Predictive Value of Circulating Tumor DNA (ctDNA) and Neutrophil To Lymphocyte Ratio (NLR) in Patients with Colon and Gastric Cancer: Case Reports

1. Introduction

In 2019, the World Health Organization (WHO) published global statistics indicating that cancer is one of the leading cause of death in 112 countries (1). These data highlight the growing prevalence of cancer as a significant challenge in the medical field, with expert projections suggesting an increase in this burden over the next two decades (2). Some studies have even postulated that cancer will soon emerge as the leading cause of mortality in all countries across the twenty-first century (1). Cancers of the gastrointestinal tract represent a significant global health burden, accounting for approximately one-quarter of all tumors and more than one-third of cancer-related deaths (1).

An accurate prognosis is fundamental to the management of an oncologic patient, as it forms the basis for decision-making. A range of clinical and paraclinical tools can be employed to ascertain a prognosis (3). It is established that in cases where cancer has advanced, TNM staging is not sufficient for an accurate prognosis. Consequently, other complementary methods has been identified as a valuable addition to the existing approach. Examples include the assessment of patient performance status and the analysis of biomarkers (4,5). The advent of targeted biomarker determinations has brought with it a great deal of hope in the context of the rise of precise medicine (6,7,8).

The neutrophil-to-lymphocyte ratio (NLR) is a quantitative measure of the ratio of neutrophils to lymphocytes in the peripheral blood. It is a valuable tool for the assessment of the inflammation, as it conjugates two distinct aspects: the innate immune response, which is primarily mediated by neutrophils, and the adaptive immune system, which is orchestrated by lymphocytes (9). The utility of this ratio, which is, in fact, an effective and cost-efficient biomarker, has been extensively investigated by scientific researchers, with findings indicating its potential applications in a range of pathological conditions, including autoimmune, cardiovascular, and oncological disorders (10). In oncology, the NLR has been identified as a significant biomarker, playing a dual role in both prognostication and prediction (9).

Tumor-circulating DNA (ctDNA) is a subset of cell-free DNA (cfDNA) present in the blood of cancer patients. ctDNA is released by tumor cells, either directly or following apoptosis or necrosis (11). The somatic genomic alterations identified in these fragments are identical to those observed in the patient’s tumor (12).

The principal advantage of ctDNA testing is that it offers a real-time representation of the tumor burden and its genomic profile, given that ctDNA has a relatively short half-life (13). The determination of this biomarker is conducted through liquid biopsy, an approach that is non-invasive. This biomarker offers high specificity and sensitivity, which contribute to the accuracy of the test results. ctDNA can be detected not only in blood samples, but also in urine, cerebrospinal fluid, ascites fluid, and other biological matrices. The information obtained from this test is extensive and includes details about mutations, methylation patterns, and microsatellite instability (14).

The utility of this biomarker is applicable to all stages of the disease, including the early and metastatic stages. Its assessment has demonstrated its utility in key areas, including early screening, diagnosis, molecular typing, prognosis, detection of minimal residual disease after curative-intent therapy and recurrence monitoring. Furthermore, this information is beneficial when selecting an appropriate course of cancer treatment (12) (14) (15).

The role of ctDNA has been established as useful in the treatment of specific solid tumors, such as non-small cell lung cancer, breast cancer, head and neck cancers, malignant melanoma, and colorectal cancer (8). The clinical utility of ctDNA as a biomarker for gastric cancer is not as robust as it is for other malignancies (16).

We report two clinical cases to assess if there is a correlation between the clinical course of patients and the results of NLR and ctDNA testing. The study included two patients with a confirmed diagnosis of colorectal or gastric cancer undergoing treatment, with dynamic NLR and ctDNA determinations.

2. Case series

The patients reported in this article were treated at our institution (Fundeni Clinical Institute, Bucharest). CtDNA testing was performed using the platform Signatera. This platform employs whole exome sequencing using multiplex PCR amplification to identify somatic variants and clonal variations, generating a test that 16 tumor-specific variants (17). Regarding NLR, a value of >3.0 was considered pathological.

Case 1

We report the case of a 53-year-old man who was diagnosed with a descending colonic tumor in January 2023. The tumor was identified during routine colonoscopy at 50 cm from the anal external orifice. The histopathology examination established the diagnosis of colonic adenocarcinoma (G2). Preoperative imaging revealed no evidence of distant metastasis. In January 2023, the patient underwent left hemicolectomy with mechanical anastomosis. Examination of the hemicolectomy specimen confirmed the following histopathology findings: pT3, pN2b, M0; LVI+, PNI+, TBS+, Td+. The immunohistochemical examination demonstrated proficiency in MMR (MLH1+, MSH2+, MSH6+, PMS2+).

The patient received adjuvant treatment with mFOLFOX6 regimen (12 cycles). After the completion of adjuvant therapy, the patient underwent the first ctDNA testing, which yielded a positive result with a value of 2.02 MTM/ml, while the NLR was 1.9, a value that is considered within the normal range. During the follow up period, the patient underwent a second ctDNA testing and the result remained positive, but the value decreased to 0.59 MTM/ml, while the NLR was 3.2.

In February 2024, imaging revealed the presence of two liver metastases, identified in segments IVb and VIII, indicating disease progression. This finding coincided with the third determination of the ctDNA value, which showed the highest value thus far (5.51 MLM/ml). Notably, the NLR exhibited a normal value of 1.8. The case was reviewed by a multidisciplinary team and surgical intervention was recommended. In March 2024, an atypical hepatectomy for segments IVb and VIII was successfully completed. At this point, the ctDNA assay demonstrated the lowest recorded value to date (0.19 MTM/ml), while the NLR demonstrated no fluctuation, remaining at 1.8.

In August 2024, imaging revealed a new liver metastasis in segment VI. The progression of the disease was also noted by one of the biomarkers, with ctDNA increasing to a value of 0.75 MTM/ml. NLR demonstrated an increase, reaching a value of 2.3. However, this increase did not reach the established cut-off value. This prompted the recommendation of stereotactic body radiotherapy (SBRT) as a potential course of treatment. In September 2024, the patient received external stereotactic radiotherapy for the liver metastasis.

During the evaluation of the patient, a total of five ctDNA and NLR determinations have been conducted. The correlations between ctDNA and NLR results and the clinical evolution of the patients are depicted in Figure 1 .

Figure 1. Correlations between ctDNA values, NLR and the clinical evolution of the patient with colon cancer

Case 2

We report the case of a 60-year-old male who was diagnosed in December of 2023 with gastric cancer after undergoing an upper digestive endoscopy with biopsy sampling. The subsequent histopathology report indicates a diffuse, poorly differentiated (G3) gastric adenocarcinoma with the cell component exhibiting the characteristics of “in signet ring”. Additional testing revealed a HER2 negative (score 0), Ki67 80%, and stable microsatellite status (MSS) tumor. Pretherapeutic assessment indicated stage III disease – cT4N1M0. Therefore, the recommended course of action was the FLOT regimen, comprising of four preoperative administrations, re-evaluation, and surgical intervention dependent upon the patient’s response to the neoadjuvant chemotherapy.

After the administration of neoadjuvant treatment, the case was discussed in a multidisciplinary team and surgical intervention was recommended. In May 2024, the surgical procedure was performed, consisting of total gastrectomy with esophagojejunostomy and D2 lymphadenectomy. The initial testing of the biomarkers was conducted two weeks following the surgical procedure and yielded a positive ctDNA rest result (0.45 MTM/ml), and the NLR was also identified as pathological, exhibiting a ratio value of 3.1.

After surgery, the decision was made to complete the therapy with four additional cycles of FLOT. The second ctDNA test result was still positive, but with a decreasing value (0.13 MTM/ml). The NLR was observed to normalize, reaching a value of 2.6.

A follow-up consisting of imaging and biomarkers evaluation was conducted in November 2024. Imaging evaluation showed no signs of residual disease, but both biomarkers showed pathologic values. The ctDNA assay recorded a value of 1.26 MTM/ml, while the NLR showed a value of 3.3 (Figure 2).

Figure 2. Correlations between ctDNA values, NLR and the clinical evolution of the patient with gastric cancer

3. Discussion

In the case of the patient with colon cancer, the ctDNA determinations were observed to be consistently positive, with the ctDNA value trends correlating with the imaging data. The assay peak occurred during the same period as the initial disease progression, after which the values began to decline. The ctDNA values subsequently increased at the time of the second disease progression.

Regarding the NLR determinations, it was observed that only one of the five determinations corelated with the ctDNA test results. At the first imaging showing progressive disease, the NLR exhibited a ratio of 1.8, which was within the normal range. At the second imaging showing progressive disease, the NLR value increased to 2.3.

These findings suggest the potential the efficacy of ctDNA determination in terms of real-time monitoring of tumor response to  treatment. Our case is hypothesis generating as no definitive conclusion can be drawn from a single case. A similar ctDNA dynamic was observed in the cohort of 168 patients published by Hao et al. (18).

In the case of the patient diagnosed with gastric cancer, it was observed that both ctDNA and NLR biomarkers had decreased values during the course of adjuvant therapy. Of particular interest is the observation that, although these values were declining, the ctDNA values remained positive, whereas the NLR assay indicated a normalization of the values. The results obtained by our research team are in agreement with those previously reported by Grizzi and colleagues (19). The ctDNA analysis demonstrated its efficacy in monitoring the efficacy of the administered treatment, and our findings indicate a reduction in the levels of ctDNA following the initiation of adjuvant therapy.

One of the key issues surrounding the ctDNA assay is that, while it facilitates accurate genotyping through the utilization of a reduced quantity of genetic material, the integration of this testing within practical, real-world contexts remains a significant challenge (15), particularly for gastric cancer (19).

Although ctDNA testing is not yet standard of care, the European Society for Medical Oncology (ESMO) guidelines recommend its use in several specific settings. For patients diagnosed with metastatic colorectal cancer who have not received prior chemotherapy, and when tissue testing is not feasible or when an urgent therapeutic decision is needed,  ESMO advises ctDNA testing for the determination of KRAS/NRAS/BRAFV600E/MSI status. Another recommendation is also proposed for pre-treated patients who are being considered for anti-EGFR rechallenge. For gastric cancer, ctDNA testing is recommended when tissue cannot be collected or when an urgent therapeutic decision is required (20).

A Japanese study about the utility of ctDNA testing in patients with colorectal cancer and molecular residual disease (MRD) demonstrated that patients with sustained clearance have a better outcome than those with temporary clearance. Furthermore, the study showed that in patients with MRD who received adjuvant chemotherapy, a decrease in ctDNA levels of at least 50% at 6 months was associated with better disease-free survival (DFS) compared with those who experienced an increase or decrease of less than 50%. The study’s findings suggested that the determination of MRD by ctDNA and the dynamics of ctDNA levels in response to adjuvant chemotherapy play a prognostic role in patient outcome (21).

Another similar study concluded that ctDNA MRD testing is not yet ready for prime time for making therapeutic decisions in colon cancer, thereby underscoring the need for additional clinical trials. The study suggested that ctDNA testing may have a role in the selection of adjuvant therapy, however, it also noted the existence of sensitivity and specificity issues (22).

The results of ctDNA testing may be flawed due to both sampling and patient related errors. False negative results may result from prolonged storage time exceeding 5 days. Also, ctDNA values may be influenced by  smoking, pregnancy, heart disease, inflammation, and physical activity. In addition, it has been observed that ctDNA values demonstrate diurnal fluctuations  (23).

A substantial body of evidence from scientific publications indicates a potential correlation between NLR and advanced cancer. This ratio may sometimes correlate with the global inflammatory status induced by the tumor and may represent a potential prognostic and predictive biomarker for oncological patients (24).

Although there is no consensus regarding the precise cut-off for the NLR, the presence of pathological values of the NLR has been linked to a poor prognosis in cancer patients (9). The following definition of NLR values is provided for clarity: the normal value ranges from 1-2, with values greater than 3 or less than 0.7 being pathological. It is noteworthy that the range between 2.3 and 3 represents a grey zone, serving as a potential early warning indicator for cancer development (25). It should be noted that NLR values can be affected by a range of factors, including age, corticosteroid therapy, hormonal status, the presence of immune-depleting diseases, and the administration of cytotoxic agents or granulocyte colony-stimulating factor (G-CSF) treatments (9).

In the context of gastrointestinal cancers, elevated NLR values facilitate the identification of patients with advanced tumors and the detection of recurrences following surgical intervention. Furthermore, the absence of normalization of this ratio at three-month intervals post-operatively may indicate the recurrence or persistence of disease (26).

A review of the literature revealed comparable correlations between ctDNA and NLR in patients with pancreatic cancer. McLellan and his team observed that ctDNA positivity was frequent in patients with an NLR higher than 5. On the other hand, for patients with positive ctDNA, but an NLR below 5, PFS and OS were shorter, suggesting that ctDNA may be more useful than NLR in this setting (27). Another publication including patients with borderline resectable or locally advanced pancreatic cancer  also demonstrated the lack of an association between NLR and ctDNA values (28).

The study of Kam and Masood suggested that ctDNA may be more useful in esophageal cancer surveillance than NLR (29). Similar discrepancy between ctDNA and NLR was observed in a larger study including patients with metastatic colorectal cancer (30).

4. Conclusion

The two clinical cases reported suggest that ctDNA testing may be more accurate than NLR assessment for monitoring treatment response in patients with colon or gastric cancers. These case reports are hypothesis generating and the predictive values of these biomarkers should be investigated in further clinical trials.

Abbreviations

cfDNA=cell-free DNA

ctDNA=Circulating tumor DNA

DFS=disease-free survival

ESMO=European Society for Medical Oncology

G-CSF=granulocyte colony-stimulating factor

MRD=molecular residual disease

NLR=neutrophil/lymphocyte ratio

WHO=World Health Organization

Statements

Author Contributions: Conceptualization, P.Ș., M.P., I.D., F.B., M.M., A.-M.D., A.-E.R., E.P., C.T.; methodology, P.Ș., M.P., I.D., F.B., M.M., A.-M.D., A.-E.R., E.P., C.T.; software, P.Ș., M.P., I.D., F.B., M.M., A.-M.D., A.-E.R., E.P., C.T; validation, M.P., I.D., F.B., M.M., C.T; formal analysis, M.P., I.D., F.B., M.M., C.T; investigation, M.P., I.D., F.B., M.M., C.T; resources, P.Ș., M.P., I.D., F.B., M.M., A.-M.D., A.-E.R., E.P., C.T.; data curation, M.P., I.D., F.B., M.M., C.T ; writing—original draft preparation, P.Ș., A.-M.D., A.-E.R., E.P.; writing—review and editing, M.P., I.D., F.B., M.M., C.T;

visualization, P.Ș., M.P., I.D., F.B., M.M., A.-M.D., A.-E.R., E.P., C.T.; supervision, M.P., I.D., F.B., M.M., C.T; project administration, P.Ș., M.P., I.D., F.B., M.M., A.-M.D., A.-E.R., E.P., C.T.; All authors have read and agreed to the published version of the manuscript.

Data Availability: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.

Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results

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