Original StudyNodal Occult Metastases in Intermediate- and High-Risk Prostate Cancer Patients Detected Using Serial Section, Immunohistochemistry, and Real-Time Reverse Transcriptase Polymerase Chain Reaction: Prospective Evaluation With Matched-Pair Analysis
Introduction
Lymph node (LN) metastases (LNM) are the strongest predictor of a poor prognosis in patients with prostate cancer (PCa) after radical prostatectomy (RP) together with primary and secondary Gleason grade, extracapsular extension, and seminal vesicle invasion.1, 2 Preoperative nomograms that consider established markers such as prostate-specific antigen (PSA), stage, number of positive cores, and Gleason score (Gs) can provide an estimate of the risk of LNM, but are still imperfect in individual patients.3 Despite recent advances in imaging techniques,3, 4, 5 pelvic LN dissection (PLND) remains the gold standard procedure for LN staging in PCa.6 According to the most used guidelines, a survival advantage with more extensive lymphadenectomy has been suggested by several studies, possibly because of the elimination of microscopic metastases. Therefore, an extended PLND is preferred when LN dissection is indicated.7, 8
The incidence of LNMs has dramatically decreased in the PSA era, but it still ranges from 1% to 45%, depending on the patient population, on the extent of PLND, and on the quality of histological analysis.6 Methods of nodal identification of LNs using microscopy vary between pathologists: standard pathological evaluation (SPE) is usually limited to a few cross-sections per LN and therefore might not identify LNM with a small diameter, thus resulting in understaging of a substantial number of patients.9 To date there has been no consensus about the number of sections per LN to be examined. Furthermore, counting the LNs in the pelvis presents more of a challenge than at many other sites, because of the large number of LNs showing replacement by adipose tissue.10
To overcome these limitations, many histological methods have been shown to significantly improve the detection rate of nodal metastases in the LNs. Palpation and meticulous dissection of the resection specimen to isolate single LNs or defatting of the connective tissue contributes to a better LN staging.9, 10 In addition, the number of sections examined per LN might improve the detection rate of nodal metastases.11 Immunohistochemistry (IHC) staining of paraffin-embedded sections using PSA and cytokeratin antibodies has been shown to be superior to routine hematoxylin and eosin (H & E) staining for the detection of small PCa metastases.12 Finally, reverse transcriptase (RT) polymerase chain reaction (PCR) has been used in the detection occult metastases (OCM) in the LNs of many organs and is actually considered the most sensitive method for detecting small numbers of metastatic cells.13
From a clinical point of view, the exact LN staging in the presence of macroscopic metastases is of utmost importance to the patient prognosis14, 15; even if it is not completely clear if the presence of LNM influences patient prognosis independently from the Gs, the pathological stage, or the margin status, the knowledge of the LN status significantly alters the therapeutic strategy and improves the clinical decision-making regarding possible early initiation of additional regional and/or systemic therapy.16 Moreover, the significance of the presence of micrometastases and its prognostic role is not completely understood. Certainly, the putative diagnostic gain of an extended evaluation of the LN specimens by increasing the number of cross-sections and applying IHC or RT-PCR has to be weighed against the consequent increase of expenses in materials and labor time.17 Moreover, the prognostic importance of the OCM in the survival of the patients still remains a matter of debate.12, 13 Attempts to test the clinical utility of these various detection strategies compared with the standard methods for staging have yielded mixed results, mainly because of the diversity of techniques used and varying numbers of patient samples examined in each study. To date, the specific contribution of these histological, immunohistochemical, and molecular techniques has not been tested in the same group of patients prospectively to our knowledge.
The aim of the present study was to prospectively evaluate the incidence of nodal OCM assessed separately using serial section (SS), IHC, and real time RT-PCR in patients submitted to RP with extended PLND for intermediate or high-risk PCa compared with standard pathological examination, and to evaluate short-term oncological outcomes of patients with OCM.
Section snippets
Study Population
Between March 2009 and October 2012, a cohort of consecutive patients with PCa submitted to RP with extended PLND for PCa at intermediate risk (defined as clinical T1c-T2 and PSA 10-19.9 ng/mL or clinical Gs = 7) or at high risk (clinical stage T3 or PSA > 19.9 ng/mL or clinical Gs = 8-10) of nodal involvement were prospectively enrolled and comprised the study population (StP). Exclusion criteria were: (1) neoadjuvant hormonal therapy; (2) extranodal involvement documented at preoperative
Matched-Pair Population
To obviate possible examination bias (eg, a greater number of positive LNs could have been detected in the StP because of better handling of the specimens and because of a greater degree of attention of the pathologist, thus determining an overdetection of nodal metastases assessed at SPE), a matched-pair population (MpP) of patients with characteristics identical to the StP and submitted to RP with extended PLND (e-PLND) between March 2008 and February 2014 at our institution was selected:
Immunohistochemistry Assay
Immunohistochemistry was performed in all of the SSs using primary antibodies, anti-PSA (Prostate Specific Antigen, polyclonal rabbit, dilution 1:2000, Dako, Glostrup, Denmark) and anti-cytokeratins (MNF116 clone, dilution 1:150, Dako) according to manufacturer's instructions. Briefly, sections underwent deparaffinization, rehydratation, endogenous peroxydase activity blocking, and enzymatic digestion pretreatment (only for cytokeratin staining), before being incubated at room temperature for 1
Results
Fifty-four consecutive patients comprised the StP. Overall, 27 (50.0%) patients were at intermediate risk of nodal involvement and 27 (50.0%) were at high risk in the StP, with a mean preoperative risk of LN involvement of 26.9% according to the Briganti nomogram. Complete patient characteristics of the StP compared with the MpP are reported in Table 1: pathological characteristics of the StP were identical to the MpP. Overall, 1064 LNs were removed and processed in the StP, with 2126 H & E
Discussion
As with other tumors, the micrometastatic process of PCa precedes the development of a macroscopic, detectable lesion by many years.19 The number of positive LNs has an important prognostic role and patients with low-volume metastasis might have significantly greater survival rates than those with high-volume metastasis.14, 15 Thus, better LN evaluation might improve patient prognosis. However, the number of LNs obtained in a PLND varies widely among centers, which is a function of surgical
Conclusion
The current pathological handling of the LNs is inaccurate. Serial sectioning, immunohistochemical, and molecular analysis of LNs can detect a not negligible percentage of patients who harbor micrometastatic PCa missed at routine pathological examination and can enhance the accuracy of lymphadenectomy as a staging method. Real-time RT-PCR contributes the most to the detection of OCM in patient and LN analysis. The clinical effect of micrometastases in PCa and the potential therapeutic role of
Acknowledgment
This work was generously supported by the SAMUR ONLUS (Advanced Studies on Urological disease) and by the “Fondazione del Monte di Bologna e Ravenna” Foundation.
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