Prostate-specific membrane antigen (PSMA) positron emission tomography (PET)/CT is a modern imaging tool used in the diagnosis of prostate cancer (PCa). About 10% of patients who undergo PSMA PET/CT have a biopsy confirmed, primary PCa without PSMA expression on PET/CT (non-PSMA_PET-expressing PCa) according to a recent systematic review [1]. However, the definition of non-PSMA_PET-expression in these studies was poorly defined and no immunohistochemical studies for confirmation of PSMA protein expression were performed. The aim of this retrospective study was to report the prevalence, characteristics, and immunohistochemical assessment of non-PSMA_PET-expressing hormone-sensitive PCa in a cohort of 362 patients who underwent PSMA PET/CT and robot-assisted radical prostatectomy (RARP).

This study was approved by the local Institutional Review Board (IRBdm19-348). All patients had biopsy confirmed, hormone-sensitive, D’Amico intermediate- to high-risk PCa and were surgically treated in a high-volume RARP centre between 2016 and 2020. The patients underwent PSMA PET/CT with the radiotracers ⁶⁸Ga-PSMA-11, ¹⁸F-DCFPyL or ¹⁸F-PSMA-1007. PET images from skull to mid-thigh were performed at a median (interquartile range [IQR]) of 49 (45–56) min post-injection after a median (IQR) bolus injection of 101 (93–110) MBq fixed-dose for ⁶⁸Ga-PSMA-11, 73 (57–83) min post-injection after a median (IQR) injected dose of 239 (202–299) MBq for ¹⁸F-DCFPyL, and 80 (74–92) min post-injection after a median (IQR) dose of 281 (252–304) MBq for ¹⁸F-PSMA-1007. All PSMA PET/CTs were centrally reviewed by experienced nuclear medicine specialists. Non-PSMA_PET-expressing PCa was defined as no focal tracer uptake in the prostate exceeding the background activity of normal prostate tissue, in accordance with Prostate Cancer Molecular Imaging Standardised Evaluation (PROMISE) criteria and the European Association of Nuclear Medicine PSMA (E-PSMA) guidelines [2]. All patients underwent a bi- or multiparametric MRI at 1.5 or 3 Tesla. All MRI scans were centrally reviewed by experienced uroradiologists. Clinical, radiological, and histopathological data were collected from the prospectively maintained institutional database. Immunohistochemical staining was performed on all non-PSMA_PET-expressing tumours and a 2:1 matched group of PSMA_PET-expressing tumours. Matching was based on radiotracer, pathological tumour stage, and Gleason score. The staining was performed with an anti-PSMA monoclonal mouse antibody (Clone 3E6; DAKO, North America Inc., Carpinteria, CA, USA). One uropathologist (E.B.) assessed the tumour area in a 20 mm² region of interest in the slide and the proportion of tumour with commonly used different staining intensities: no expression, weak expression, moderate expression, intense expression. The overall staining intensity was calculated with the histoscore (H-score), ranging from 0 to 300 [3]. Differences in non-PSMA_PET-expressing PCa and PSMA_PET-expressing PCa regarding clinical, radiological, pathological and immunohistochemical data were analysed with Pearson chi-square test, Fisher’s exact test, Student’s t-test, or Mann–Whitney U-test. Statistical significance was set on P = 0.05.

The prevalence of non-PSMA_PET-expressing PCa was 4.1% (15/362). Comparable prevalence was reported for the different radiotracers: ⁶⁸Ga-PSMA-11: 3.5% (eight of 228); ¹⁸F-DCFPyL: 5.4% (six of 112); ¹⁸F-PSMA-1007: 4.5% (one of 22). The initial serum PSA level was significantly lower in patients with non-PSMA_PET-expressing PCa than in those with PSMA_PET-expressing PCa, at a median (IQR) of 6.8 (4.1–9.4) vs 11 (7.5–21) ng/L (P = 0.001, Table 1). There were no differences between both cohorts for clinical tumour stage, biopsy Gleason score, molecular imaging nodal (miN1) or distant metastasis stage (miM1), or D’Amico high-risk PCa. Also, there were no differences in Prostate Imaging Reporting and Data System (PI-RADS) scores or MRI tumour volumes (median 2.0 vs 2.5 mL, P = 0.47) [4].

There were no differences between the cohorts for pathological tumour stage, RARP specimen Gleason score, positive surgical margin rate, presence of intraductal growth or cribriform growth patterns. In patients who underwent an extended pelvic lymph node dissection, lymph node metastases (pN1) were found in one of 14 (7%) patients with non-PSMA_PET-expressing PCa vs 81/301 (27%) patients with PSMA_PET-expressing PCa (P = 0.13).

None of the investigated tumours, including non-PSMA_PET-expressing tumours, fully lacked immunohistochemical PSMA protein expression. Non-PSMA_PET-expressing tumours had a significantly smaller proportion of tumour cells with either moderate or intense PSMA protein expression compared to PSMA_PET-expressing tumours, at a median (IQR) of 35 (15–70)% vs 85 (70–96)% (P = 0.001). The overall staining intensity (H-score) was significantly lower in non-PSMA_PET-expressing tumours compared to PSMA_PET-expressing tumours, at a median (IQR) of 135 (90–170) vs 210 (170–285) (P = 0.001).

This study is the first to show the characteristics of patients with non-PSMA_PET-expressing PCa and the first to report on the immunohistochemical analysis of these tumours. With a definition that adheres to recently proposed guidelines, the prevalence of non-PSMA_PET-expressing PCa in the present study was lower (4.1%) than the previously reported 10% [1]. This is consistent with more recent studies evaluating local tumour presence on PSMA PET/CT that reported a 4.3–5.9% prevalence [5, 6]. Previous authors tried to determine a cut-off value for PSMA_PET-expressing PCa based on the maximum standard uptake value (SUV_max). However, SUV_max measurement is influenced by a number of tracer-related and technical factors (e.g. dosage, injection time, voxel size, number of iterative updates of ordered subset expectation maximisation). Due to the multicentre aspect of this study, technical inconsistencies were present, probably leading to unreliable SUV_max measurements. Therefore, SUV_max was omitted from the analysis. We showed that, except for a lower serum PSA level, non-PSMA_PET-expressing PCa and PSMA_PET-expressing PCa have no different clinical, radiological, and histopathological characteristics. Moreover, as the majority of non-PSMA_PET-expressing tumours proved to be (plain) adenocarcinoma after histopathological examination, it is to be doubted that these tumours represent de-differentiated, neuroendocrine PCa [7, 8]. In fact, non-PSMA_PET-expressing tumours are presumably hormone-sensitive PCas that have similar preoperative and histopathological features compared to PSMA_PET-expressing PCas. It needs to be acknowledged that the aetiology of non-PSMA_PET expression is probably multifactorial and not just a consequence of dismal prognostic tumour features. We showed that non-PSMA_PET expression is associated with decreased immunohistochemical PSMA protein expression when compared to PSMA_PET-expressing PCa. The proportion of cells with moderate and intense immunohistochemical PSMA protein expression was significantly lower in the non-PSMA_PET-expressing tumours compared to the PSMA_PET-expressing tumours, as well as the overall PSMA-staining intensity (H-score). However, none of the non-PSMA_PET-expressing tumours fully lacked immunohistochemical PSMA protein expression. Therefore, it is likely that technical factors with respect to PSMA PET/CT imaging itself or due to the biodistribution of the radiotracer may have partially contributed to the phenomenon of non-PSMA_PET expression.

This study is limited by the retrospective nature of the cohort, the different radiotracers and the different scanning protocols used. However, to reduce bias, all scans were centrally reviewed, and all non-PSMA_PET-expressing scans were re-reviewed by an experienced nuclear medicine specialist (M.D.) who had access to pre- and postoperative data. Also, no difference in the prevalence of non-PSMA_PET-expressing PCa was found when comparing the different radiotracers. Due to the low prevalence of non-PSMA_PET expression, the group size was small, which limits the statistical power of the analyses.

The detection of lymph node metastases with PSMA PET/CT may be unreliable in those with non-PSMA_PET-expressing PCa. Clinicians could consider performing a bone scan in patients with a high a priori risk of distant metastases (PSA level of >20 ng/L or Gleason score 9–10) in the case of non-PSMA_PET-expressing PCa to determine the eligibility of curative treatment. Future studies should focus on the intermediate- to long-term oncological outcomes of patients with non-PSMA_PET-expressing PCa.

In conclusion, the prevalence of non-PSMA_PET-expressing PCa in a large contemporary cohort was low. Apart from a lower serum PSA level, patients with non-PSMA_PET-expressing tumours had similar clinical, radiological, and histopathological features to those with PSMA_PET-expressing tumours. Non-PSMA_PET-expressing tumour cells had significantly lower immunohistochemical PSMA protein expression than PSMA_PET-expressing tumour cells.

前列腺特异性膜抗原 (PSMA) 正电子发射断层扫描 (PET)/CT 是一种用于诊断前列腺癌 (PCa) 的现代成像工具。根据最近的一项系统评价 [ 1 ] ，接受 PSMA PET/CT 的患者中约有 10% 的活检证实，在 PET/CT 上没有 PSMA 表达的原发性 PCa（非 PSMA _PET表达 PCa） 。然而，在这些研究中对非 PSMA _PET表达的定义不明确，并且没有进行用于确认 PSMA 蛋白表达的免疫组织化学研究。这项回顾性研究的目的是报告非 PSMA _{PET的患病率、特征和免疫组化评估}-在接受 PSMA PET/CT 和机器人辅助根治性前列腺切除术 (RARP) 的 362 名患者队列中表达激素敏感性 PCa。

这项研究得到了当地机构审查委员会 (IRBdm19-348) 的批准。2016 年至 2020 年期间，所有患者均经过活检证实、激素敏感、D'Amico 中度至高风险 PCa，并在高容量 RARP 中心接受了手术治疗。患者接受了 PSMA PET/CT 与放射性示踪剂⁶⁸ Ga-PSMA -11、18 F- ^DCFPyL或¹⁸ F-PSMA-1007。从头骨到大腿中部的 PET 图像在中位数 (IQR) 推注 101 (93-110) MBq 固定剂量后的中位数（四分位距 [IQR]）注射后 49 (45-56) 分钟进行对于⁶⁸ Ga-PSMA-11，在中位数 (IQR) 注射剂量为 239 (202-299) MBq 后 73 (57-83) 分钟注射后^{18F-DCFPyL 和18} F-PSMA-1007的中位数 (IQR) 剂量为 281 (252-304) MBq 后 80 (74-92) 分钟。所有 PSMA PET/CT 均由经验丰富的核医学专家集中审查。_{根据前列腺癌分子成像}标准化评估 (PROMISE) 标准和欧洲核医学协会 PSMA ( E- PSMA) 指南 [ 2]。所有患者都接受了 1.5 或 3 特斯拉的双参数或多参数 MRI。所有 MRI 扫描均由经验丰富的泌尿放射科医生集中审查。从前瞻性维护的机构数据库中收集临床、放射学和组织病理学数据。对所有不表达 PSMA _{PET的肿瘤和表达 PSMA PET}的肿瘤的 2:1 匹配组进行免疫组织化学染色。匹配基于放射性示踪剂、病理肿瘤分期和 Gleason 评分。用抗 PSMA 单克隆小鼠抗体（Clone 3E6；DAKO，North America Inc.，Carpinteria，CA，USA）进行染色。一位泌尿病理学家 (EB) 评估了 20 mm ^{2内的肿瘤区域}切片中感兴趣区域和肿瘤的比例常用不同的染色强度：无表达、弱表达、中度表达、强表达。使用组织评分（H 评分）计算整体染色强度，范围从 0 到 300 [ 3 ]。用 Pearson 卡方检验、Fisher 精确检验、Student t检验或 Mann-Whitney U检验分析非 PSMA _PET表达 PCa 和 PSMA _PET表达 PCa 在临床、放射学、病理学和免疫组织化学数据方面的差异。统计学显着性设定为P  = 0.05。

非 PSMA _PET表达 PCa 的患病率为 4.1% (15/362)。不同放射性示踪剂的患病率相当：⁶⁸ Ga-PSMA-11：3.5%（228 个中的 8 个）；¹⁸ F-DCFPyL：5.4%（112 个中的 6 个）；¹⁸ F-PSMA-1007：4.5%（22 个之一）。非 PSMA _PET表达 PCa患者的初始血清 PSA 水平显着低于 PSMA _PET表达 PCa 患者，中位数 (IQR) 为 6.8 (4.1-9.4) 对 11 (7.5-21) ng/大号（磷 = 0.001，表 1)。两个队列在临床肿瘤分期、活检 Gleason 评分、分子成像淋巴结 (miN1) 或远处转移分期 (miM1) 或 D'Amico 高危 PCa 方面没有差异。此外，前列腺成像报告和数据系统 (PI-RADS) 评分或 MRI 肿瘤体积（中位数 2.0 vs 2.5 mL， P  = 0.47）没有差异[ 4 ]。

病理肿瘤分期、RARP 标本 Gleason 评分、手术切缘阳性率、导管内生长或筛状生长模式的存在在队列之间没有差异。在接受扩大盆腔淋巴结清扫术的患者中，在 14 名（7%）非 PSMA _PET表达 PCa 患者中发现淋巴结转移（pN1）与 81/301（27%）名 PSMA _PET表达患者PCa ( P  = 0.13)。

没有一个研究的肿瘤，包括非 PSMA _PET表达肿瘤，完全缺乏免疫组织化学 PSMA 蛋白表达。_{与表达 PSMA PET 的肿瘤相比，不表达 PSMA PET}的_肿瘤具有中度或强烈 PSMA 蛋白表达的肿瘤细胞比例明显更小，中位数 (IQR) 为 35 (15–70)% vs 85 (70– 96)% ( P  = 0.001)。_{与表达 PSMA PET}的肿瘤相比，非 PSMA _PET表达肿瘤的总体染色强度（H 评分）显着降低，中位数（IQR）为 135（90-170）与 210（170-285）（P  = 0.001)。

该研究首次展示了非 PSMA _PET表达 PCa 患者的特征，并首次报告了这些肿瘤的免疫组织化学分析。根据最近提出的指南的定义，本研究中表达非 PSMA _PET的 PCa 的患病率 (4.1%) 低于先前报道的 10% [ 1 ]。这与最近在 PSMA PET/CT 上评估局部肿瘤存在的研究一致，该研究报告的患病率为 4.3-5.9% [ 5, 6 ]。_{以前的作者试图根据最大标准摄取值 (SUV max} )确定表达 PSMA _PET的 PCa 的截止值。但是，SUV _max测量受到许多示踪剂相关和技术因素的影响（例如剂量、注射时间、体素大小、有序子集期望最大化的迭代更新次数）。由于本研究的多中心方面，存在技术上的不一致，可能导致 SUV_最大测量值不可靠。因此，分析中省略了SUV _{max 。}我们发现，除了较低的血清 PSA 水平外，非 PSMA _PET表达 PCa 和 PSMA _PET表达 PCa 没有不同的临床、放射学和组织病理学特征。此外，作为大多数非 PSMA _PET- 表达肿瘤在组织病理学检查后被证明是（普通）腺癌，这些肿瘤是否代表去分化的神经内分泌 PCa [ 7, 8 ] 值得怀疑。事实上，与表达 PSMA _{PET的 PCas 相比，不表达 PSMA PET}的肿瘤可能是激素敏感的 PCas，其具有相似的术前和组织病理学特征。需要承认的是，非 PSMA _PET表达的病因可能是多因素的，而不仅仅是预后不佳的肿瘤特征的结果。_{我们发现，与 PSMA PET}相比，非 PSMA _PET表达与免疫组织化学 PSMA 蛋白表达降低有关-表达PCa。_{与表达 PSMA PET}的肿瘤相比，表达非 PSMA _PET的肿瘤中具有中度和强烈免疫组织化学 PSMA 蛋白表达的细胞比例以及整体 PSMA 染色强度（H 值）显着降低。然而，没有一个非 PSMA _PET表达肿瘤完全缺乏免疫组织化学 PSMA 蛋白表达。因此，与 PSMA PET/CT 成像本身相关的技术因素或由于放射性示踪剂的生物分布可能部分导致了非 PSMA _PET表达的现象。

这项研究受到队列的回顾性、不同的放射性示踪剂和使用的不同扫描方案的限制。然而，为了减少偏差，所有扫描都经过集中审查，所有非 PSMA _PET表达扫描都由可以访问术前和术后数据的经验丰富的核医学专家 (MD) 重新审查。此外，在比较不同的放射性示踪剂时，未发现表达非 PSMA _PET的 PCa 的流行率没有差异。由于非 PSMA _PET表达的低流行率，组规模很小，这限制了分析的统计能力。

用 PSMA PET/CT 检测淋巴结转移在那些不表达 PSMA _PET的 PCa 中可能是不可靠的。在非 PSMA _PET表达的 PCa 的情况下，临床医生可以考虑对具有高先验远处转移风险（PSA 水平 >20 ng/L 或 Gleason 评分 9-10）的患者进行骨扫描，以确定是否符合根治性治疗。_{未来的研究应侧重于非 PSMA PET}表达 PCa患者的中长期肿瘤学结果。

总之，在一个大型当代队列中，非 PSMA _PET表达的 PCa 的患病率很低。除了较低的血清 PSA 水平外，非 PSMA _{PET表达肿瘤患者与 PSMA PET}表达肿瘤患者具有相似的临床、放射学和组织病理学特征。_{与表达 PSMA PET}的肿瘤细胞相比，不表达 PSMA _PET的肿瘤细胞具有显着降低的免疫组织化学 PSMA 蛋白表达。