal dominant familial cancer syndrome (retinal angiomas, hemangioblastomas, pheochromocytomas, and CCRCC). VHL syndrome patients are born with a germline VHL mutation affecting all their cellular elements. Inactivation or silencing of the remaining wild-type allele in renal location facilitates the formation of CCRCC in VHL patients. The fact that similar defects in VHL gene are found to be responsible for approximately 60% to 90% of sporadic CCRCC308,309 greatly widened the implication of our understanding the molecular mechanisms of VHL inactivation.310
VHL can be thought of as a cellular oxygen sensor. Under normoxic conditions, HIF-1α is inhibited by normal VHL protein that facilitates elimination of HIF-1α.311 Under hypoxic condition, HIF-1α escapes destruction by VHL and is allowed to exert its crucial role in inducing angiogenesis factors (VEGF), cell growth factors (TGF-α and β), and factors involved in glucose uptake and acid-base balance (GLUT-1 and CAIX, respectively) (Fig. 11). A defective VHL function in CCRCC lead to abnormal accumulation of HIF-1α even under normal condition, in turn resulting in the overexpression of the above proteins that are normally inducible only during hypoxia. The overexpressed VEGF, PDGF-B, and TGF-β act on neighboring vascular cells to promote tumor angiogenesis. The augmented tumor vasculature provides additional nutrients and oxygen to promote the growth of tumor cells. Furthermore, TGF-α acts in an autocrine manner on the tumor cells by signaling through the epidermal growth factor receptor (EGFR), which promotes tumor cell proliferation and survival.
More recently, mutations in a number of genes affecting chromatin remodeling have been identified in CCRCC. The most prevalent of these is the PBRM1 gene, which is mutated in approximately 40% of CCRCC tumors.310,312
Currently, several agents targeting the vHL pathway, particularly components of VEGF signaling (sunitinib, sorafenib, pazopanib, and bevacizumab) and the mTOR C1 complex (temsirolimus and everolimus) are approved by the US FDA for the treatment of advanced kidney cancer.310,313–329
Currently, the majority of advanced CCRCC patients receive VEGF pathway antagonist, either sunitinib or pazopanib, as first-line therapy for metastatic disease, based on the demonstration that these agents prolong PFS compared with interferon-α or placebo, respectively. Despite the availability of a number of agents with activity, the most effective second-line therapy, as well as the optimal sequencing of these agents, remains unclear. Both sorafenib and pazopanib are associated with an improved PFS compared with placebo in patients who have previously received cytokine therapy. In patients who have progressed on first-line therapy with a VEGF pathway antagonist, everolimus was hitherto the only agent shown to offer clinical benefit (modest prolongation of PFS compared with placebo in a randomized phase III study).315,316 Axitinib, a potent, selective, second-generation VEGF receptor (VEGFR 1, 2, 3) inhibitor, was recently shown to offer a superior PFS compared with sorafenib, a first-generation VEGFR and RAF inhibitor, in the second-line setting in a phase III AXIS trial.314
Analysis of vHL mutation status as well as plasma CAIX, VEGF, sVEGFR2, tissue inhibitor of metalloproteinase-1 (TIMP-1), and Ras p21 was performed in the TARGET trial of sorafenib versus placebo in advanced RCC.330 On multivariate analysis that included ECOG performance status, MSKCC score, and the biomarkers assayed, only baseline TIMP-1 levels were prognostic for survival. No predictive markers were identified.329 Choueiri et al331 evaluated tumor CAIX expression (IHC) in 94 patients treated with antiangiogenic therapies. However, CAIX expression was neither prognostic nor predictive of response to sunitinib. For sorafenib-treated patients, high CAIX expression (>85%) was associated with decreased tumor size in response to treatment.
Other targeted agents being pursued in advanced CCRCC include temsirolimus (CCI-779), a selective mTOR inhibitor where partial responses was noted in 7% of patients and minor responses in 26%. The median survival rate was 15 months. The notable activity of the drug in patients with poor prognostic features prompted a phase III trial.300,332 Cho and Chung333 examined expression of CAIX, phosS6, phosAkt, and PTEN in 20 patients with advanced CCRCC treated with temsirolimus in a phase II clinical trial. These investigators found a positive significant association between phos S6 expression and objective response to temsirolimus. A similar trend was associated with positive expression of phosAkt.
Hereditary papillary renal cancer, the familial form of type 1 PRCC, is characterized by activating germline mutations of the MET oncogene located on chromosome 7q31. Somatic MET mutations have also been detected in 5% to 13% of sporadic PRCC.334 The MET oncogene encodes a transmembrane RTK for hepatocyte growth factor (HGF) (Fig. 12). MET undergoes autophosphorylation when stimulated by its ligand, HGF, leading to downstream activation of an intracellular cascade including PI3K and mTOR. MET has been implicated in motility, proliferation, angiogenesis, and cell survival. Several strategies targeting the MET pathway are being explored in PRCC, including antibodies against HGF and MET, as well as inhibitors of MET kinase activity. Foretinib is a dual MET and VEGFR2 kinase inhibitor that is being currently evaluated in a phase II study of patients with PRCC (NCT00726323). Unlike previous trials of MET pathway antagonists, this trial is restricted to patients with papillary histology (both type 1 and 2 histologies are included). The presence of MET pathway activation is analyzed to determine whether MET status impacts on response to the agent. Foretinib seems to be well tolerated with the majority of patients experiencing some degree of tumor regression on interim analysis.310
Testicular germ cell tumors (GCT) are the most common malignancy in men aged 15 to 44 years. In 2012, 8590 cases were diagnosed in the United States alone. GCT incidence increases after puberty and peaks in the third decade of life. A significant geographical variation in GCT risk is observed where the lifetime risk is estimated to be 0.4% to 0.7% in males born in the United States and 1% in Nordic European regions, whereas risk is lowest in Asia and Caribbean. A puzzling steep increase in the incidence in the United States and Northern Europe has been observed up to 2007.
The great advances in our understanding of the biology of GCT and the unparalleled strides in refining the treatment of this group of testicular tumors is one of the great success stories in oncologic diseases with only 360 deaths due to GCT recorded in the United States in 2012.119
Emerging Molecular Diagnostic Applications
Tremendous advances have been achieved in our understanding of the biology and genetic molecular events involved in the pathogenesis of GCT. Current GCT treatment success rates are the envy of other solid tumors disciplines. Nevertheless, given the young age of affected GCT patient population and the gravity of potential side effects (eg, secondary malignancy) associated with current treatment protocols, new biomarkers that will help better identify patients in need of such aggressive treatment are invaluable. Likewise, identification of new molecular targets of GCT therapy would be advantageous.335–339
As shown in Figure 13,340 during fetal life, after an initiating event in diploid primordial germ cell, loss of imprinting and aneuploidization lead to the formation of the IGCNU precursor lesions. The extensive chromosomal instability in IGCNU leads to further transformation into invasive GCT primarily through loss of DNA and gain of 12p resulting in the development of seminoma. Additional events such as loss of N-myc and c-kit activity and activation of pRb, HER2, and p53 result in histogenetic differentiation toward NSGCT.340,341
Overrepresentation of chromosome 12p region is a consistently present structural chromosomal aberration in GCT. Isochromosome 12p is present in up to 80% of GCT including extratesticular tumors. Therefore, the identification of isochromosome 12p by interphase FISH-based cytogenetics is occasionally resorted to in cases where above-mentioned IHC approach fails to resolve the differential diagnosis of GCT versus somatic carcinoma.342–346
Recently, our group performed the first comprehensive genome-wide analysis of copy number variation and loss of heterozygosity in 25 primary seminomas using high-resolution single nucleotide polymorphism array.347 Our study confirmed several previously reported genomic alterations and discovered 8 novel alterations including amplifications and homozygous deletions (Table 4). Moreover, a comparison of genomic alterations of early-stage and late-stage seminoma identified copy number variations that seems to correlate with progression, which included deletions in chromosomes 4q, 5p, 9q, 13q, and 20p, and amplifications in chromosomes 9q and 13q associated with advanced stage (stages II/III vs. stage I). A deletion of chromosome 13q13.3, which encodes for DCLK1 and SOHLH2, was altered in 20% of the samples. SOHLH2 is a germ cell–specific transcription factor that may be a critical regulator of early germ cell development (Table 4).
At the epigenetic level, alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5 mC) are a hallmark of cancer cells, including testicular GCTs. Smiraglia et al348 have demonstrated significant epigenetic differences between seminomas and nonseminomas using restriction landmark genomic scanning. Seminomas show almost no CpG island methylation, in contrast with nonseminomas that show CpG island methylation at a level similar to other solid tumors. The group proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone global 5 mC erasure (loss of imprinting), and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure. More recently, our group used immunohistochemical staining against 5 mC to evaluate global methylation in GCT.349 We found staining for 5 mC to be undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous GCTs. Our findings supported that testicular GCT are derived in most cases from IGCNU cells that have undergone developmentally programmed 5 mC erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. In such proposed linear pathogenesis model, it seems that IGCNU and subsequently developed seminoma cells remain unmethylated, whereas nonseminomatous histologic types seem to arise mainly after a de novo methylation of seminoma cells destined to give rise to nonseminomas (Fig. 14).
Most recently, we found a differential methylation pattern between seminomas and nonseminomatous GCT using methylation-specific PCR. Although MGMT, VGF, ER-β, and FKBP4 seem to be predominantly methylated in nonseminomas, APC and hMLH1 genes are methylated in both groups.349–351
Among therapy predictive markers, DNA damage detection and apoptosis initiation programs are thought to play a strong role in the exquisite chemosensitivity enjoyed by GCT. Wild-type p53 overexpression have been associated with chemosensitivity in GCT, whereas overexpression of MDM2 (p53 inhibitor) was associated with resistance to therapy.336,352–354 In contrast, defective mismatch repair pathway leading to microsatellite instability (MSI) has been related to resistance in a subset of cisplatin refractory seminoma. Mayer et al355,356 were able to illustrate a correlation between IHC loss of MLH1, MSH2, and MSH6, and MSI in 50% of unstable tumors demonstrating for the first time a positive correlation between MSI and treatment resistance in GCT. Mutations of BRAF are associated with MSI and have also been shown to be frequent in germ cell tumors with cisplatin chemotherapy resistance.357,358
In another study, Dimov et al359 were able to show that high levels of Topo II-α is expressed by most seminomas, embryonal carcinomas, yolk sac tumors, and choriocarcinomas suggesting a possible mechanism for the sensitivity of these components to Topo II-α inhibitors. Interestingly, teratomas with mature and immature elements expressed low levels of Topo II-α, which might have contributed to their relative chemoresistance further implying that the variable chemoresponsiveness of testicular GCTs could have an underlying molecular basis.359
Mazumdar et al360 recently suggested that a subgroup of embryonal carcinoma (with or without other histologies) with a cluster profile of high Ki67, low apoptosis, and low p53 had a better survival beyond what would be predicted by their IGCCCG prognostic category reflecting their increased tendency to respond to treatment.360
EGFR is a receptor tyrosine kinase that has been extensively targeted therapeutically in solid tumors, including lung and colonic adenocarcinoma. Immunohistochemical EGFR expression is observed in almost half of chemorefractory metastatic embryonal carcinomas and seems to correlate with EGFR gene amplification or polysomy observed by FISH. The therapeutic role of EGFR-targeted therapy remains to be determined.361
As mentioned above, so far, conflicting results have been found regarding the role of the presence of exon 11 and 17 c-kit-activating mutations in predicting incidence risk of bilateral seminomas in patients with prior orchiectomy.362–366
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molecular; genetics; biomarkers prognostics; urologic; neoplasms
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