Towards mid-2018, a 40- to 45-year-old patient underwent colonoscopy because of persistent constipation, with a diagnosis of stenotic colorectal adenocarcinoma on splenic flexure. The chest and abdominal CT scan showed a metastatic disease, in particular a lesion on the left lobe of the liver of about 107 × 67 × 94 mm and six lesions on the right lobe, the bigger of which was about 21 mm (Figure 1A,B). There was also peritoneal carcinomatosis. At diagnosis CEA was 1.8 μg/L (<5 μg/L) and Ca 19–9 14.5 KU/L (<37 KU/L). The molecular characterization was performed on the primary tumor tissue using a MassARRAY Sequenom analysis (Myriapod Colon status, Diatech Pharmacogenetics, Jesi, Italy), revealing a RAS and BRAF wt status. Consequently, the patient received a first-line treatment with FOLFOXIRI (Irinotecan 150 mg/mq over 1 h day 1, oxaliplatin 85 mg/mq over 2 h day 1, calciolevofolinate 200 mg/mq over 2 h day 1, 5 fluorouracil 2400 mg/mq over 48 h) plus Panitumumab (6 mg/kg over 1 h day 1) every 2 weeks. After four cycles of treatment, CT scan evaluation showed disease progression, in particular on the left lobe of the liver where the bigger metastasis was 180 mm (Figure 1C), while there was an improvement on the primary tumor because the colorectal cancer step from 96 × 89 mm to 61 × 54 mm. Because of the young age of the patient, a liquid biopsy sample was made to obtain useful information for new drugs. RAS and BRAF status were analyzed by a Real-Time PCR method (Easy-KRAS status, Easy-NRAS status, Easy-BRAF status, Diatech Pharmacogenetics). Results showed the presence of a BRAF mutation (V600E). Concomitantly, the patient had a performance deterioration with severe abdominal pain and received a second-line treatment with FOLFIRI (Irinotecan 180 mg/mq over 90 min day 1, calciolevofolinate 200 mg/mq over 2 h day 1–2, 5 fluorouracil 400 mg/mq 3′-4′ day 1, 5 fluorouracil 2,400 mg/mq 48 h day 1) plus aflibercet (4 mg/kg over 1 h) every 2 weeks. Unfortunately, the patient rapidly progressed with a decline of performance status and died after 1 month from chemotherapy. Based on the results obtained on liquid biopsy about V600E BRAF mutation, the same analysis was repeated on DNA of the tumor tissue using a more sensitive methodology (Real-Time PCR), but a wt BRAF status was confirmed. These results suggest two hypotheses: 1) BRAF mutation was already present in the primary tumor tissue but heterogeneously, and tumor tissue analysis was not able to evidence the mutation; 2) BRAF mutation was induced during treatment as a secondary resistance mechanism.

At the end of 2014, a 55 to 60-year-old patient underwent pancolonscopy because of rectal bleeding, with a diagnosis of rectal adenocarcinoma placed at 8, 9 cm from anal rhyme. The abdominal and chest CT scan showed a localized disease on the proximal rectum. The pelvic NMR described a rectal cancer and the disease was a cT3N + M0. At diagnosis, CEA was 10.1 μg/L (<5 μg/L). The patient received a neo-adjuvant treatment inside a clinical trial, with FOLFOX4 (Oxaliplatin 85 mg/mq over 2 h day 1, calciolevofolinate 100 mg/mq over 2 h day 1–2, 5 fluorouracil 400 mg/mq 3′-4′ day 1–2, 5 fluorouracil 600 mg/mq 22 h day 1–2 every 2 weeks) for 4 cycles and hypofractionated radiotherapy (25 Gy/5 fr) that was administered between the second and the third cycle of chemotherapy, After the neoadjuvant treatment CEA was 5.7 µg/Land the patient underwent a surgical resection of rectum (ypT2N1cR0). After surgery, the patient performed an adjuvant therapy with FOLFOX4 for eight cycles and at the end of therapy, CEA was 4.8 μg/L. In early 2017 disease relapse was shown, with bilaterally pulmonary metastases at CT scan and CEA increased to 8.7 μg/L (Figure 2).

Molecular characterization performed using MassARRAY Sequenom (Myriapod Colon status, Diatech Pharmacogenetics) on the tumor tissue of the surgical resection of 2015 showed a BRAF and RAS wt status. Then the patient started a first-line treatment with FOLFIRI (Irinotecan 180 mg/mq over 90 min day 1, calciolevofolinate 200 mg/mq over 2 h day 1–2, 5 fluorouracil 400 mg/mq 3′-4′ day 1, 5 fluorouracil 2,400 mg/mq 48 h day 1 every 2 weeks) plus cetuximab (400 mg/mq over 2 h the first time and then 250 mg/mq over 1 h) for eight cycles with evidence of SD at CT scan and a slight increase of CEA that was 15.6 μg/L. Maintenance treatment with weekly cetuximab was then carried out but interrupted after only 2 months when a CT scan showed a progression disease on the lungs where the bigger lesion step from 12 × 10 to 15 × 14 mm on the inferior left lobe and CEA became 48.5 μg/L. Concomitantly we performed a liquid biopsy analysis and RAS and BRAF status were analyzed using a Real-Time PCR method (Easy-KRAS status, Easy-NRAS status, Easy-BRAF status, Diatech Pharmacogenetics). From this analysis, a KRAS G13D mutation was identified. In view of this result, KRAS status was determined in the primary tissue of 2015 using the Real-Time PCR assay, revealing a borderline KRAS G13D. These results highlight that the KRAS G13D was already present sub-clonally at the baseline, not identified using the standard mutation analysis methodology, and was responsible for the inefficacy of the anti-EGFR agent. After these evaluations, the patient received a second-line treatment with FOLFOX4 with an SD and improvement of CEA that went down to 10.8 μg/L after five cycles. After ten cycles of treatment an abdominal and chest CT scan showed a dimensional increase of most of the known lung lesions. The patient received Regorafenib (160 mg/die q 21) as third-line treatment for 9 months with stable disease as a better response but a progressive increase of CEA from 35 μg/L to 269 μg/L. At progression, Regorafenib was stopped and the patient started Trifluridine/Tipiracil (700 mg/mq q 28) for three cycles, with the radiological progression of disease and CEA increasing to 413 μg/L. The patient subsequently received metronomic capecitabine plus hyperthermia with further progression and was referred to palliative care. The patient died about 5 years after the first diagnosis of tumor.

In early 2018, a 55 to 60-years-old patient with persistent constipation and bleeding underwent colonoscopy, with a diagnosis of distal rectal adenocarcinoma with a single synchronous hepatic metastasis (about 25 mm) on IV–VIII segment (Figure 3A). CEA was 2.6 μg/L at diagnosis. A pelvic NMR described a rectal cancer extended from 2 cm above the anal opening to 10 cm, which was defined as cT3N1M1 (Figure 3B). Molecular characterization performed by MassARRAY Sequenom (Myriapod Cancer status, Diatech Pharmacogenetics) of the primary tissue revealed a RAS and BRAF wt status. The patient received a first-line treatment with FOLFOX6 (oxaliplatin 85 mg/mq over 2 h day 1, calciolefolinate 200 mg/mq over 2 h day 1, 5 fluorouracil over 3′-4′ day 1, 5 fluorouracil over2400 mg/mq over 48 h) plus panitumumab (6 mg/kg) every 2 weeks for five cycles and then capecitabine (825 mg/mq BID daily for 6 weeks) with concomitant radiotherapy. At the end of chemoradiotherapy, an abdominal and chest CT scan showed a progression of the liver disease on the IV-VIII segment from 24 × 20 mm to 42 × 36 mm (Figure 3C) while the pelvic NMR described a regression of the primary tumor (Figure 3D). The pathology report from an anterior resection of the rectum described a complete pathologic regression (pT0N0R0) of the primary tumor. It was not possible to perform the liver resection at the same time because of the appearance of a mixed acidosis that would have greatly prolonged the intervention. Therefore, we decided to restart treatment with FOLFOX6 and panitumumab. After 4 cycles, there was an increase of the liver disease up to 55 × 45 mm on IV-VIII segment, and the pathologic report from left hepatectomy reported intestinal adenocarcinoma with abundant necrosis. Molecular characterization of the metastasis performed by Next Generation Sequencing (NGS) (Oncomine Focus Assay, Thermofisher Scientific, Monza, Italy) revealed NRAS Q61K and APC c.4199C > A mutations. Liquid biopsy performed at the same time by Real-Time PCR (Easy-NRAS status, Diatech Pharmacogenetics) revealed the presence of NRAS Q61K mutation. These results revealed that in the metastatic lesion, a NRAS N61Q clone was selected and was responsible for the hepatic progression, and liquid biopsy was able to identify such mutation. The repetition of molecular analysis on DNA of the primary tissue, using a Real-Time PCR-based method, revealed a borderline presence of the NRAS Q61Q mutation. This result reinforced the hypothesis that the mutation was present sub-clonally in the primary tissue, but the low percentage of mutated cells did not obstacle the responsiveness to the anti-EGFR treatment. Conversely, the mutation was selected in the metastatic lesion, rendering it not sensitive to treatment. Liquid biopsy was able to identify the mutation.

An FDG-PET scan performed after liver surgery revealed an early liver and lung progression of disease, so a second-line treatment was started with FOLFIRI (Irinotecan 180 mg/mq over 90 min day 1, calciolevofolinate 200 mg/mq over 2 h day 1–2, 5 fluorouracil 400 mg/mq 3′-4′ day 1, 5 fluorouracil 2400 mg/mq 48 h day 1 every 2 weeks) plus bevacizumab (5 mg/kg over 90′ the first time, then 5 mg/kg over 1 h next) every 2 weeks After six cycles of treatment, CEA became positive until 7.2 μg/L and an abdominal and chest CT scan confirmed a progression of disease with multiple lung and liver metastases. Then the patient was treated with 4 cycles of Trifluridina/Tipiracil (700 mg/mq q 28) but there was again a progression of disease. The patient unfortunately died 2 months after.

At the end of 2018, a 45- to 50-year-old patient underwent colonoscopy for abdominal pain with a diagnosis of adenocarcinoma on the right colon. The basal abdominal and chest CT scan described a liver metastasis on V, VI segment about 30 mm and a doubt lesion about 6 mm on the superior left lobe of the lung. At diagnosis CEA was 1.6 μg/L (<5 μg/L) and Ca 19–9 14.5 KU/L (<37 KU/L). The patient underwent right hemicolectomy and liver resection with a diagnosis of poorly differentiated carcinoma G3 of the colon, with focal aspects of squamous differentiation, and the presence of carcinomatous lymphangitis and hepatic metastasis from poorly differentiated carcinoma. Tissue molecular characterization was performed on specimens of the primary lesion in another center and revealed a RAS and BRAF wt status. Liquid biopsy analysis performed by a Real-Time PCR method (Easy-KRAS status, Easy-NRAS status, and Easy-BRAF status, Diatech Pharmacogenetics) confirmed a RAS and BRAF wt status.

In early 2019, the patient received first-line chemotherapy with FOLFOX6 (oxaliplatin 85 mg/mq over 2 h day 1, calciolefolinate 200 mg/mq over 2 h day 1, 5 fluorouracil over 3′-4′ day 1, 5 fluorouracil over 2400 mg/mq over 48 h) plus panitumumab (6 mg/kg) every 2 weeks for five cycles, and the CT scan confirmed a lung metastatic lesion increased to 14 × 13 mm. An FDG-PET scan confirmed the single lesion. Then the patient was operated on in the superior left lobe of the lung, and the disease was about 15 mm. The diagnosis was poorly differentiated adenocarcinoma, with large necrotic areas compatible with metastases from carcinoma of the large intestine. Upon immunohistochemical investigation, neoplastic cells showed minimal expression of CDX2 and were negative for TTF1, neuroendocrine markers, and cytokeratins 7 and 20. The growth fraction was 70%. Molecular analysis on this lesion, made using an NGS Focus Oncomine assay (Thermofisher) revealed the presence of PIK3CA c.3073A > G and MAP2K1 c.607G > A mutations. Liquid biopsy performed at the same time using the Oncomine Colon cfDNA Assay (Thermofisher Scientific) revealed the same MAP2K1 mutations, together with an APC mutation (c.4463T > G) that was not covered by the Focus Oncomine assay performed on tissue. It was not possible to verify the presence of the PIK3CA mutation as that specific mutation was not analyzed in the Oncomine Colon cfDNA Assay. After surgery, post-operative chemotherapy with FOLFOX6 was administered. After 4 cycles of treatment an abdominal and chest CT scan did not describe metastatic disease, whereas a brain CT scan performed for referred headache, photophobia and dizziness unfortunately showed a cerebellar lesion about 35 mm on the right (Figure 4A) and another lesion about 20 × 20 mm on temporal left site (Figure 4B).

The patient received craniotomy with cerebellar right resection but the second lesion was not removed. The report was metastatic localization of poorly differentiated adenocarcinoma with large necrotic areas, compatible with known colic primitiveness. Upon immunohistochemical investigation, the cells were only focally positive for CDX2 and negative for CK20 and CK7, TTF1, and Chromogranin. The cell proliferation index, assessed by immunohistochemistry using Ki67/Mib1, was approximately 70%. Molecular analysis of this lesion, made by NGS Oncomine Focus assay, revealed the same spectrum of mutation found in the lung lesion (PIK3CA c.3073A > G, MAP2K1 c.607G > A). At the end of 2019, mediastinal lymph nodes and liver metastases appeared on FDG-PET scan, in the same period the patient received brain tomotherapy (2,500 cGy 5 Fr). In early 2020, a second-line treatment with FOLFIRI (Irinotecan 180 mg/mq over 90 min day 1, calciolevofolinate 200 mg/mq over 2 h day 1–2, 5 fluorouracil 400 mg/mq 3′-4′ day 1, 5 fluorouracil 2400 mg/mq 48 h day 1 every 2 weeks) was administered for only two cycles but it was stopped because of grade 2 diarrhea and decline of performance status with clinical progression of disease.

By this case report, it has been highlighted that, in this case, liquid biopsy was able to identify the same spectrum of mutation found in tissue, permitting to evidence the presence of mutations carried by the metastatic lesions.