Fig. 5.1
Recurrence-free progression following radical prostatectomy stratified by prostatectomy grade. Green line: Gleason score 6, Grade Group 1. Orange line: Gleason score 3 + 4, Grade Group 2. Dark blue line: Gleason score 4 + 3, Grade Group 3. Red line: Gleason score 8, Grade Group 4. Purple line: Gleason score 9,10, Grade Group 5. RFP recurrence-free progression (From Epstein et al. [22]. Reprinted with permission from Elsevier)
The major consequence of the new grading system relative to the issue of whether Gleason score 6 should be called cancer is that the new system addresses the issue of renaming Gleason score 6 not cancer to changing the grading system to more accurately reflect the indolent nature of Gleason score 6 prostate cancer. A Gleason score 6 out of 10 prostate cancer would in the new system be “Grade Group” 1 out of 5. Patients could be reassured that they have a Grade Group 1 tumor on biopsy that is the lowest-grade tumor possible which in most cases can be followed with active surveillance. In a recent survey of 7 focus groups with 37 prostate cancer patients from 2015 to 2016, the majority of patients (84%) agreed that it would be clearer if grades were reported on a scale of 1–5 instead of 6–10 [29]. Eighty-eight (88%) would prefer to hear they have “Group 1” rather than “Gleason score 6,” and 80% would feel more comfortable choosing active surveillance with “Group 1” vs. “Gleason score 6.” However, follow-up is still needed with Grade Group 1 prostate cancer on biopsy as in approximately 20% of cases there is higher-grade cancer in the prostate that has not been sampled [21].
Molecular Genetics of Gleason 6 Prostate Cancer
Somatic Genetic Alterations
Molecular Subtypes of Prostate Cancer Identified by “Omics”
Over the last several years, a number of studies have characterized the molecular “taxonomy” or “landscape” of prostate cancer using high-throughput genomic analyses of hundreds of specimens, including both primary and metastatic tissue samples [30–35]. While these studies have validated many findings from prior studies, they have also provided a more comprehensive picture of “molecular subtypes” of prostate cancer and revealed a number of previously unrecognized driver genes and pathways (Fig. 5.2). For example, by profiling large number of primary and metastatic tumors, the “long tail” of prostate cancer mutations has been better identified. The long tail is characterized by the finding that a number of recurrent somatic driver mutations occur at low frequency (e.g., <5%). Interestingly, mutations within DNA repair genes (~19%), which were only partially appreciated from prior work (e.g., see [36]), occur relatively often as somatic alterations and, at least in those patients that progress to metastatic castrate-resistant disease, also occur relatively frequently in the germline (~12%) [37].
Fig. 5.2
Molecular subtypes of prostate adenocarcinoma . Somatic genetic and DNA cytosine methylation changes are shown indicating the seven major mutually exclusive subtypes of prostatic adenocarcinoma defined by the TCGA. Note that Gleason score 6 tumors are present scattered throughout all subtypes (Adapted from Cancer Genome Atlas Research Network [30], with permission from Elsevier)
In a recent publication resulting from The Cancer Genome Atlas (TCGA) Research Network , seven major molecular subtypes of primary prostatic adenocarcinoma were identified [30] (Fig. 5.2). The first four were defined by different ETS family gene fusions including (1) ERG, (2) ETV1, (3) ETV4, and (4) FLI1, and the remaining were defined by (5) SPOP mutations , (6) FOXA1 mutations , and (7) IDH1 mutations . Together, these subtypes encompassed 74% of all primary adenocarcinomas of the prostate with the remainder still undefined using their stratification criteria. As shown in Fig. 5.2, Gleason score 6 tumors are scattered throughout all of these subtypes indicating that Gleason score 6 tumors do not appear to arise via alterations in distinct molecular pathways from those of higher-grade lesions. When one specifically considers the first four subtypes, approximately 50% of prostatic adenocarcinomas from men of European decent harbor a clonal somatic rearrangement resulting in ETS family member gene fusions, with TMPRSS2-ERG being the most common. In terms of Gleason score and disease stage, approximately 50% of all primary prostate cancers, including tiny “insignificant” Gleason score 6 cancers at RP [38], as well as 50% of castrate-resistant lethal metastatic prostate adenocarcinomas (CRPC), have TMPRSS2-ERG or other ETS-related gene fusions [31]. Thus, the most common clonal somatic genetic alteration in prostate cancer occurs with nearly equal frequency in Gleason score 6 and higher-grade tumors. Interestingly, African American men have a lower frequency (approximately 50% of the frequency of those of European descent) of these rearrangements [39].
While the total number of overall mutations, as well as the frequency of mutations in genes encompassing the long tail, is higher in castrate-resistant metastatic prostate cancer than in primary tumor samples, considering all of the aberrations indicated above (including the long tail mutations), most somatic alterations in prostate cancer can now be clustered into major pathways (Fig. 5.3) including AR associated (FOXA1, ZBTB16, NCOR1, NCOR2), PI3 kinase pathway associated (PIK3CA, PIK3CB, AKT1), RAF fusions (BRAF, RAF1), WNT pathway (APC < CTNNB1, RNF43, RSP02, ZNRF3), DNA repair (BRCA2, ATM, CDK12, MLH1, MSH2), cell cycle (RB1, CDKN1B, CDKN2A, CCND1), chromatin modifiers (KMT2C, KMT2D, KDM6A, CHD1), and others (SPOP, MED12, ZFHX3, ERF, GNAS). Although the overall number of cases is still relatively small, when mutations in these genes occur in men with clinically localized disease, they do not tend to be highly enriched in different Gleason grade tumors and some can be found in Gleason score 6 tumors, with no apparent striking difference in the overall number of such lesions in Gleason score 6 and higher prostate tumors [30, 32, 33]. One notable exception is found in those cases that harbor TP53 mutations or deletions. These tend to occur relatively infrequently in primary prostate tumors , but when present in such tumors, they are more common in higher-grade, higher-stage lesions [40], with a further increase seen in castrate-resistant metastatic disease [31, 35].
Fig. 5.3
Major pathways associated with genomic alterations in prostate cancer. Recent whole genome profiling studies reveal that many of the mutations identified in prostate cancer can be grouped into specific pathways (Adapted from Spratt et al. [34], with permission from Nature Publishing Group)
Other Rearrangements and Copy Number Alterations
Chromoplexy consists of a series of DNA breakage and joining events in which a number of DNA segments from different genomic locations become ligated together [32]. While not specifically addressed in the manuscript, when one examines the data, this alteration was reported to occur similarly in Gleason score 6 tumors and higher-grade prostate cancers [32, 41]. Other well-characterized and extensively documented alterations, such as deletions on chromosome 8p resulting in loss of one allele of NKX3.1, deletions involving PTEN on chromosome 10q23, and gains of 8q24/MYC, occur in Gleason score 6 lesions, albeit at a reduced rate compared to higher-grade and more aggressive lesions [16, 42–50]. Thus, these more traditionally examined alterations are more common in higher-grade tumors but do occur with at least some frequency in Gleason score 6. Taken together, these results suggest common molecular pathways and mechanisms for the development of a number of somatic genetic alterations between Gleason score 6 and higher-grade tumors.
Overall Percent of Genome Altered
Some types of rearrangements lead to large-scale copy number alterations with gains and losses of relatively large segments of genomic material. Such copy number alterations are common in prostate cancer [51], and a number of studies have demonstrated that copy number changes tend to increase in extent and number with grade and disease aggressiveness [32, 51–53]. Further, a subset of prostatic carcinomas, predominantly Gleason score 6 tumors, has been deemed nearly free of large-scale copy number alterations [52, 53]. These “quiet” genomes in some Gleason score 6 lesions may be molecularly distinct from other Gleason score 6 lesions and higher-grade more aggressive tumors. Nevertheless, it is also clear that at least some of the Gleason score 6 score tumors analyzed to date do show relatively large numbers of copy number alterations such that this feature alone cannot entirely separate prostate cancers by grade [32, 51–53]. Given the current evidence suggesting tumors with “quiet” genomes are likely to be nonaggressive, it is possible that in the future if a method for routine measurement of copy number could be employed clinically (e.g., using a clinical grade test in a CLIA-certified laboratory) and one could rule out the presence of any other higher-grade or separate tumor (e.g., with multiparametric MRI), then a Gleason score 6 tumor could be considered nonmalignant or premalignant if copy number alterations in such a lesion were shown to be minimal.
Somatic Epigenetic Changes
Somatic DNA methylation of the CpG island within the GSTP1 gene occurs in approximately 90% of all prostatic adenocarcinomas, regardless of grade or stage [54]. A number of other genes are also hypermethylated frequently in prostate cancer (e.g., APC, RASSF1, MDR1, EDNRB, HOXD3, TGFB2), and although hypermethylation of some occurs more frequently in higher-grade lesions (EDNRB, HOXD3, APC, TGFB2) or those with biochemical recurrence (APC, PTGS2, HOXD3, TGFB2), most occur commonly both in Gleason score 6 and higher-grade lesions [55–57]. These results further support the overall concept of similar molecular alterations occurring in Gleason score 6 and higher-grade lesions. The finding of IDH1 mutations in prostate cancer by the TCGA is novel, and, as in other cancers with IDH1 and IDH2 mutations, tumors from these patients are apparently enriched for very high numbers of somatic CpG methylation events [30]. At this time, however, there are not enough cases with IDH1 mutations to determine whether they occur with greater or reduced frequency in Gleason score 6 tumors. When looking at the evidence from more recent genome-wide studies, it does not appear that Gleason score 6 tumors have highly distinctive somatic CpG island DNA alterations as compared with those of other Gleason scores [30, 58], although a number of CpG methylation events in specific loci do appear to add value in distinguishing lethal metastatic disease from those without biochemical progression 5 years after prostatectomy [58].
Clonal Relationships Suggest a Common Origin for Gleason Score 7 Tumor Components
Whether Gleason score progresses remains an open debate. If some higher-grade tumors can arise as a progression event from a Gleason pattern 3 lesion, then if this occurs at a non-negligible rate, it would provide an additional argument against renaming Gleason score 6 cancers as non-cancer. Recent studies examining clonal relationships between prostatic tumors that are composed of both Gleason patterns 3 and 4 indicate they are clonally linked [59, 60]. Further, in at least a few cases, it has been shown that the Gleason pattern 4 lesions harbored an additional “hit” by showing a deletion in the PTEN gene , while the clonally related adjacent Gleason pattern 3 lesion did not show the PTEN alteration [60]. This suggests that a Gleason pattern 4 lesion can evolve from a Gleason pattern 3 tumor. This fits well with findings that PTEN alterations usually occur during disease progression as a subclonal molecular alteration, subsequent to TMPRSS2-ERG fusion, when such lesions are found together in the same tumor [32, 61–63]. While these results are compelling, this does not preclude the finding that at times it appears that high-grade prostate tumors may arise de novo in the prostate [64, 65].
RNA Expression Profiles
Using various types of large-scale gene expression profiling techniques over the last several years, a number of groups have examined the relation between gene expression profiles and Gleason grade [66–68]. While statistically significant differences have been found to be able to classify tumors of different Gleason scores using gene expression signatures and may add new prognostic information beyond GS, no specific genes or pathways have emerged that can strongly distinguish in a diagnostic sense among the different grades on an individual patient tumor basis. Hence, these methods cannot definitely classify a given tumor as indolent Gleason score 6, and none of these have been developed into a clinical grade test such that further development of these at this time does not seem practical for clinical implementation as of now. In terms of commercial activity, several companies have employed RNA expression classifiers, usually consisting of quantitative assessments of the relative RNA levels of tens to dozens of genes, to the problem of augmenting prognostic power for prediction of a number of different outcomes, independent of Gleason score (reviewed in [69]). Each of these can now be applied to clinical formalin-fixed paraffin-embedded specimens (either radical prostatectomy or biopsy as per specific clinical question being addressed), and each has shown promise in various clinical disease states to add some value to the prognostic ability of Gleason score and other standardly collected clinical-pathological parameters [69]. However, none of these can be used to determine if an individual tumor can be considered an indolent GS 6 cancer such that they could be used to help reclassify some Gleason score 6 tumors as non-cancer. Irshad et al. specifically addressed the question of identifying a gene expression signature for indolent vs. aggressive prostate cancer and were able to synthesize it down to a few protein-based IHC markers [70]. If further validated and developed into a clinical grade assay, such a test may add value in terms of this question.
Other Tissue-Based Molecular Markers
Trock et al. recently found that patients harboring pure Gleason score 6 tumors in their prostatectomy samples have a lower rate of PTEN loss in Gleason pattern 3 areas than patients with Gleason score 7 do in their Gleason pattern 3 regions (either 4 + 3 = 7 or 3 + 4 = 7). Further, there was also a greater rate of chromosome 8p loss and chromosome 8q24 gain in Gleason pattern 3 regions from patients with a Gleason score 7 tumor. The Gleason pattern 4 regions showed higher rates of changes at all three examined loci [71]. Lotan et al. reported that tumors that were only Gleason score 6 on biopsy that had lost PTEN by IHC had an increased rate of upgrading at prostatectomy compared to those without PTEN loss [72]. Taken together, these findings indicate that Gleason pattern 3 lesions are different molecularly depending on whether they are present in the setting of a Gleason score 6 tumor or in the setting of a Gleason score 7 tumor. These studies also suggest that appropriate molecular markers may be applied to help determine if patients with Gleason score 6 biopsies are at higher risk for harboring a previously unsampled higher-grade tumor in the prostate. Immunohistochemistry for PTEN has been extensively validated analytically and is currently employed in a number of Clinical Laboratory Improvement Amendments 1988 (CLIA) certified anatomic pathology laboratories. Another promising tissue-based approached implemented an 8-marker multiplex immunofluorescence assay that may also prove useful in determining whether a given Gleason score 6 lesion in a needle biopsy is at risk for being associated with a poor outcome or more aggressive disease [73]. Another possible avenue may also be application of the percent of the genome with copy number alterations as mentioned above [52, 53], if this technology is further developed into a clinical grade test. Therefore, it is hoped that in the future in addition to histological grading, molecular markers along with improved imaging can be applied that will facilitate the overall determination of the aggressiveness of a given Gleason score 6 lesion and aid in the selection of patients for AS. If clinicians could be confident (e.g., ≥95%) that a given patient only harbored a Gleason score 6 lesion with molecular properties also consistent with indolent disease, then it may be appropriate to reclassify this lesion as a non-cancer.
Conclusions/Summary
There is no strong molecular evidence suggesting that Gleason score 6 as opposed to higher-grade tumors commonly arise as unique and distinct molecular subtypes, as in the case of urinary bladder cancer . In fact, a number of molecular alterations are shared between Gleason score 6 and higher-grade tumors such as ETS family member gene fusion events, point mutations in a number of genes, chromoplexy, and somatic CpG hypermethylation of specific genes. While some of these changes are substantially less common in Gleason score 6 tumors, they nonetheless support similar pathways of tumor development overall. Further, at least at times, it appears that Gleason pattern 3 and 4 regions within a given tumor can be clonally related and some Gleason pattern 4 lesions may evolve from Gleason pattern 3.
On a practical matter, it is possible that since a number of tissue-based biomarkers can add prognostic value beyond Gleason score, the appropriate application of such markers, along with improved imaging, can help better classify patients with an indolent Gleason score 6 tumor only. While we would not advocate changing the label of cancer for Gleason score 6 lesions at this time, these approaches may indeed facilitate the safe management of patients on active surveillance. In terms of molecular markers and the known biology of this disease, we consider overall copy number alteration burden at this time to be the most promising molecular feature that could potentially be employed to help determine if a given lesion has a “quiet” genome and could be considered indolent. Others include PTEN immunohistochemistry, commercial RNA expression signatures, and multiplex immunofluorescent assays. It should also be emphasized, however, that even if such markers or signatures are employed using validated clinical tests and such testing favors an indolent lesion, we are still left with the sampling problem in that one may have simply missed a more aggressive lesion that is present. Beyond this, even if it is clear that at a given point in time there is only a Gleason score 6 lesion (perhaps with multiparametric MRI imaging largely ruling out other higher-grade lesions) with an indolent biomarker signature, and hence one can conclude that this lesion can safely be labeled a “non-cancer,” this does nont preclude the possibility that an additional clinically meaningful cancer will not develop in the future. We are concerned that if the label of cancer is removed, then many patients with Gleason score 6 tumors will be lost to follow-up. While it is not clear at present if patients with indolent Gleason score 6 tumors are at risk for developing higher-grade tumors over time, most would agree that long-term follow-up of such patients is prudent at this time.
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