Gao C L, Dean R C, Pinto A, Mooneyhan R, Connelly R R, McLeod D G, Srivastava S, Moul J W
Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Urol. 1999 Apr;161(4):1070-6.
The reverse transcriptase polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) expressing cells in the blood circulation has been under intense investigation since 1992. Although it has been suggested that this technology could be used as molecular staging for occult prostatic hematogenous metastases, we have been unable to confirm RT-PCR PSA positivity of peripheral blood to predict stage or recurrence in radical prostatectomy cases. We performed bone marrow RT-PCR PSA assay on a large cohort of radical prostatectomy cases and evaluate the use of this assay in improving prostate cancer staging and detecting early recurrence.
Unilateral anterior iliac crest bone marrow aspirates were performed on 116 patients immediately before radical prostatectomy between February 1995 and September 1997. Radical prostatectomy specimens were processed as whole mounts. A sensitive nested RT-PCR assay with specific primers derived from the PSA sequence was used, which enabled us to detect PSA expressing LNCaP prostate cancer cells at the sensitivity of 1 cancer cell per 10 million lymphocytes (1/10(7)). A minimum of 3 RT-PCR PSA reactions were performed on all patients and at least 2 positive tests were required to define positivity. Patients were followed for PSA recurrence (mean followup 14.7 months).
PSA expressing cells were detected in bone marrow of 51 of 116 patients (44.0%) when at least 2 of 3 RT-PCR PSA assays per patient were positive. A much higher rate of RT-PCR PSA positivity was noted (77/116 patients, 66.3%) when any RT-PCR PSA positivity was considered. In 10 randomly selected cases the RT-PCR product was confirmed as PSA by deoxyribonucleic acid sequencing. Of 51 bone marrow RT-PCR positive cases 25 (49%) had organ confined disease and 26 (51%) had nonorgan confined disease. Similarly, bone marrow RT-PCR PSA was not associated with age, race, grade, pretreatment PSA or prostatic acid phosphatase value, clinical stage or margin status. However, the 2-year disease-free survival was 96.6% in RT-PCR negative patients versus 77.5% in RT-PCR positive patients (p = 0.054), and bone marrow RT-PCR PSA was an independent prognostic factor in multivariate analysis including PSA, Gleason grade and pathological stage.
Bone marrow RT-PCR PSA positivity in this study did not predict pathological stage, grade or margin positivity as determined from whole mount prostate cancer specimens. Furthermore, no relationship with age, grade or serum markers and bone marrow RT-PCR PSA positivity was noted. However, bone marrow RT-PCR PSA was associated with early disease recurrence. Further studies and longer followup are warranted to define the metastatic potential of the PSA expressing cells in the bone marrow of prostate cancer patients.
自1992年以来，针对血液循环中表达前列腺特异性抗原（PSA）细胞的逆转录酶聚合酶链反应（RT-PCR）检测法一直受到深入研究。尽管有人提出这项技术可用于隐匿性前列腺血行转移的分子分期，但我们无法证实外周血RT-PCR PSA阳性可预测根治性前列腺切除术病例的分期或复发情况。我们对一大组根治性前列腺切除术病例进行了骨髓RT-PCR PSA检测，并评估该检测法在改善前列腺癌分期及检测早期复发方面的应用。
1995年2月至1997年9月期间，在116例患者行根治性前列腺切除术之前，立即从单侧髂前嵴进行骨髓穿刺抽吸。根治性前列腺切除标本制成整装切片。采用一种敏感的巢式RT-PCR检测法，其引物来源于PSA序列，这使我们能够以每1000万个淋巴细胞中检测到1个癌细胞（1/10⁷）的灵敏度检测表达PSA的LNCaP前列腺癌细胞。对所有患者至少进行3次RT-PCR PSA反应，且至少需要2次阳性检测结果才能判定为阳性。对患者进行PSA复发情况随访（平均随访14.7个月）。
当每位患者3次RT-PCR PSA检测中至少有2次为阳性时，在116例患者中的51例（44.0%）骨髓中检测到表达PSA的细胞。若考虑任何一次RT-PCR PSA阳性，则RT-PCR PSA阳性率更高（77/116例患者，66.3%）。在随机选取的10例病例中，通过脱氧核糖核酸测序确认RT-PCR产物为PSA。在51例骨髓RT-PCR阳性病例中，25例（49%）为器官局限性疾病，26例（51%）为非器官局限性疾病。同样，骨髓RT-PCR PSA与年龄、种族、分级、治疗前PSA或前列腺酸性磷酸酶值、临床分期或切缘状态无关。然而，RT-PCR阴性患者的2年无病生存率为96.6%，而RT-PCR阳性患者为77.5%（p = 0.054），并且在包括PSA、Gleason分级和病理分期的多变量分析中，骨髓RT-PCR PSA是一个独立的预后因素。
本研究中骨髓RT-PCR PSA阳性并不能预测从整装前列腺癌标本确定的病理分期、分级或切缘阳性情况。此外，未发现年龄、分级或血清标志物与骨髓RT-PCR PSA阳性之间存在关联。然而，骨髓RT-PCR PSA与疾病早期复发相关。需要进一步研究及更长时间的随访来确定前列腺癌患者骨髓中表达PSA细胞的转移潜能。
