In chemo-naive patients, surgical resection of the primary tumor resulted in significant decrease of CTCs (Table 2), with postoperative median numbers of 2 tCTCs/mL of blood (IQR 1 to 4, P < 0.001, Fig. 2B), 2 eCTCs/mL of blood (IQR 1 to 3, P < 0.001, Supplementary Figure 6A, http://links.lww.com/SLA/B453), and 0 mCTCs/mL of blood (IQR 0 to 1, P < 0.001, Supplementary Figure 6B, http://links.lww.com/SLA/B453). A similar significant CTCs population reduction was observed in neoadjuvant patients measuring postoperatively at 2 tCTCs/mL of blood (IQR 1 to 4, P < 0.001, Fig. 2C), 2 eCTCs/mL of blood (IQR 1 to 3, P < 0.001, Supplementary Figure 7A, http://links.lww.com/SLA/B453), and 0 mCTC/mL of blood (IQR 0 to 1, P = 0.023, Supplementary Figure 7B, http://links.lww.com/SLA/B453).
In addition, patients who were explored and found to have occult abdominal metastatic disease (OMD) intraoperatively had significantly higher CTCs numbers than patients who underwent surgical resection (Table 2): 20 tCTCs/mL of blood (IQR 12 to 24, P < 0.001, Supplementary Figure 8A, http://links.lww.com/SLA/B453), 15 eCTCs/mL of blood (IQR 9 to 18, P < 0.001, Supplementary Figure 8B, http://links.lww.com/SLA/B453), and 3 mCTC/mL of blood (IQR 2 to 6, P < 0.001, Supplementary Figure 8C, http://links.lww.com/SLA/B453). In patients with OMD and patients whose tumor was not resected due to local extension and major vessel involvement, no significant difference between pre- and post-exploration CTCs was identified (tCTCs P = 0.16, eCTCs P = 0.09, mCTCs P = 0.10).
Fifty-nine patients (44%) presented with disease recurrence within 1 year from surgery. Multiple-site metastases was the most common pattern (n = 23, 39%), followed by local recurrence (n = 15, 25%) and liver metastases (n = 11, 19%). Preoperative CTCs were significantly higher in the recurrence cohort (tCTCs P < 0.001, eCTCs P = 0.002, mCTCs P = 0.005; Table 3). The difference was also significant in the postoperative CTCs populations. In order to clearly identify the proportion of epithelial/mesenchymal phenotype cells in the samples, we calculated an mCTCs-to-eCTCs ratio
: it was significantly higher in the preoperative samples of patients who presented with early recurrence (median 0.23, IQR 0.14 to 0.33) than in patients who did not recur within a year (median 0.10, IQR 0 to 0.22, P = 0.008).
We further sought to identify preoperative factors, including demographic and clinical parameters combined with CTCs dynamics, which will allow identification of aggressive disease biology and increased probability of early recurrence. To increase the accuracy of the predictive model, we performed a distinct univariable and multivariable logistic regression analysis for chemo-naive and neoadjuvant patients (Table 4). In addition, the multivariable analysis was performed separately for each of 2 mutually exclusive CTCs populations (eCTCs and mCTCs) and the total number of CTCs (tCTCs). On univariate analysis for the chemo-naive cohort, CA19-9, which is the primary clinical biomarker for PDAC, was not predictive of 1-year recurrence at an optimal cut-off value of 178 U/mL [OR 1.18 (95% CI 0.86–1.62), P = 0.30]. On the contrary, all 3 different CTCs populations were independently associated with an increased risk of early-recurrence in their separate multivariable models: mCTCs had the highest predictive ability with an area under the curve (AUC) of 0.69, 52% sensitivity, and 82% specificity (Supplementary Figure 9, http://links.lww.com/SLA/B453). Similarly, in patients who underwent neoadjuvant therapy, all 3 CTCs populations were the only independent parameters associated with early recurrence: in this cohort, the tCTCs predictive model had the highest accuracy with an AUC of 0.75, 72% sensitivity, and 75% specificity.
In the studied cohort, mortality within 1 year from surgery was 31% (n = 51). In chemo-naive patients, preoperative or post-operative eCTCs and mCTCs numbers were not associated with 1-year disease-specific mortality (P = 0.65, P = 0.89 and P = 0.33, P = 0.43, respectively). However, in the neoadjuvant cohort, the predictive role of CTCs was present: postoperative numbers (POD 4 to 7) of all 3 populations (tCTCs ≥4/mL, eCTCs ≥3/mL, and mCTCs ≥1/mL of blood) were associated with early death from PDAC (P = 0.006, P = 0.009, and P = 0.018, respectively - Supplementary Figure 10, http://links.lww.com/SLA/B453).
ratio was 0.23 (IQR 0.14 to 0.33), whereas at the time of recurrence, the median ratio value was 0.42 (IQR 0.29 to 0.57, P < 0.001). However, the
ratio was not specific enough to distinguish patients who presented with local versus distant metastases (P = 0.303).
The utilization of CTCs as a “liquid biopsy” to assess the tumor burden and guide clinical decisions has developed into an emerging field of study.26 In pancreatic cancer patients, CTCs have been previously introduced as possible biomarkers for recurrence and survival.18,27 However, biological hurdles, such as extensive cell heterogeneity and physical challenges, associated with cell kinetics within circulation limit that potential.28,29 In this prospective study, we attempted to characterize the physical attributes and phenotypes of CTCs in PDAC patients, longitudinally assess their dynamics along the course of the disease, and develop a comprehensive model for their potential clinical significance. The two main pillars of our study were a 2-step isolation technique (size-based positive selection and immunofluorescent biomarker staining) and the stratification of identified cells into an epithelial and epithelial/mesenchymal phenotype.
In the studied cohort, more than 95% of resectable PDAC patients had at least 1 identified CTC/mL of peripheral blood, whereas the median number of total CTCs across all preoperative samples was 11/mL of blood. Experimental models have calculated the cell dissemination rate from a tumor to vary between 3 and 4 × 106 per g/day,30 yet most are passively shed and less than 0.01% have metastatic potential.27 On the basis of our observations, the median number of CTCs in the peripheral circulation of a patient with resectable PDAC can be estimated at approximately 50,000 at any given time. In addition, most of the CTCs were isolated as single cells with a median size comparable to that of WBCs. Previous studies have also identified the presence of CTCs clusters or microemboli in patients with PDAC.18 We observed small clusters of <5 cells in 1% of the peripheral blood samples. In PDAC, disseminated cells travel through the portal vein into the liver, and subsequently enter the right side and later the peripheral circulation. Even though CTCs clusters are able to pass through capillary-sized vessels,31 it is unlikely that they make it to the periphery, due to extreme hydrodynamic flow forces and shear stress.32 The occurrence of EMT and the loss of epithelial marker expression, such as E-cadherin, which sustains cell adhesions may have an additional negative effect. However, it is possible that large cohesive cell cohorts disseminating from PDAC become trapped in the hepatic lobule network and may eventually give rise to liver metastases, according to the mechanism of collective migration.33 Identification of CTCs clusters in the portal vein can support this hypothesis and provide an explanation for the increased incidence and aggressiveness of liver metastatic disease in PDAC.34,35
Most identified CTCs expressed exclusively an epithelial phenotype and most likely were the result of tumor apoptosis in the circulation. A variable number of CTCs expressing a mixed epithelial/mesenchymal phenotype (mCTCs) was also present in preoperative samples, yet in a smaller percentage (n = 94, 70%). On the contrary, CTCs with purely mesenchymal traits were not identified in any of the patients. These findings concede with the recently introduced “partial EMT” concept, where CTCs acquire mesenchymal traits, while retaining specific epithelial characteristics.13,36 This mixed phenotype allows a cellular plasticity that appears to be critical in the establishment of metastatic colonies.37,38 An ongoing study for genetic characterization of CTCs from our group will provide further insight on the genetic link between the primary tumor and metastatic lesions. KRAS mutations have been previously identified in PDAC CTCs verifying their malignant origin39,40; however, comprehensive DNA sequencing on a single cell level continues to be a challenging task.41
ratio: patients who recurred early had significantly higher ratio than those who did not (P = 0.008). Moreover, patients who were found to have occult metastases also had significantly higher CTCs and
ratio at the time of recurrence was significantly higher than the preoperative sample, suggesting a more aggressive biology, likely associated with treatment resistance and clonal drift.46 In current clinical practice, disease surveillance is performed with repeated imaging and CA19-9 assessment. A limited number of studies have evaluated CA19-9 as a longitudinal follow-up biomarker. CA19-9 appears to have a mediocre sensitivity and specificity and is not useful for patients who do not express the Lewis antigen.4 Our developed CTCs model can identify recurrence with an accuracy of 80%, more than 2 months earlier from imaging evidence of disease. However, specificity was not high enough to predict the site of recurrence (local vs distant).
Scientific interest on the importance of circulating biomarkers has erupted in recent years, primarily regarding the role of circulating tumor DNA (ctDNA). Recent studies have shown the significant potential of ctDNA on early cancer detection and follow-up of disease.6,47 However, ctDNA represents collectively all indolent or aggressive clones shedding in the circulation from CTCs, the primary tumor, or metastases. On the contrary, each CTC represents a unique clone and may provide direct insight on the clonal prevalence in the metastatic mechanism. Data from this study suggest that significant potential for CTCs as a PDAC biomarker is present. However, as cell kinetics and survival are significantly affected within the circulation, the only way to overcome these obstacles is to develop optimal isolation and enrichment assays.48 Longitudinal assessment of ctDNA in PDAC is currently underway in our institution and we will soon be able to make a direct comparison between the 2 biomarkers or combine them for increased sensitivity and specificity.
The authors would like to acknowledge Lindsay Manos, Lara Espin, Christi Walsh, Caitlin Brown, and Tiffany Zavadsky for their help in patient follow-up, Hao Zhang for his valuable assistance in flow cytometry, and Maritina Iliadi for figures layout and editing.
Dr Nipun B. Merchant (Miami, FL):
I want to congratulate Dr Wolfgang for presenting another very provocative study advancing the field of pancreas cancer as the group from Johns Hopkins continues to do.
In reviewing this manuscript, perhaps one of the most important findings in their study cohort, unrelated to this study, is that 44% of patients had recurrence of disease within 1 year of surgery, and that, too, with the majority of patients having recurrence at multiple sites, and 31% of patients died within 1 year of surgery. This clearly emphasizes the fact that we still have not gotten a handle on this disease and further underscores the need for biomarkers of early detection as well as therapeutic response.
A rate-limiting step for biomarker development in pancreas cancer is a limited and difficult access to tissue, particularly for longitudinal analysis, highlighting the need for liquid biopsy based biomarker development.
Dr Wolfgang and colleagues have presented a prospective, longitudinal study showing that 2 major CTC subtypes, epithelial and mesenchymal, can be identified in all pancreas cancer patients and that surgical resection of the primary tumor results in significant reduction of CTC burden. Patients who presented with early disease recurrence in less than 12 months had significantly higher preoperative as well as postoperative CTC numbers, and patients who received neoadjuvant chemotherapy had significantly lower CTCs than untreated surgical resectable patients suggesting that these cells have a potential to be used as a biomarker of therapeutic response.
In their multivariable analysis, pre-op numbers of all CTC subpopulations were the only predictors of early recurrence within 12 months of surgery, and a risk assessment score accurately identified the disease recurrence 2 months earlier than detected on imaging.
This is an excellent paper, and I think the way we need to move forward to make progress in pancreas outcomes. However, it also generates several comments and questions. And based on this, I have some logistical, methodological, and clinical relevance questions.
Surgical resection of the primary tumor resulted in significant reduction but not disappearance of CTC burden. Do you think this could be related to the timing of your first specimen collection 4 to 6 days postoperatively?
Do CTCs ever disappear? Is persistence of CTCs over time alone a risk factor of recurrence as versus increasing number?
You did not see a statistical correlation between the number of CTCs and tumor stage, size, location, and differentiation, yet pre- and post-op CTCs and mesenchymal to epithelial CTC ratio were also significantly higher in the early recurrence cohort. Can you comment on this?
In treatment-naive patients, pre- or post-op CTC numbers were not associated with 1-year disease-specific survival even though they decreased between pre- and post-op levels. Yet, in those who received neoadjuvant therapy, the predictive role of CTCs was present. What do you attribute this difference to?
You emphasize CTCs mesenchymal phenotype, those that undergo EMT, thereby having an increased risk of metastatic potential. Both post-op samples and treatment-naive and neoadjuvant-treated patients decreased to no mesenchymal CTCs in many patients, yet before recurrence, the mesenchymal-to-epithelial CTC ratio increased. How does this occur, and where do these cells come from?
I am also intrigued by the fact that there was no difference seen in the mesenchymal-to-epithelial ratio from local versus metastatic recurrence. As mesenchymal CTCs would have a higher likelihood of hematogenous spread, one would think that you will have a higher likelihood of detection in the blood in metastatic patients.
You show that CTCs that you isolate appear to be tumor derived as you have sequenced KRAS, TP53, and SMAD4 from them. What are the advantages of isolating CTCs compared with other emerging biomarkers such as circulating tumor DNA? Also, how can you determine that there are no false-positive cells or artifacts?
Lastly, what are some of the biological hurdles such as extensive cell heterogeneity and physical challenges associated with the kinetics within the circulation that have to be overcome before we can see CTCs as a robust biomarker in pancreas cancer?
I want to congratulate you and your colleagues for continuing to push the field forward in pancreas cancer, and I would like to thank the Association for the privilege of the floor.
Response From Christopher L. Wolfgang:
Thank you, Dr Merchant, for the nice discussion and insightful questions. It is an honor to have you discuss our paper. I will summarize and put some of these questions together.
The first question was regarding the fact that in some cases the CTCs do not fall to zero following surgical resection.
The findings of residual CTCs on post-op day 4 to 6 was not surprising. Initially, we thought that maybe this was the result of residual cells that would clear over time. On the basis of animal models, CTCs have a half life of approximately, at most, 1 to 2 hours, and in some instances measured even lower. So, based on this work, if you had a surgical resection, you would you expect that within post-op day 4 or 7 that you would see CTCs go to 0, and we did not see that. I was very surprised to see that over time, as shown in the manuscript, that a given patient settles out to a new baseline and remains there, even in the absence of recurrence. This baseline may be 0, but in many cases, it is not. This is exciting because I believe we are looking directly at the dormant subclinical disease that can relapse in the future. In the patients who made it to 1 year without recurrence, some have as many as 5 CTCs at baseline. In a few years, we will be able to answer your question of whether or not such patients are at risk of late recurrences as our cohort matures. It brings up another interesting question of where is the reservoir of this subclinical disease? One potential site is the bone marrow and liver, which we are planning on evaluating in a subsequent study.
What I think is interesting about this study is that we are looking at something we have never seen before. I think this is subclinical or dormant disease that we never had an opportunity to evaluate before this work. What will be interesting is what happens in the population that did not recur within 1 year, and the ones that we see the residual CTCs. Potentially, we know that these patients are still at risk of recurring. Will they have a resurgence in those cells?... and will those cells be the ones that cause the metastatic disease? These questions will be something that we will be able to determine as this study progresses.
The next question related to the lack of a statistical correlation with the pathological features and the number of CTCs. In the beginning, I was a bit troubled by this. We know that there are pathological features that predict relatively aggressive behavior, but if you think about it, it is imprecise, and there are factors that we do not know that predict. For example, we have all had node-positive, margin-positive patients who lived a long time and margin-negative patients who died within a year. And what I think that we are seeing here is that the CTCs are a very sensitive window into the tumor biology, and maybe are completely independent of those pathological features, suggesting that this may be a more direct assessment of tumor biology. We will have to see how that plays out over time.
There was a question then about the lack of correlation with survival in the chemo-naive patients, whereas there was a correlation in the neoadjuvant patients, and that there was correlation, however, with both recurrences for both neoadjuvant and adjuvant.
This is a very interesting observation. CTCs correlated with recurrence at 1 year but not survival for chemo-naive patients. The mathematic explanation is that they are still alive, but with disease.
The biological explanation is less clear but much more interesting. One possible explanation, which is highly unsettling, is that upfront chemo subjected the systemic disease to a natural selection process and simply caused clonal selection for more aggressive clones. That does not mean neoadjuvant is worse treatment … we just selected for aggressive clones before surgery as opposed to after surgery. In other words, choose your poison … select for the bad clones before surgery or after surgery. It is a little disheartening. Now this is just a theory, but as time goes on, we will evaluate this question in this cohort. The bright spot in this potential explanation is now that we can directly assess these clones (with CTCs analysis), we may be able to mitigate the clonal selection with new cocktails of existing drugs. Such as one would do for drug-resistant bacteria with treatment based on culture data.
The next question was regarding the fact that there was no difference seen in the mesenchymal to epithelial ratio from local versus metastatic recurrence.
Good question and I do not know the answer. However, based on published work and work we are doing, my bias is that even so-called local recurrence is systemic. That is … it is systemic disease that comes back through circulation or perhaps perineural invasion and grows aggressively in the already primed stroma of the tumor bed.
The next question was basically the differences among the various forms of liquid biopsy and why we chose CTCs.
ctDNA represents an average of everything that is going on throughout the entire body with respect to the cancer, that is, it measures collectively the indolent clones, aggressive clones, and even perhaps is weighted toward what is dead. In contrast, each CTC represents a unique clone, it is not an average. Moreover, it is most likely a direct assessment of what causes metastases, not indirect like ctDNA.
That said, we have a ctDNA arm of this study not part of this manuscript. We will be able to make a direct comparison or perhaps be able to make a composite marker as future studies from this trial.
Finally, the biological hurdles and challenges…in particular tumor heterogeneity.
Yes…the heterogeneity poses logistical (and cost in assessment) problems, but with careful assessment and data management these can be overcome. The heterogeneity of what we see is exciting because it mirrors what we see clinically with the biological behavior. The heterogeneity in CTCs tell me we have the necessary data points to measure the diverse biologies of patients’ tumors. I think we will see that at the end of the day we do not need to eradicate all clones, just the bad one. Perhaps not even eradicate it but develop a vaccine toward that clone that converts the disease into a chronic condition.
The other “physical challenge” is what method of isolation to use. They all have their weakness and strengths. We used ISET because we did not want to bias ourselves by a priori antibody-based selection. However, we compromised by biasing ourselves to CTCs greater than 8 microns in size. I believe this calculated decision may have paid off because I am not sure we would have identified the mCTC otherwise. We have ongoing work using a new flowcell device developed in the applied physics laboratory at Johns Hopkins and we also use flow cytometry at times to test certain aspects of CTCs.
Dr Keith D. Lillemoe (Boston, MA):
Chris, great paper, really advancing this field and probably all of oncology.
Based on your data, it would seem that using this type of data for clinical decision making, must be getting close. In your patient example, could you have started chemotherapy earlier and maybe altered the outcome in terms of survival? Have you started using these data to alter your management of the patients?
Second of all, the one finding that probably affects surgeons most, is the dramatic data, that early recurrence could be predicted by CTCs. Will it ever get to the point where people will be turned down for surgery because of a high level of preoperative CTCs either before or after neoadjuvant therapy?
Response From Christopher L. Wolfgang:
Thanks, Dr Lillemoe. Very insightful. Are we altering therapy in response to the CTCs? Hopefully, one day we will be doing that, but not at this point.
What we envision for the next trial is something pretty simple but in the vein of precision medicine. That is to simply switch from FOLFIRINOX to Gem-based chemo or vice versa upon an increase in CTCs but no clinical evidence or relapse or progression.
Regarding will we use this to exclude patients from surgery, I do not think we should use these results to make that decision. I think it is being able to alter neoadjuvant approaches, targeted approaches, with surgical resection to get the best outcome based on looking at the circulating disease, because right now we do not. We focus on the local disease and we fail systemically. Now, we have a tool to look at the systemic disease and use that in patients who go to surgical resection.
Dr Selwyn Vickers (Birmingham, AL):
Chris, really a nice paper and great presentation. Two quick questions. As you know, there are data being very clear now that organoids may be a really powerful predictor of response. Can you grow organoids from these CTCs in order to predict disease response to therapy?
Second, has there been an inherent value, as you can now isolate these cells versus their surface markers to doing single-cell exome sequencing to determine the further correlation with biology?
Response From Christopher L. Wolfgang:
Great question. The organoid question first. We actually have an organoid effort and group in our laboratory. One of our surgeons went to train with Dave Tuveson for exactly this reason. Right now, we are able to establish organoids from primary tumors, but after 3 years of trying, it has become exceedingly difficult to create tissue culture from CTCs, and it probably means that only a small percentage of circulating cells are capable of originating metastases. If you do a calculation you can estimate that at any one time in an individual, there are probably about 50,000 circulating cells. The probability of drawing 10 circulating cells of blood and getting that cell that can create metastases and then having the right conditions is pretty low. But, ultimately, that would be the perfect goal, so we are attempting to do that.
Probably the thing to focus on are CTC clusters, which I had not talked about here, which are probably the most aggressive clones.
Yes, we are planning on doing molecular analysis on these cells such as RNA seq.
Dr Andrew Lowy (La Jolla, CA):
It strikes me that this mesenchymal population is what others would probably call pancreatic cancer stem cells, which have been characterized by a number of groups. So, I wonder if you have looked at co-expression of markers specifically for stem cell populations that have been shown to be enriched in proteins such as CXCR4, Musashi, and second, whether you would apply single-cell sequencing to your CTCs to try to look for potential novel targets, particularly for these presumably highly drug-resistant cells.
Response From Christopher L. Wolfgang:
Excellent question. In our previous work on these mesenchymal cells, they do have tumor-initiating markers such as aldehyde dehydrogenase, CD133, CD44, and ALDH - so they do overlap with what would be defined as tumor-initiating cells.
Just real quickly, because it was asked before by Dr Vickers on the single cell sequencing, we are doing that, but, as you know, right now, maybe only 10% or 15% of the mutations are targetable. So, it is important to do. We have the ability to do it. But I think there is only a minimal impact in how we change therapy. RNA, we are working on those data now.
Dr Pierre-Alain Clavien (Zurich, Switzerland):
Chris, congratulations for this great work. I have only one short question, which relates to an ethical issue. Through the search for CTC dynamics, you are extracting information about the disease and putative poor natural history in patients who are doing well. Did you inform the patient about the CTC findings and possible poor prognosis? And if so, what was the recommendation given to them?
Response From Christopher L. Wolfgang:
That is a great question. We have actually talked about this at the onset of the study, if we were to find something clinically important, what would we tell patients? We inform patients that all of the data that we generate are available to them, and we can talk to them about it.
As one of the clinicians caring for these patients, I am blinded to the data, and we have a system in the laboratory that the people collecting the clinical data are separated from the CTC analysis, whereas the ones doing the analysis are blinded from the clinical data, so that we actually have an unbiased study.
When people request their CTC results and the clinician will be unblinded and review it with them. We inform them that we do not know what it means right now. It does not alter the clinical management.
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