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Molecular Load of Pathologically Occult Metastases in Pelvic Lymph Nodes Is an Independent Prognostic Marker of Biochemical Failure After Localized Prostate Cancer Treatment

Anna C. Ferrari, Nelson N. Stone, Ralf Kurek, Elizabeth Mulligan, Roy McGregor, Richard Stock, Pamela Unger, Ulf Tunn, Amir Kaisary, Michael Droller, Simon Hall, Heiner Renneberg, Kenneth J. Livak, Robert E. Gallagher, John Mandeli

From the New York University Cancer Institute; Mount Sinai School of Medicine, New York; Albert Einstein Cancer Center, Bronx, NY; Stadtische Kliniken, Offenbach, Germany; Royal Free Hospital, London, United Kingdom; and Applied Biosystems Inc, Foster City, CA

Address reprint requests to Anna C. Ferrari, MD, New York University Cancer Institute, New York University Medical School, 160 E 34th St, 8th Floor, New York, NY 10016; e-mail: anna.ferrari{at}nyumc.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 
PURPOSE: Thirty percent of patients treated with curative intent for localized prostate cancer (PC) experience biochemical recurrence (BCR) with rising serum prostate-specific antigen (sPSA), and of these, approximately 50% succumb to progressive disease. More discriminatory staging procedures are needed to identify occult micrometastases that spawn BCR.

PATIENTS AND METHODS: PSA mRNA copies in pathologically normal pelvic lymph nodes (N0-PLN) from 341 localized PC patients were quantified by real-time reverse-transcriptase polymerase chain reaction. Based on comparisons with normal lymph nodes and PLN with metastases and on normalization to 5 x 10⁶ glyceraldehyde-3'-phosphate dehydrogenase mRNA copies, normalized PSA copies (PSA-N) and a threshold of PSA-N 100 or more were selected for continuous and categorical multivariate analyses of biochemical failure-free survival (BFFS) compared with established risk factors.

RESULTS: At median follow-up of 4 years, the BFFS of patients with PSA-N 100 or more versus PSA-N less than 100 was 55% and 77% (P = .0002), respectively. The effect was greatest for sPSA greater than 20 ng/mL, 25% versus 60% (P = .014), Gleason score 8 or higher, 21% versus 66% (P = .0002), stage T3c, 18% versus 64% (P = .001), and high-risk group (50% v 72%; P = .05). By continuous analysis PSA-N was an independent prognostic marker for BCR (P = .049) with a hazard ratio of 1.25 (95% CI, 1.001 to 1.57). By categorical analysis, PSA-N 100 or more was an independent variable (P = .021) with a relative risk of 1.98 (95% CI, 1.11 to 3.55) for BCR compared with PSA-N less than 100.

CONCLUSION: PSA-N 100 or more is a new, independent molecular staging criterion for localized PC that identifies high-risk group patients with clinically relevant occult micrometastases in N0-PLN, who may benefit from additional therapy to prevent BCR.

	INTRODUCTION

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An increasing number of patients diagnosed with localized prostate cancer (PC) are cured by radical prostatectomy (RP) or radiation therapy (XRT). However, 30% of patients experience biochemical recurrence (BCR; ie, increasing serum prostate-specific antigen [sPSA] levels), and 50% of these patients die of androgen-independent progression despite systemic therapy.¹ Prognostic factors, including sPSA, clinical tumor stage (TS), and Gleason score (GS), have been formulated as tables, nomograms, and risk-group categories for estimating the probability of non–organ-confined disease at diagnosis and disease progression after primary therapy.^2-6 Low-risk group patients have an 85% to 90% cure rate; however, over 50% of high-risk patients experience BCR of whom 90% die of PC within 10 years.^7-9

Although helpful, risk factor analysis does not definitively establish the presence of metastases from which BCR later evolves.^10,11 The prognostic importance of pathologically detected pelvic lymph node (PLN) metastases has, however, been established by markedly inferior final outcome.^12,13 By logical extension, occult micrometastatic PC in pathologically N0-PLN might also be associated with a reduced prognosis.

Previous nonquantitative reverse transcriptase polymerase chain reaction (RT-PCR) studies of PSA, prostate-specific membrane antigen (PSMA), and kallikrein-2 mRNAs provided evidence for micrometastases in N0-PLN.^14-17 One retrospective study of archival N0-PLN in pathologic TS3c disease found a significant association between RT-PCR positivity for kallikrein-2 mRNA and the development of metastasis and decreased survival.¹⁸ Similar studies using peripheral blood mononuclear cells and bone marrow have not provided information of prognostic value.^17,19-22 From these considerations, we prospectively tested the hypothesis that quantification of PSA mRNA copies in N0-PLN might reveal occult metastases predictive for subsequent progression.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 
Patients and Investigators
Bilateral N0-PLN were prospectively (1996 to 2002) and consecutively collected from 341 patients undergoing definitive treatment for localized PC:175 patients at the Klinikum Frankfurt am Main (KO), Offenbach, Germany; 130 patients at the Mount Sinai School of Medicine (MSSM), New York, NY; and 36 patients at the Royal Free Hospital (RFH), London, United Kingdom. All investigators completed protection of human research subjects education requirements. All patients signed institutional review board approved consent. Study eligibility required clinical stage T1-T3 without evidence of metastasis (N0, M0). Patients with pathologic N1 disease were excluded.

Clinical Evaluation and Staging
All patients had PSA screening, digital rectal examination, and transrectal ultrasound-guided biopsy (six cores). One investigator (N.S.) performed digital rectal examination and transrectal ultrasound seminal vesicle (SV) biopsies in XRT TS 2 or 3, or GS 7 or higher, or PSA greater than 20 ng/mL patients. Staging followed the American Joint Committee on Cancer 1992 criteria.²³ Required computed tomography of the abdomen/pelvis and bone scan excluded M1 patients. N stage was defined by frozen and final pathology of all lymph node (LN) groups sampled. Bilateral obturator and internal iliac LN were removed by laparoscopy before XRT.²⁴ In RP patients, bilateral obturator and the external, internal, and common iliac LNs were obtained by extended lymphadenectomy,²⁵ but only bilateral obturator and internal iliac were processed from metastases-free patients for quantitative real-time RT-PCR (Q-PCR) analysis. Frozen blocks containing three to five LN per group were sectioned for histology. In the absence of metastasis, 10 contiguous microtome slices were dropped in RNA buffer and stored at –20°C. The rest of the lymph node tissue was processed for final pathology. All prostate sections were assessed for tumor extension into SV and capsule. Since 75% of patients had RP, the pathologic TS was chosen. In the 25% of patients who had XRT, we combined clinical TS with the SV biopsy results. Thus, virtually all TS3c cases were identified regardless of treatment. RP margins were not measured at KO and, consequently, could not be analyzed. GS was calculated by one pathologist (P.U.) from the two most prominent patterns in the prostatectomy or biopsy specimens. sPSA was determined by the Abbott (Abbott Laboratories, Abbott Park, IL; MSSM and RFH) or Hybritech (Beckman Coulter Inc, Fullerton, CA; KO) assay.

Definitive Treatment
Eighty-eight high-risk patients treated with XRT received 3 months each of neoadjuvant, concomitant, and adjuvant luteinizing hormone-releasing hormone (LHRH) agonist with antiandrogens (MSSM). XRT consisted of seed implants (100 Gy palladium-103) using a real-time technique followed 2 months later by 45 Gy of conformal external-beam irradiation given in 180 Gy fractions to a target of 1.5 cm outside of the prostate.²⁶ One hundred seventy-five RP patients received 3 months of neoadjuvant LHRH agonist with cyproterone acetate (KO). Seventy eight RP patients did not receive hormone therapy (38 patients at RFH; 40 patients at MSSM). All surgeries were nerve-sparing radical retropubic prostatectomies.

Clinical Follow-Up
Disease assessment every 6 months included sPSA, clinical progression, or/and initiation of hormone therapy. Median follow-up was 48 months (range, 6 months to 102 months).

Sample Processing and Q-PCR Procedures
Total RNA, extracted with RNAzol kit (Biotecx Laboratories Inc, Houston, TX) was converted to complementary DNA (cDNA) in a 25 µL reaction containing 5 ug RNA, random-hexamer primers (Pharmacia, Piscataway, NJ), and Superscript-RNAse-H^-RT (Invitrogen, Carlsbad, CA). Q-PCR was performed in a 50 uL reaction, containing test cDNA, 1x TaqMan Universal PCR Master Mix (ABI, Foster City, CA), primers (900 nmol/L), and probe (200 nmol/L) using an ABI PRISM 7700 DNA Sequence Detection System.²⁷ The primers and VIC–labeled probe for the housekeeping gene glyceraldehyde-3'-phosphate dehydrogenase (GAPDH) were proprietary (ABI). For PSA, forward and reverse primers were 5'-CCCACTGCATCAGGAACAAA-3' and 5'-GAAATACCTGGCCTGTGTCTTCA-3'. These PSA-specific primers amplified an 81 nucleotide sequence spanning the exon 2/3 junction in PSA mRNA. The FAM-labeled, minor-groove-binding PSA probe sequence was 5'-TGAAACAGGCTGTGCCGACCCAGC-3' (ABI). GAPDH and PSA copy numbers were determined by reference to standard curves constructed from serial dilutions of linearized plasmid DNAs containing reporter gene inserts. Standard curves were log-linear over a 2-2 x 10⁷ copy range. The positive control was a dilution of one LNCaP cell /10⁶ peripheral blood mononuclear cells; the negative control lacked cDNA template.

Q-PCR Sample and Patient Assessment
PSA copy number was normalized to 5 x 10⁶ GAPDH copies for the same RNA (PSA-N), based on preliminary studies evaluating assay sensitivity and signal-to-noise issues (Table 1). To accomplish this, 1 µL of the RT reaction (0.2 ug RNA-equivalent of cDNA) was used to determine GAPDH copies and then a volume containing 5 x 10⁶ GAPDH copies was used for PSA Q-PCR (279 patients). Reported PSA-N values were further based on the maximum-mean PSA copy number, the highest of the two means of triplicate Q-PCR determinations from the left- and right-sided LN RNAs. In 38 patients, the GAPDH copies varied from 10⁶ to 4.6 x 10⁶ or 5.3 x 10⁶ to 10⁷. In these samples, PSA-N was calculated by dividing the maximum-mean PSA copies by the GAPDH copies and multiplying by 5 x 10⁶. One of 24 patients with less than 10⁶ GAPDH, had 790 PSA copies, and was included in the analysis; 23 patients were excluded because of low sensitivity (six RP patients without hormone therapy, 17 patients with hormone therapy, which included one XRT patient and 16 RP patients).

Biochemical Failure Criteria
BCR was based on published guidelines: for RP patients, as the first date that the sPSA reached 0.2 ng/mL or higher, confirmed by a second determination^28,29; for XRT patients, from the first of three consecutive rises rather than the midpoint between the nadir and first rise.³⁰

Statistical Analysis
The choice of the 100 PSA-N threshold was based on the distribution of the PSA-N values assessed by univariate analysis. PSA-N 100 was the lower limit for the upper one sixth of the sample and the upper limit of the central two thirds reference interval values. For univariate analyses, known prognostic factors were grouped. There were three groups for baseline sPSA ( 10, > 10-20, > 20 ng/mL), and GS (< 7, 7, 8), and four groups for TS ( 2a, 2b, 2c-3a, b, 3c). Kaplan-Meier curves of biochemical failure-free survival (BFFS) were calculated for patients with PSA-N less than 100 and 100 or greater copies and compared by the log-rank test within each risk factor and within defined risk-group categories.⁴ The proportions of patients with PSA-N greater than 100 copies were compared within each risk-group by ² tests.

To evaluate the ability of PSA-N to predict time to BCR after adjusting for known prognostic factors, two separate Cox regression time-to-biochemical-failure analyses were performed. In the first analysis, the prognostic factors were grouped and treated as categorical variables, with PSA-N categorized as PSA-N less than 100 or PSA-N 100 or greater. The relative risk was defined as the proportional increase in BCR expected with a PSA-N 100 or greater compared with PSA-N less than 100, after adjusting for the other variables in the model. In the second analysis, the prognostic factors were treated as ordinal and continuous variables. The log of both sPSA and PSA-N were used. All statistical tests performed were two sided with significance level .05 using SAS statistical software version 9.1 (SAS Institute, Cary, NC).

	RESULTS

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Patient Characteristics
The 341 patients were predominantly white (95%), younger than 65 years of age (59%), with sPSA less than 10 ng/mL (54%; Table 2). Three quarters of patients had RP; one quarter of patients had XRT; 85% and 89%, respectively, achieved nadir sPSA 0.1 ng/mL or less. The treatment groups were balanced in the distribution of sPSA and TS. However, the proportion of patients with GS 6 or less was higher (39% v 17%) and with 8 or more was lower (22% v 39%) in the RP and XRT groups, respectively (P = .0002). Overall, 56.6% of patients had high-risk features: 44.5% in the high risk group and 12.1% in TS3c.

BFFS analysis with 4-years follow-up was performed on a subgroup of 297 patients for whom assessable Q-PCR and sufficient PSA follow-up data were available. Overall, BFFS was 77% in patients with PSA-N less than 100 versus 55% in patients with PSA-N 100 or more (P = .0002; Fig 1). When similarly analyzed by individual risk factors, statistically significant BFFS differences were greatest in patients with high risk features (Figs 2, 3, and 4): sPSA greater than 20 ng/mL, 60% versus 25% (P = .014); GS 8 or higher, 66% versus 21% (P = .0002); and TS3c disease, 64% versus 18% (P = .001). A significant association of PSA-N 100 or more was also noted with sPSA 10 to 20 ng/mL (P = .045).

View larger version (7K): [in this window] [in a new window]   Fig 1. Kaplan-Meier biochemical failure-free survival (BFFS; %) curves for patients with normalized prostate-specific antigen copies (PSA-N) less than 100 versus 100 or greater in pathologically N0 pelvic lymph nodes (N0-PLN). All assessable N0-PLN patients.

View larger version (7K): [in this window] [in a new window]   Fig 2. Kaplan-Meier biochemical failure-free survival (BFFS; %) curves for patients with normalized prostate-specific antigen copies (PSA-N) less than 100 versus 100 or greater in pathologically N0 pelvic lymph nodes (N0-PLN). Serum prostate-specific antigen categories: (A) less than 10; (B) 10 or higher to 20; and (C) 20.

View larger version (7K): [in this window] [in a new window]   Fig 3. Kaplan-Meier biochemical failure-free survival (BFSS; %) curves for patients with normalized prostate-specific antigen copies (PSA-N) less than 100 versus 100 or greater in pathologically N0 pelvic lymph nodes (N0-PLN). Gleason score categories: (A) less than 7; (B) 7; and (C) 8 or higher.

View larger version (13K): [in this window] [in a new window]   Fig 4. Kaplan-Meier biochemical failure-free survival (BFFS; %) curves for patients with normalized prostate-specific antigen copies (PSA-N) less than 100 versus 100 or greater in pathologically N0 pelvic lymph nodes (N0-PLN). Tumor stage: (A) 2a or less; (B) 2b; (C) 2c to 3a-b; and (D) 3c.

View this table: [in this window] [in a new window]   Table 4. Four-Year BFFS by PSA-N < 100 or 100 Copies in N0-PLN and Risk-Group Category

View this table: [in this window] [in a new window]   Table 5. Proportion of Patients With PSA-N 100 Copies by Risk Factor Category

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

These findings support our hypothesis that PSA-N could identify clinically significant occult micrometastases in N0-PLN not directly assessable by established risk factors. However, like established risk factors, the independent prognostic value of the PSA-N 100 or more criterion was enhanced in combination and revealed a subgroup of high risk-group patients who have an additional risk of BCR.^2-4 Early implementation of systemic therapy in these cases may increase the estimated 40% to 50% BFFS at 5 years.^13,31-35 Conversely, those patients at relatively lower risk of BCR may benefit from more intensive locoregional treatment. These findings suggest that PSA-N greater than 100 meets a crucial criterion for a useful new risk factor: the augmentation of predictive capacity of other established risk factors.³⁶

The importance of the quantitative relationship of PSA-N to its predictive value is further supported by our concurrent finding that a previously reported semi-quantitative conventional RT-PCR assay for sensitively detecting PSA mRNA in N0-PLN,¹⁶ using the same RNAs used in this study, was not informative about BCR risk (our unpublished results). A similar lack of quantitative precision likely limited the predictive value of previous studies using nonquantitative RT-PCR analysis.^18,37,38 Q-PCR provides quantitative accuracy due both to the methodology and to the normalization of each PSA copy value to a more constantly expressed housekeeping gene control (eg, GAPDH) for RNA integrity and amplification efficiency.²⁷ In addition, we performed Q-PCR using small amounts of high-quality RNA from fresh-frozen obturator and internal iliac LN samples. These considerations demonstrate that the technical aspects of determining PSA-N are practically accomplishable under quite standard clinical circumstances with higher sensitivity and less time commitment than previously applied immunohistochemistry.³⁸

We acknowledge that PSA-N 100 or more as a novel risk factor needs additional validation in additional prospective studies. BCR is not the optimal end point, because it can precede the development of metastases and death by many years.³⁹ Nevertheless, BCR is commonly accepted as the earliest sign of disease recurrence, and it continues to be utilized as the first step to assess the potential clinical relevance of new markers.⁴⁰ Surrogate end points that have been more tightly linked to metastasis and death after BCR include PSA doubling time, GS 8 or higher, and time from surgery to BCR,^9,39,41-44 as recently documented with follow-up of 10 years post-RP and 6 years post-BCR.⁴⁰ In our study, two of these end points could not be reliably used because neoadjuvant/adjuvant hormone therapy might have variably prolonged time to BCR and might have affected the first-order PSA kinetics required to calculate PSA doubling time.^41,45 Notably, we did observe a strong association between PSA-N 100 or more and GS 8 or higher supporting the possibility that PSA-N 100 or more in our cases will ultimately be linked to definitive disease progression with additional follow-up.

We found that the proportion of patients with high risk factors and PSA-N 100 or more was consistently higher than those with lower risk factors but this did not reach statistical significance (Table 5); this contrasts with the strong association of high risk factors with BCR. Also, in the smaller high risk-group category, the proportion of cases with PSA-N 100 or more was essentially the same as lower-risk group categories (see Results). These findings suggest that the initial dissemination of PC cells to PLN from the primary may not have greatly differed between risk groups. Additional study is required to understand these relationships, which may have involved several differences related to inherent PC cell biology in the primary tumor, the N0-PLN microenvironment, the host immune response to micrometastases, and the limited follow-up time.^46,47 In any event, these considerations do not detract from the proposed utility of PSA-N 100 or more in N0-PLN of high risk patients as a marker for disease recurrence based on our statistical analysis of postcausal clinical events.

Another consideration is that our patient population contained a disproportionate number of high risk patients (56.6%) compared with published series (5% to 35% high risk patients).^{13,31-33,39,48,49} This disproportion may actually have assisted with the evaluation of PSA-N 100 as a risk factor because it likely increased the number of BCR cases. Consistent with this interpretation, BFFS after 4-year follow-up was 72% compared with 78% to 82% with 5 years observation in large RP series containing smaller numbers of high risk patients.^13,33,50,51 A final consideration is the heterogeneity of therapy in our study group. Regarding RP versus XRT, some imbalances in individual risk factors were observed, however, there was no significant difference in BFFS (P = .34), which seems most relevant for the validity of assessing these treatment groups in common. Regarding hormone therapy, we determined that 3 months of neoadjuvant hormone therapy did not suppress PSA-N (see Results). Thus, it is improbable that these limited biases compromised assessment of the PSA-N criterion.

In conclusion, we have developed an assay that reveals and quantifies clinically relevant occult micrometastases in pathologically negative PLN at the time of primary PC therapy. In combination with established clinical risk factors, PSA-N 100 or more divides patients categorized as high risk into two further subgroups with significantly different relative risks of BCR. This certification and grading of metastases at the time of primary therapy in an already unfavorable risk setting provides a more evidence-based approach for assessing risk of subsequent BCR and for making clinical decisions regarding possible early initiation of additional regional and/or systemic therapy in order to inhibit disease progression.

	Authors' Disclosures of Potential Conflicts of Interest

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Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Kenneth J. Livak Applied Biosystems (N/R) Applied Biosystems (B)

Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C) $100,000 (N/R) Not Required

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 

Conception and design: Anna C. Ferrari, John Mandeli Financial support: Anna C. Ferrari, Robert E. Gallagher Administrative support: Anna C. Ferrari Provision of study materials or patients: Anna C. Ferrari, Nelson N. Stone, Ralf Kurek, Roy McGregor, Richard Stock, Ulf Tunn, Amir Kaisary, Michael Droller, Simon Hall, Heiner Renneberg, Kenneth J. Livak, Robert E. Gallagher Collection and assembly of data: Anna C. Ferrari, Nelson N. Stone, Ralf Kurek, Elizabeth Mulligan, Roy McGregor, Ulf Tunn, Amir Kaisary, Michael Droller, Simon Hall, John Mandeli Data analysis and interpretation: Anna C. Ferrari, Pamela Unger, Robert E. Gallagher, John Mandeli Manuscript writing: Anna C. Ferrari, Robert E. Gallagher, John Mandeli Final approval of manuscript: Anna C. Ferrari, Nelson N. Stone, Ralf Kurek, Elizabeth Mulligan, Roy McGregor, Richard Stock, Pamela Unger, Ulf Tunn, Amir Kaisary, Michael Droller, Simon Hall, Heiner Renneberg, Kenneth J. Livak, Robert E. Gallagher, John Mandeli Other: Elizabeth Mulligan [Performance of all experimental assays]

 

	ACKNOWLEDGMENTS

 
We thank James F. Holland, MD, Distinguished Professor of Neoplastic Diseases, the Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, for encouragement and generous support.

	NOTES

 
Supported by United States Public Health Service research Grants No. CA9813501 (A.C.F), CA86794 (R.E.G.), and the T.J. Martell Foundation for Cancer Leukemia and AIDS Research (A.C.F.).

Presented in part at the 40th Annual Meeting of American Society of Clinical Oncology, New Orleans, LA, June 5-8, 2004, and in an oral presentation at the American Society of Clinical Oncology, Prostate Cancer Symposium, Orlando, FL, February 17-19, 2005.

Authors' disclosures of potential con- flicts of interest and author contributions are found at the end of this article.

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Submitted July 25, 2005; accepted April 24, 2006.

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