Overview
At present, more and more patients with malignant tumors are undergoing the detection of circulating tumor cells (CTC) in the peripheral blood and ctDNA-MRD (minimal residual disease) during the process of their disease diagnosis and treatment. Due to more research on ctDNA-MRD,clinically, more attention may be paid to ctDNA-MRD. In actual clinical work, not every patient can have CTC and ctDNA detected in their peripheral blood. Here, this article reports two patients with malignant tumors (lung cancer and rectal cancer). A relatively large number of CTCs were detected in their peripheral blood (detected by the CTCBIOPSY system of Chipscreen Biosciences), but no ctDNA was detected (negative). Therefore, it is recommended to conduct combined CTC and ctDNA detection for patients with malignant tumors to improve the detection efficiency of MRD (minimal residual disease) in tumor patients.
Basic patient information
Case One
The patient is a 68-year-old male. Two weeks before the operation, a pulmonary nodule was detected during the examination. The CT examination on 2024-08 showed that: a nodule was seen in the posterior basal segment of the right lower lung lobe, with a size of approximately 24 mm × 18 mm, showing continuous enhancement, and the bronchial tract could be seen inside, with an irregular edge; a nodule was seen in the dorsal segment of the left lower lung lobe, with a size of approximately 6 mm × 4 mm. In September 2024, a thoracoscopic right lower lobectomy and a thoracoscopic mediastinal lymph node resection were performed. The rapid intraoperative pathology showed that: (right lower lung lobe) invasive carcinoma with poor tissue differentiation.
Pathological Diagnosis after Surgery:
(1)Gross Examination
Specimen Name: Lobectomy Specimen; Orientation Mark: Marked with a tied thread; Specimen Size: 18 cm × 11 cm × 3 cm; Tumor Location: (Right Lower Lung); Gross Type: Peripheral Type; Tumor Size: 3.1 cm × 2.8 cm × 1.5 cm.
(2)Microscopic Findings
Tumor Type: Invasive Non-mucinous Adenocarcinoma. Whether there is a combination with other types of tumors awaits supplementary reports from immunohistochemistry. Pathological Subtype: Mainly characterized by complex glandular structures, micropapillary structures, and solid structures. Acinar, papillary, and lepidic structures can be seen. Pathological Grading: Poorly Differentiated (Grade 3); Airway Dissemination: Present; Vascular Invasion: Vascular invasion can be seen; Nerve Invasion: Nerve invasion is present; Pleural Involvement: Requires special staining for further clarification; Margin Status: No cancer involvement was seen at the bronchial/stapled margin.
Pathological Diagnosis of Lymph Nodes: ① Cancer metastasis was seen in (Lymph Node 7) (1/4); ② No cancer metastasis was seen in (Lymph Node 9) (0/1); ③ No cancer metastasis was seen in (Lymph Node 10) (0/1); ④ No cancer metastasis was seen in (Lymph Node 11) (0/1); ⑤ No cancer metastasis was seen in (Lymph Node 12) (0/1).
Immunohistochemistry Results: Tumor Cells in (Right Lower Lung Lobe): CK-pan (+), CX7 (+), Napsin A (+), TTF-1 (+), CK5/6 (-), P63 (-), P40 (-), Syn (-), CgA (-), CD56 (-), Nut (-), Ki67 (Approximately 50% + in the hot spot area), BRG1 (+), INI-1 (+).
Combined with HE sections, this case is an invasive adenocarcinoma, Grade III. Postoperative Pathological Staging: pT2N2M0, Stage IIIA. According to the guidelines, adjuvant treatment is required after surgery.
Lung cancer ctDNA-MRD (Molecular Residual Disease) detection was performed in September 2024, and peripheral blood circulating tumor cells (CTC) detection was performed in October 2024.
ctDNA Detection
The next-generation sequencing technology (NGS) was used to detect four types of variations (including point mutations, small insertions and deletions, copy number variations, and currently known fusion genes) of more than 300 genes related to tumor development. Specific detection details: the entire coding regions of more than 50 genes, the intron or fusion breakpoint regions or promoter regions of more than 10 genes, and the partial exon regions of more than 280 genes. The ctDNA detection was carried out one month after the operation.
The total amount of DNA was 67 ng; the average sequencing depth exceeded 30,000. The detection result was: 0 somatic variations were detected. This result indicates that the ctDNA is negative.
CTC Detection
The CTCBIOPSY® method was used for the detection of peripheral blood circulating tumor cells (CTC). The detection was performed one month after the operation. The result showed that the sample volume of this patient was 5 ml of peripheral blood, with a single CTC count of 5 and a CTM count of 0.
Summary
The postoperative pathological staging of this patient is pT2N2M0, Stage IIIA. The lung cancer MRD (molecular residual disease) detection was carried out in September 2024, and the result was MRD negative. The peripheral blood circulating tumor cells (CTC) detection was carried out in October 2024, with CTC = 5 and CTM = 0. For the sake of easy understanding, it can be considered that this patient is CTC positive and ctDNA-MRD negative.
Case Two
The patient is a 47-year-old male. One year ago, he had diarrhea and an increased frequency of bowel movements without any obvious precipitating factors. The frequency of bowel movements increased further after eating greasy, cold, or raw foods. In the nearly 2 – 3 months before the operation, the frequency of bowel movements increased significantly, with an average of 6 to 7 times a day. Occasionally, mucus stools and bloody stools were seen. A colonoscopy examination was performed on 2023-09. A new growth was seen encircling half of the lumen 15 cm from the anus. Biopsy of this area was prone to bleeding. The pathological diagnosis of the biopsy was: atypical hyperplasia of glandular epithelium and canceration (15 cm from the anus). Subsequently, an extended radical resection of rectal cancer under laparoscopy was performed on 2023-10.
Postoperative Pathological Diagnosis:
(1)Gross Examination
Operation and Specimen Name: Radical Resection of Rectal Cancer (Dixon Operation); Specimen Site: Rectum; Number and Size of Tumors: One tumor, measuring 4.5 × 4 × 1.5 cm; Gross Type: Ulcerative Type.
(2)Microscopic Findings
Histological Type: Adenocarcinoma; Pathological Grading: Moderately Differentiated (G2); Tumor Infiltration Level in the Intestinal Wall (T Staging): The tumor penetrated the muscularis propria and reached the adipose connective tissue around the intestinal wall (T3); Lymph Node Status (N Staging): Cancer metastasis was seen in the perirectal lymph nodes (4/29); Intravascular Tumor Thrombus: Present; Nerve Invasion: Present; Circumferential Resection Margin: Negative; Other Margins: No tumor involvement was seen in the upper and lower intestinal margins and the separately submitted anastomotic ring (N2a); Distant Metastasis (M Staging): Distant metastasis could not be determined (Mx); Primary Tumor Staging (pTNM, AJCC 8th Edition): pT3N2aMx, Stage IIIB.
Peripheral blood circulating tumor cells (CTC) detection was performed in November 2023, and lung cancer ctDNA-MRD detection was performed in December 2023.
ctDNA Detection
The detection method was the same as above. The peripheral blood ctDNA was detected in December 2023. The average sequencing depth of this patient was lower than 30,000. The detection results showed that 0 somatic variations and 0 germline mutations were detected.
CTC Detection
The method was the same as above. The CTC detection was carried out in November 2023. The results showed that when the sample volume of this patient was 5 ml of peripheral blood, the single CTC count was 8 and the CTM count was 0.
Summary
The postoperative staging of this patient is pT3N2aMx, Stage IIIB. The peripheral blood circulating tumor cells (CTC) detection was carried out in November 2023, with CTC = 8 and CTM = 0. The peripheral ctDNA-MRD detection was carried out in December 2023: no ctDNA was detected, that is, the ctDNA was negative. For the sake of easy understanding, it can also be considered that this patient is CTC positive and ctDNA-MRD negative.
Discussion
1.Domestic and foreign experts basically recognize that ctDNA is one of the ideal biomarkers for MRD detection and believe that ctDNA detection is the most effective method to discover MRD at present. Some domestic and foreign guidelines (such as CSCO guidelines, NCCN guidelines, etc.) also recognize the clinical significance of ctDNA-MRD detection. Therefore, more doctors use ctDNA results to reflect MRD.
2.For ct-DNA-MRD, the general understanding is that the detection of minimal residual disease (MRD) based on circulating tumor DNA (ctDNA) can identify the recurrence of postoperative early-stage cancer patients earlier than traditional clinical or imaging methods, thus providing an opportunity for early intervention. Actually, both peripheral blood CTC and ctDNA are indicators reflecting minimal residual disease (MRD). Since more scholars tend to use ctDNA to reflect MRD, there may be insufficient recognition of peripheral blood CTC among these scholars.
3.In fact, the detection results of both are greatly affected by the detection techniques. It seems that the ctDNA detection technique based on next-generation sequencing (NGS) is even better. The detection depth of NGS is represented by the limit of detection (LOD). Studies have found that NGS can stably detect ctDNA with an abundance of ≥0.02%. The NGS detection method can discover the recurrence of the disease earlier than imaging, which is beneficial for patients to adjust their treatment plans in a timely manner. Therefore, the recognition of the NGS technique seems to be higher as well. Interestingly, based on the current clinical sample detection results, the CTC detection technique of the CTCBIOPSY® method doesn’t seem to be bad either.
4.Judging from the detection results of the above two patients, both detection schemes are limited by the detection equipment and methods and may have certain limitations. From the limited data, there may be an overlap in the positive detection rates of the two in tumor patients. Therefore, it would be better to combine the two. As early as 2022, some scholars reported a study. Among 28 patients with liver cancer recurrence, the positive detection rate of CTC was 75%, the positive detection rate of ctDNA was 70.4%, and the combined positive detection rate of CTC and ctDNA was 85.7%. This study shows that the combined detection of the two may be more beneficial for patients with liver cancer recurrence [1]. Therefore, it is recommended that for patients with malignant tumors, in addition to detecting ctDNA, the peripheral blood CTC should also be detected simultaneously.
5.The detection results similar to those of these two patients have brought some confusion to the actual clinical work. For example, will patients with a relatively large number of CTCs in the peripheral blood experience recurrence? And when will it occur? Regarding the CTC detection results similar to those of these two patients, some scholars believe that although CTCs are detected in the peripheral blood of these patients, these CTCs are inert cells and cannot reflect the changes in the patient’s condition. To better explain and utilize these results, a lot of work needs to be done. In addition, the comparative study of the clinical values of CTC and ctDNA awaits further development.
6.From the current clinical practice, the false negative rate of MRD detection is the primary problem that urgently needs to be solved when applying MRD in clinical settings. Studies have found that at the landmark of MRD, the ctDNA allele fraction of most recurrent patients is lower than 0.01%. This indicates that the minimum detection limit needs to be reduced to below 0.01% in order to minimize the false negative rate in ctDNA-MRD detection [2]. Currently, lower detection limits have begun to be applied in clinical practice.
7.Currently, in clinical practice, there are more studies on ctDNA compared with CTC. Based on the existing research results, there are corresponding expert consensuses for both CTC and ctDNA, such as “Expert Consensus on Laboratory Detection of Peripheral Blood Cell Immune Function in Solid Tumors”, “Consensus on Detection of Molecular Residual Disease in Solid Tumors”, “Chinese Expert Consensus on Detection and Clinical Application of Molecular Residual Disease in Gastric Cancer (2023 Edition)”, etc.
8.For these two patients with malignant tumors, peripheral blood CTC and ctDNA detections were carried out simultaneously. The results showed that CTC was positive while ctDNA was negative. Similar patients are not uncommon in clinical practice and deserve more attention.
References
- Zhao L, Jiang L, Liu Y, et al. Integrated analysis of circulating tumour cells and circulating tumour DNA to detect minimal residual disease in hepatocellular carcinoma. Clin Transl Med. 2022 Apr;12(4):e793.
- Moding EJ, Nabet BY, Alizadeh AA, Diehn M. Detecting liquid remnants of solid tumors: circulating tumor DNA minimal residual disease. Cancer Discov. 2021 Dec 1;11(12):2968-2986.
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