A near-universal principle of cancer staging recognizes the established relationship between regional lymph node metastases and prognostic risk.4, 23
In colon and rectal cancer, lymph node metastasis is the single most important prognostic characteristic, representing pathologic evidence of dissemination of tumor cells beyond their primary location. Clinically, approximately 50% of stage III patients will suffer disease recurrence.1, 2, 4, 9, 23, 25–27
Because up to 25% of patients without histological evidence of nodal involvement also suffer recurrent disease, it is presumed that many such patients harbor occult metastases not identified at the time of primary resection.1, 2
Under-staging by conventional methods reflects the combination of insufficient numbers of nodes for review, the analysis of only small volumes of individual lymph node tissue missing metastatic tumor cells28
, and the sensitivity of histopathology, which reliably detects only 1 cancer cell in 200 normal cells29
. Molecular staging could overcome limitations in the detection of occult lymph node metastases by incorporating all available tissue into analyses, and increasing detection sensitivity through quantifiable disease-specific molecular markers1, 10
which nominally identify a single cancer cell in 1 million normal cells30
In this study, prospective detection of occult metastases by GUCY2C qRT-PCR appeared to be an independent prognostic marker of risk. Molecular staging revealed that about 13% of pN0 patients were free of tumor cells, while about 87% had GUCY2C results that suggested occult metastases. Even in the context of shorter follow-up, which could introduce a bias against the utility of GUCY2C in this setting, pN0(mol+) patients exhibited a significantly greater risk of earlier disease recurrence and reduced disease-free survival, the primary and secondary outcomes of the study, compared to pN0(mol−) patients. While enrollment was sufficient to satisfy requirements for these outcomes, confidence intervals around estimates in multivariable analyses were broad. Future studies with greater numbers of patients should provide more precise estimates of the prognostic utility of GUCY2C qRT-PCR.
Although a high proportion of pN0 patients exhibit GUCY2C expression, indicating occult metastases, most pN0 patients will not recur.3, 23
Similarly, not all stage III patients who have histopathologically-detectable lymph node metastases ultimately develop recurrent disease.3, 23
Reconciliation of this apparent inconsistency relies on the recognition that nodal metastases, regardless of methods used to detect them, do not assure recurrence but, rather, indicate risk. In support of this concept, our study suggests recurrence rates for pN0(mol+) patients with occult metastases that are nearly identical to those for stage III pN1 patients3
, the lowest stage in which all patients have histopathologically-detectable metastases (see , Supplemental Fig. 4
There is a well-established relationship between burden of disease, quantified as the number of lymph nodes harboring tumor cells by histopathology, and prognostic risk in colorectal cancer patients. Assuming there are adequate numbers of nodes to review 3, 23, 25, 26
, stage III patients with ≥4 involved lymph nodes exhibit a recurrence rate that is approximately 50–100% greater than those with ≤3 involved nodes.3, 23
As in histology-based analyses, one limitation of the present study is the variable number of lymph nodes available for molecular staging from individual patients, reflecting the requirement for fresh dissection of surgical specimens. Additionally, lymph nodes <5 mm were excluded from molecular analyses, reflecting size limits for tissue bisection, although they are a particularly rich source of tumor metastases.31, 32
These considerations suggest that the precision of staging by molecular analyses will benefit from optimum lymph node sampling to incorporate tumor burden into prognostic risk stratification.1, 2, 27
An analysis of the subset of pN0 patients providing ≥12 lymph nodes for GUCY2C qRT-PCR, applying standard AJCC definitions for pN1 and pN23, 23
, revealed that those with 0–3 involved nodes exhibited a prognostic risk similar to pN0(mol−) patients (5.9% v 8.3%, respectively; Supplemental Fig. 6
). Conversely, those with ≥4 involved nodes exhibited a risk (≤3 versus ≥4, p=0.027) identical to patients with stage III pN1 disease (Supplemental Fig. 3
). Improved prognostic risk stratification by integrating detection of occult metastases and estimates of tumor burden underscores the essential importance of adequate lymph node sampling for optimum molecular1, 2, 27
, as well as histopathological3, 23, 25, 26
, staging of patients with colorectal cancer.
Beyond the number of involved lymph nodes, there is an evolving relationship between the volume of cancer cells in individual nodes, disease burden, and prognostic risk.3, 28
While metastatic foci ≥0.2 mm are associated with increased disease recurrence, the relationship between individual tumor cells or nests smaller than 0.2 mm and prognostic risk remains undefined.3
The emergence of qRT-PCR provides an unprecedented opportunity for cancer cell enumeration in tissues. However, the superior sensitivity of RT-PCR30
, with its optimum tissue sampling and capacity for single cell discrimination, could identify occult cancer cells in lymph nodes below the threshold of prognostic risk3
, limiting the specificity of molecular staging. In that context, the current study was not designed to identify the quantitative threshold defining risk. Indeed, one limitation of this study was the requirement to define a priori
the diagnostic limit of GUCY2C. In future studies, it will be essential to more precisely define the quantitative relationship between marker expression and disease risk that incorporates tumor burden to optimize prognostic sensitivity and specificity.
The presence of tumor cells in regional lymph nodes also directs therapy in patients with colon cancer. While adjuvant chemotherapy provides a survival benefit to patients with stage III disease, its utility in patients with pN0 colon cancer remains uncertain, with marginal survival benefits in stage II patients in some, but not all, clinical trials.3, 5–9, 23, 33, 34
This uncertainty of treatment benefit in stage II patients is echoed in the dynamic evolution of treatment guidelines, in which adjuvant therapy has become discretionary in stage II patients with clinicopathologic features of poor prognostic risk, including T4 stage, intestinal obstruction, and intestinal perforation.9, 33, 35, 36
It is tempting to speculate that heterogeneous responses to therapy in pN0 patients reflect, in part, heterogeneity with respect to occult nodal metastases. Moreover, standard of care includes adjuvant chemotherapy for stage III pN1 patients, a cohort with a recurrence rate identical to pN0(mol+) patients (see , Supplemental Fig. 4
). These considerations highlight the importance of advancing beyond the present study to refine the prognostic specificity of molecular staging employing GUCY2C qRT-PCR to more precisely stratify risk in pN0 patients and better inform the use of adjuvant chemotherapy.
Molecular staging represents one component of a comprehensive diagnostic, prognostic and predictive paradigm to personalize management strategies for individual patients.37, 38
It provides adjunctive clinicopathological information that supplements, but does not replace, complimentary anatomical, microscopic, and morphological staging modalities. Beyond enhancing these current approaches, molecular staging offers a unique opportunity to prioritize future complex resource-intensive analyses of primary tumors that will optimize patient management. In that context, analyses of primary tumors to define mutations, gene expression and epigenetic profiles, and proteomic signatures to stratify risk, predict responses to chemotherapy, and personalize interventions, may best be applied to pN0(mol+), rather than pN0(mol−), patients.39–43
These considerations underscore the present and future importance of integrating molecular approaches incorporating specific markers of disease, like GUCY2C, and powerful detection methods like qRT-PCR, into analytical paradigms directing the management of patients with colorectal cancer.