The Clinical Significance of Perineural Invasion by Prostate Cancer on Needle Core Biopsy: Involvement of Single Versus Multiple Sextant Sites

On approval by the institutional review board, including the request to waive the documentation of patient consent, we assessed consecutive patients who had undergone systematic sextant prostate needle core biopsy followed by robotassisted radical prostatectomy (performed within 6 months after biopsy) at our institution between 2010 and 2016 where subspecialty-based surgical pathology practice had been performed. Within our surgical pathology database, we identified a total of 538 men who met the inclusion criteria for PNI on sextant biopsy specimens from 6 sites/regions (ie, right apex, right mid, right base, left apex, left mid, left base) with 2 or more cores per site (ie, 12 cores in 468 men, 13 cores in 53 men, 14–17 cores in 17 men) apart from targeted biopsy specimens. We then counted the number of cancer-positive or PNI-positive sites (ie, 1–6). Patients showing PNI on targeted biopsy specimens, as well as those undergoing neoadjuvant therapy before prostatectomy, were excluded from analysis. In those with multiple biopsy procedures (eg, active surveillance cases), data only from the latest biopsy specimen was used.

In all available biopsy specimens, we confirmed PNI that was defined as the presence of cancer directly adjacent to a nerve but was not necessarily encircled completely by tumor glands/cells¹⁴ ; indirect contact of tumor with a nerve was disregarded. Gleason score/Grade Group (GG) in biopsy and prostatectomy specimens was re-evaluated by a senior author (H.M.) based on the recommendations by the International Society of Urological Pathology criteria^15,16  as well as the Genitourinary Pathology Society.¹⁷  We also retrieved clinicopathologic findings, such as age at biopsy, preoperative prostate-specific antigen (PSA) value, cT and cN staging category (before prostatectomy), pT and pN staging category (after prostatectomy), surgical margin status, and estimated cancer length (on biopsy specimens) or volume (on prostatectomy specimens) (see Supplemental Table 1 in the Supplemental Digital Content, containing 4 figures and 14 tables, at https://meridian.allenpress.com/aplm in the October 2022 table of contents), as well as follow-up data. Biochemical recurrence after prostatectomy in patients with no adjuvant therapy was defined as a single PSA level of 0.2 ng/mL or higher, while PSA failure in those undergoing adjuvant therapy immediately after prostatectomy (ie, before disease progression) was defined as an increase in PSA value of 2 ng/mL or higher or 50% or more over nadir or the introduction of salvage therapy.^18,19  PSA recurrence in those both with and without adjuvant therapy was considered as disease progression.

Data were analyzed, using the Student t test (when the normality of the distribution was verified) or Mann-Whitney U test (when the normality of the distribution was rejected) for continuous variables, as well as the X²  test or Fisher exact test for noncontinuous variables. The rates of progression-free survival and cancer-specific survival were calculated by the Kaplan-Meier method, and comparisons were made by the log-rank test. In addition, the Cox proportional hazards model was used to determine statistical significance of prognostic factors in a multivariate setting. All statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software) and EZR software,²⁰  a graphical user interface for R version 4.0.2 (The R Foundation for Statistical Computing). P values less than .05 were statistically significant.

In 538 cases that met the inclusion criteria, prostatic adenocarcinoma was found in 1 (n = 41), 2 (n = 78), 3 (n = 154), 4 (n = 106), 5 (n = 78), or 6 (n = 81) sextant sites of the biopsy specimens, while PNI was present in 1 (n = 331), 2 (n = 132), 3 (n = 47), 4 (n = 19), 5 (n = 5), or 6 (n = 4) sites. To more accurately compare a single site versus multiple sites of PNI, we excluded 41 men showing cancer at only 1 site, because all of these cases had a single PNI site while cancer should have been present at multiple sites in all biopsy specimens with multiple PNI sites (eg, ≥2 cancer sites in 2-site PNI cases, 6 cancer sites in 6-site PNI cases). Thus, we examined, in a retrospective, blinded manner, a total of 497 sets of prostate biopsy specimens exhibiting PNI at 1 to 6 sites (and cancer at ≥2 sites) with corresponding radical prostatectomy specimens (Supplemental Tables 1 and 2).

We first compared 497 cases with a single site versus multiple sites with PNI (Table 1). Compared with a single PNI, multiple PNIs were associated with a significantly higher PSA level (P = .005) or significantly higher tumor grade on a biopsy specimen (P = .01). Similarly, multiple PNIs were significantly associated with adverse histopathologic features on radical prostatectomy, including higher tumor grade (P = .04), higher pT staging category (P < .001), lymph node metastasis (P < .001), positive surgical margin (P = .01), and larger estimated tumor volume (P < .001). When 1 versus 2 to 6 PNIs were compared in smaller groups of patients (ie, cancer at 3–6 [n = 419; Supplemental Table 3], 4–6 [n = 265; Supplemental Table 4], 5–6 [n = 159; Supplemental Table 5], or 6 [n = 81; Supplemental Table 6] biopsy sites), there were significant differences in PSA (3–6 sites), prostatectomy GG (6 sites), pT status (3–6/4–6/5–6 sites), pN status (3–6 sites), and tumor volume (3–6/4–6 sites).

We further compared subgroups of patients with cancer at 2 to 6 biopsy sites based on the highest biopsy GG, such as GG1 (Supplemental Table 7), GG2 (Supplemental Table 8), GG1–2 (Supplemental Table 9), GG3 (Supplemental Table 10), GG1–3 (Supplemental Table 11), GG4 (Supplemental Table 12), GG5 (Supplemental Table 13), and GG4–5 (Supplemental Table 14). In these subgroup analyses, significant differences in prostatectomy GG (GG1–3), pT (GG2, GG1–2, GG3, GG1–3), surgical margin status (GG3, GG1–3, GG5), and tumor volume (GG2, GG1–2, GG3, GG1–3) were observed.

We next compared 1 to 2 sites versus 3 to 6 sites with PNI from biopsy specimens showing cancer only at 3 or more sites (Table 2). Three or more PNIs were significantly associated with a higher PSA level (P = .007), higher tumor grade on biopsy specimens (P = .02), higher pT category (P < .001), and larger tumor volume (P < .001). Similarly, there were significant differences in PSA (P = .03), GG on biopsy (P < .001) or prostatectomy (P < .001) specimens, and pT (P = .008) or pN (P = .01) category between 1 to 3 versus 4 to 6 PNIs in cases with cancer only at 4 or more biopsy sites (Table 3), as well as in PSA (P = .006), GG on biopsy (P = .02) or prostatectomy (P = .002) specimens, and tumor volume (P = .03) between 1 to 4 versus 5 to 6 PNIs in those with cancer only at 5 or more biopsy sites (Table 4).

Kaplan-Meier analysis coupled with a log-rank test was then performed to assess the impact of the quantification of sextant biopsy sites with PNI on the prognosis after radical prostatectomy, with mean and median follow-up of 72 and 71 months, respectively. Overall, multiple PNI sites were associated with worse prognosis. In particular, multiple-PNI patients with cancer at 2 or more (Figure 1, A; P < .001), 3 or more (Figure 1, B; P < .001), or 4 or more (Figure 1, C; P = .001) biopsy sites, but not 5 or more (Figure 2, A; P = .06) or 6 (Figure 2, B; P = .17) biopsy sites, had significantly higher risks of disease progression, compared with respective control patients exhibiting a single PNI site. Similarly, 3 or more PNIs in cases with cancer at 3 or more biopsy sites were associated with a significantly higher risk of progression, compared with 1 to 2 PNIs (Supplemental Figure 1, A and B; P = .01). However, higher numbers of PNI sites, including 4 or more PNIs in cases with cancer at 4 or more biopsy sites (Supplemental Figure 1, C and D; P = .09) and 5 or more PNIs in cases with cancer at 5 or more biopsy sites (Supplemental Figure 1, E and F; P = .16), were not strongly associated with the risk of progression. As the number of PNI sites might be closely related to tumor volume in biopsy specimens, the analysis was adjusted for actual tumor length per positive biopsy site. When divided into 4 groups based on the average tumor length on 2 cores/1 positive biopsy site, significant differences between 1 versus 2 to 6 PNIs were seen in cases showing a tumor length of >1.5/≤2.0 cm (P = .004) or >2.0 cm (P = .001), but not in those of ≤1.0 cm or >1.0/≤1.5 cm (Supplemental Figure 2). In subgroup analysis in patients with cancer at 2 to 6 biopsy sites, there were significant differences in progression-free survival rates between 1 versus 2 to 6 PNIs in those with biopsy GG2 (P = .01), GG1–2 (P = .01), GG3 (P = .004), GG1–3 (P < .001), or GG5 (P = .03) tumor (Supplemental Figure 3). To further determine the clinical significance of PNI location, we compared the outcomes of 290 patients with a single PNI site at the apex, middle, or base of the prostate. Progression-free survival was not significantly different among these 3 groups (Supplemental Figure 4). Meanwhile, only 8 (1.6%) patients died of prostate cancer during follow-up, and there were no significant differences in the risk of cancer-specific mortality between any of the 2 groups dichotomized by the number of PNI sites.

To determine if PNI quantification was an independent predictor of disease progression, multivariate analysis for variables that could only be obtained before radical prostatectomy (ie, age, PSA, GG, cT or cN stage category, total cancer length, number of PNI sites) was performed using the Cox model. Multisite PNI in patients with cancer at 2 or more biopsy sites showed significance for recurrence, compared with a single PNI site (hazard ratio [HR], 1.556; 95% CI, 1.056–2.291; P = .03) (Table 5). However, no significance was found in 1 to 2 versus 3 to 6 PNI sites (HR, 1.028; 95% CI, 0.642–1.647; P = .91) in those with cancer at 3 or more sites, while GG (P < .001), cT (P = .01), and cancer length (P < .001) were still strong factors.

Various studies have suggested that PNI in prostate biopsy specimens, which can be detected in a subset (eg, 6.7%–50%⁸) of cancer cases, is a strong indicator of higher grade and/or stage disease, especially with extraprostatic extension, and resultant poorer oncologic outcome.^5–9  Many of these studies have demonstrated the independent prognostic value of biopsy PNI to predict biochemical recurrence after definitive therapy. Meanwhile, the prognostic significance of PNI in radical prostatectomy specimens remains controversial. Specifically, several studies have suggested that PNI in prostatectomy specimens holds no value as an independent prognosticator.^12,21  By contrast, the clinical impact of PNI quantification in prostate biopsy specimens has not been fully investigated. Therefore, in the present study, we compared clinicopathologic findings, including those on radical prostatectomy, as well as long-term outcomes, in men whose biopsy specimen had exhibited PNI in 1 to 6 sextant sites of the specimen.

As previously mentioned, studies that have demonstrated a significant association of PNI on prostate biopsy specimens with the prognosis have largely focused on the presence of PNI rather than detailed quantification of PNI.^5–9  Our study detailed PNI quantification by comparing cases with a single PNI site versus multiple PNI sites. We found that multisite PNI was strongly associated with adverse clinicopathologic features, including a higher preoperative PSA level and higher tumor grade on biopsy specimens, as well as worse histopathologic findings on radical prostatectomy, such as higher tumor grade, higher pT staging category, lymph node metastasis, positive surgical margin, and larger estimated tumor volume. In particular, the findings in pT category may be inevitable because PNI is thought to be the main route of extraprostatic spread.²²  In addition, in our outcome analysis, multiple PNI sites were associated with significantly higher risks of disease progression after radical prostatectomy, compared with a single PNI site. The rates of progression-free survival were also significantly lower in subgroups of patients with GG1–2, GG2, GG1–3, GG3, or GG5 cancer showing multiple sites with PNI. Moreover, there were significant or nonsignificant differences in the prognosis between 1 to 2 versus 3 to 6 PNIs (in all cases with cancer at 3 or more sites) or 1 to 3 versus 4 to 6 PNIs (in all cases with cancer at 4 or more sites), respectively. More strikingly, multivariate analysis for only parameters that could be obtained at the time of biopsy revealed that multisite PNI (versus a single PNI site), as well as PSA, GG, clinical T stage, and cancer length, was an independent predictor of disease progression after radical prostatectomy. We further assessed the impact of the location of biopsy PNI on patient outcomes and found no significant differences in progression-free survival in those exhibiting a single PNI site at the apex, middle, or base of the prostate. These findings are supported by the fact that nerves are distributed evenly in the prostate from apex to base.²³ 

PNI quantification has been studied in radical prostatectomy specimens, demonstrating its role as a prognosticator.^10–12,24  For instance, a study involving 265 prostatectomies showed that the incidence of biochemical recurrence was significantly higher in cases with multifocal PNIs (n = 32) than in those with unifocal PNI (n = 91) (HR, 4.81; P < .001) as well as in those with unifocal PNI than in those without PNI (n = 142) (HR, 2.02; P = .012).¹⁰  In another study using 114 prostatectomy specimens where S-100 protein was immunohistochemically stained for assisting the detection of nerves, overall survival times in patients with less than 1 PNI and 1 or more PNI(s) per 5 high-power fields were 159 and 173 months, respectively (P = .047).¹¹  A more recent study with 721 cases demonstrated the prognostic value of more than 3 PNIs, as an independent factor, compared with no PNI (HR, 2.12; P = .044), but the difference between 1 to 3 and more than 3 PNIs was not assessed.¹²  In addition, 10 or more PNIs detected only in the extraprostatic tissue were found to be associated with a significantly higher risk of biochemical recurrence even in a multivariate setting.²⁴  Our present data on the quantification of biopsy PNI sites are thus compatible with previous PNI findings in radical prostatectomy specimens and may be more useful for decision making for the management of prostate cancer.

There are several limitations in our investigation. First, the present study is subject to potential selection bias due to the retrospective design, although we have analyzed consecutive patients who met the inclusion criteria. Second, we compared only radical prostatectomy cases; the clinical impact of PNI quantification in patients undergoing active surveillance, radiotherapy, hormonal therapy, or other was not evaluated. Third, we assessed the numbers of sextant sites with PNI in each specimen (ie, 1–6) but did not consider those of biopsy cores with PNI or actual PNI foci, although the latter information is often unavailable in the current surgical pathology practice. Moreover, the interobserver variability in the diagnosis of PNI was noted,⁸  and we did not use immunostaining for nerve markers in virtually all biopsy specimens. We might thus have missed PNI in some cases, which could then generate some bias. Last, biopsy specimens with multiple sites of PNI translate to those with multiple sites of cancer, thus potentially skewing the prognostic significance of multisite PNI. We adjusted for this by excluding biopsy specimens with cancer in fewer sextant sites, and multisite PNI remained prognostically significant. We additionally compared the prognosis in subgroups of patients with similar tumor volume in biopsy specimens and found that multisite PNI was associated with a significantly higher risk of tumor progression after radical prostatectomy in those showing an average tumor length per positive biopsy site (for 2 cores) of more than 1.5 and 2.0 cm or less or more than 2.0 cm, but not in those with less than or equal to a 1.5-cm tumor.

In conclusion, compared with a single site of PNI, multiple sites with PNI on sextant needle core biopsy of the prostate were associated with worse histopathologic features in radical prostatectomy specimens and poorer prognosis. Significant differences in tumor grade, pT/pN staging category, and/or tumor volume were also seen between 1 to 2 versus 3 to 6, 1 to 3 versus 4 to 6, or 1 to 4 versus 5 to 6 PNIs. These findings support the current standard and, in the pathology report, PNI may thus need to be specified, if present, in every sextant site of the biopsy specimen, especially those showing GG1–3 cancer. Further studies to validate our results are required. The quantification of biopsy cores with PNI or actual PNI foci in biopsy specimens may also need to be assessed.