A case of laparoscopic lymphadenectomy for adenocarcinoma of unknown primary incidentally detected as a solitary enlarged lymph node along the common hepatic artery.

BACKGROUND: Even in cancer of unknown primary (CUP), which is rare clinical condition, solitary anterosuperior lymph node (LN) along the common hepatic artery (No.8a LN) enlargement diagnosed as metastatic adenocarcinoma has never been reported. CASE PRESENTATION: A 68-year-old Japanese male, with a history of early gastric cancer that had been completely treated by endoscopic submucosal dissection 26 years ago, was detected a single enlarged nodule along the common hepatic artery, No.8a LN, incidentally by computed tomography performed for monitoring of interstitial pneumonia. Endoscopic ultra-sound-guided fine needle aspiration revealed that this nodule was adenocarcinoma suggestive of metastasis, but other imaging studies, including upper and lower gastrointestinal endoscopy, positron emission tomography, and ultrasonography did not detect any primary cancer. We have finally diagnosed as the LN metastasis of CUP and performed laparoscopic lymphadenectomy for this tumor. The tumor was approximately 5 cm in size, was in close proximity to the pancreas, and involved part of the right gastric artery and vein. LNs in the No.5 and No.8a areas, including this tumor, were dissected laparoscopically, and radical resection was achieved. The patient had no postoperative complication and was discharged on postoperative day 10. Immunohistopathological findings revealed that the tumor was poorly differentiated adenocarcinoma, and different from the histology of gastric cancer resected 26 years ago, although the tumor was suggestive of gastrointestinal origin. Imaging studies performed 2 and 6 months after discharge also did not reveal a primary site. CONCLUSION: We reported a case of solitary No.8a LN adenocarcinoma of CUP. For diagnostic and therapeutic purposes, radical resection is recommended for single enlarged intra-abdominal LN of CUP.

Novel and efficient method for culturing patient-derived gastric cancer stem cells.

Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.

Efficacious Combination Drug Treatment for Colorectal Cancer That Overcomes Resistance to KRAS G12C Inhibitors.

Recent advances in combinatorial chemistry led to the discovery of inhibitors targeting the KRAS G12C-mutant protein. However, efficacy of its monotherapy on colorectal cancer is limited. Thus, effective combination drugs should be explored for applicable patients with colorectal cancer to fully benefit from the KRAS G12C inhibitor treatment. Here we used a patient-derived colorectal cancer stem cell (PD-CRC-SC) spheroid culture and showed that three-drug combination of inhibitors against KRAS G12C, EGFR, and FGFR synergistically suppressed the growth of colorectal cancer cells carrying the KRAS G12C mutation. Likewise, a combination of KRAS G12C and SHP2 inhibitors was also effective. Importantly, activation of the PI3K/AKT pathway in heregulin-responsive colorectal cancer cells canceled out the effect of KRAS G12C inhibition, which was largely overcome by PI3K inhibitors. These results reveal that evaluating efficacy of combination therapies with PD-CRC-SC spheroids can be a promising strategy to find the best regimen for patients with colorectal cancer.

Chemosensitivity of Patient-Derived Cancer Stem Cells Identifies Colorectal Cancer Patients with Potential Benefit from FGFR Inhibitor Therapy.

Some colorectal cancer patients harboring FGFR (fibroblast growth factor receptor) genetic alterations, such as copy number gain, mutation, and/or mRNA overexpression, were selected for enrollment in several recent clinical trials of FGFR inhibitor, because these genetic alterations were preclinically reported to be associated with FGFR inhibitor sensitivity as well as poor prognosis, invasiveness, and/or metastatic potential. However, few enrolled patients were responsive to FGFR inhibitors. Thus, practical strategies are eagerly awaited that can stratify patients for the subset that potentially responds to FGFR inhibitor chemotherapy. In the present study, we evaluated the sensitivity to FGFR inhibitor erdafitinib on 25 patient-derived tumor-initiating cell (TIC) spheroid lines carrying wild-type RAS and RAF genes, both in vitro and in vivo. Then, we assessed possible correlations between the sensitivity and the genetic/genomic data of the spheroid lines tested. Upon their exposure to erdafitinib, seven lines (7/25, 28%) responded significantly. Normal colonic epithelial stem cells were unaffected by the inhibitors. Moreover, the combination of erdafitinib with EGFR inhibitor erlotinib showed stronger growth inhibition than either drug alone, as efficacy was observed in 21 lines (84%) including 14 (56%) that were insensitive to erdafitinib alone. The in vitro erdafitinib response was accurately reflected on mouse xenografts of TIC spheroid lines. However, we found little correlation between their genetic/genomic alterations of TIC spheroids and the sensitivity to the FGFR inhibitor. Accordingly, we propose that direct testing of the patient-derived spheroids in vitro is one of the most reliable personalized methods in FGFR-inhibitor therapy of colorectal cancer patients.

Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells.

Mismatch repair (MMR)-deficient or microsatellite instability (MSI) colorectal cancer includes two subtypes; Lynch syndrome and sporadic MSI cancer, both of which generate multiple neoantigens due to unrepaired mutations. Although such patients respond very well to immune checkpoint therapy, their diagnosis can be confused by low quality DNA samples owing to formalin fixation and/or low cancer cell content. Here we prepared high-quality DNA samples from in vitro-cultured cancer spheroids that consisted of the pure cell population. We evaluated their diagnostic power by on-chip electrophoresis, mutational burden assessment, and direct sequencing. Because formalin-fixed paraffin-embedded (FFPE) tissues are widely used as the DNA source, we compared such samples with spheroid DNA. Additionally, we performed immunohistochemistry (IHC) for MMR proteins on spheroids as well as primary tumor sections. Of 111 cases of colorectal cancer patients, we found seven MSI-high cases in which all diagnostic results agreed on spheroid-based assays, whereas the results with the FFPE DNA were less reliable though analyzable. Importantly, there was an MSS case that appeared as MSI by IHC on primary tumor sections. Based on these results, we propose to employ cultured cancer spheroids as the source of both DNA and IHC specimens for more reliable clinical diagnosis.

[A Case of Squamous Cell Carcinoma of the Esophagus with Achalasia -- Conversion from Chemoradiotherapy to Surgery].

The patient was a 67-year-old woman with achalasia and squamous cell carcinoma(SCC)of the esophagus. She presented with a difficulty in swallowing. The cancer was on the surface of the esophagus. The patient initially received systemic chemotherapy with 5-FU and cisplatin, and radiation therapy. The difficulty in swallowing persisted due to insufficiency of radiation treatment caused by achalasia. Therefore, we shifted the treatment plan from chemoradiotherapy to surgery. Endoscopic examination performed before surgery showed that there was no obvious cancer in the esophagus. We resected the esophagus routinely. On the specimen, no cancer cells were detected upon macroscopic and microscopic examinations; metastasis was not detected in the lymph node. Achalasia is a recognized risk factor for esophageal SCC. In the treatment of superficial SCC, no difference of therapeutic effect was observed between surgery and chemoradiation. However, for the treatment of certain cases of SCC with achalasia, including the treatment of achalasia itself, surgery can be the preferred option of treatment.

[Clinical Response of Metastatic Colon Cancer to Chemotherapy with S-1 and Oxaliplatin - A Case Report].

Chemotherapy with S-1 and oxaliplatin is a new treatment for metastatic colorectal cancer. We present the first case of S-1, oxaliplatin, and bevacizumab therapy in our hospital. The patient was a 69-year-old woman with ascending colon cancer and multiple lung and liver metastases. She tended to suffer from constipation; stenoses at the cecum and colon cancer were detected by colon fiberscopy. Following surgical resection of the primary tumor, the patient received systemic chemotherapy with S-1, oxaliplatin, and bevacizumab. Following chemotherapy, CT showed no cancer in the lung and cancer reduction in the liver or dissemination. The patient had diarrhea and no appetite at first, so we reduced the oxaliplatin dose by 80%. After reduction of the oxaliplatin dose, we could treat the patient with S-1 and oxaliplatin continuously with no toxicity. S-1 and oxaliplatin chemotherapy is cost-effective, and has less toxicity than other chemotherapies, if proper measures are taken. It seemed to have a non-inferior response rate and disease control compared to other chemotherapies, such as FOLFOX. Thus, this chemotherapy is a valid choice for metastatic colorectal cancer.