Molecular Deciphering of Colorectal Cancer: Exploring Molecular Classifications and Analyzing the Interplay among Molecular Biomarkers MMR/MSI, KRAS, NRAS, BRAF and CDX2 - A Comprehensive Literature Review.

Background: Colorectal cancer (CRC) exhibits molecular and morphological diversity, involving genetic, epigenetic alterations, and disruptions in signaling pathways. This necessitates a comprehensive review synthesizing recent advancements in molecular mechanisms, established biomarkers, as well as emerging ones like CDX2 for enhanced CRC assessment. Material and Methods: This review analyzes the last decade's literature and current guidelines to study CRC's molecular intricacies. It extends the analysis beyond traditional biomarkers to include emerging ones like CDX2, examining their interaction with carcinogenic mechanisms and molecular pathways, alongside reviewing current testing methodologies. Results: A multi-biomarker strategy, incorporating both traditional and emerging biomarkers like CDX2, is crucial for optimizing CRC management. This strategy elucidates the complex interaction between biomarkers and the tumor's molecular pathways, significantly influencing prognostic evaluations, therapeutic decision-making, and paving the way for personalized medicine in CRC. Conclusions: This review proposes CDX2 as an emerging prognostic biomarker and emphasizes the necessity of thorough molecular profiling for individualized treatment strategies. By enhancing CRC treatment approaches and prognostic evaluation, this effort marks a step forward in precision oncology, leveraging an enriched understanding of tumor behavior.

Evaluation of the Immunohistochemical Scoring System of CDX2 Expression as a Prognostic Biomarker in Colon Cancer.

Encoded by the CDX2 homeobox gene, the CDX2 protein assumes the role of a pivotal transcription factor localized within the nucleus of intestinal epithelial cells, orchestrating the delicate equilibrium of intestinal physiology while intricately guiding the precise development and differentiation of epithelial tissue. Emerging research has unveiled that positive immunohistochemical expression of this protein shows that the CDX2 gene exerts a potent suppressive impact on tumor advancement in colorectal cancer, impeding the proliferation and distant dissemination of tumor cells, while the inhibition or suppression of CDX2 frequently correlates with aggressive behavior in colorectal cancer. In this study, we conducted an immunohistochemical assessment of CDX2 expression on a cohort of 43 intraoperatively obtained tumor specimens from patients diagnosed with colon cancer at Colțea Clinical Hospital in Bucharest, between April 2019 and December 2023. Additionally, we shed light on the morphological diversity within colon tumors, uncovering varying differentiation grades within the same tumor, reflecting the variations in CDX2 expression as well as the genetic complexity underlying these tumors. Based on the findings, we developed an innovative immunohistochemical scoring system that addresses the heterogeneous nature of colon tumors. Comprehensive statistical analysis of CDX2 immunohistochemical expression unveiled significant correlations with known histopathological parameters such as tumor differentiation grades (p-value = 0.011) and tumor budding score (p-value = 0.002), providing intriguing insights into the complex involvement of the CDX2 gene in orchestrating tumor progression through modulation of differentiation processes, and highlighting its role in metastatic predisposition. The compelling correlation identified between CDX2 expression and conventional histopathological parameters emphasizes the prognostic significance of the CDX2 biomarker in colon cancer. Moreover, our novel immunohistochemical scoring system reveals a distinct subset of colon tumors exhibiting reserved prognostic outcomes, distinguished by their "mosaic" CDX2 expression pattern.

NGS mutational status on first diagnostic tissue, liquid biopsy and mastectomy in G2-G3 breast cancer.

Breast cancer is one of the more frequently diagnosed cancers leading to death in women, and, like other tumor types, it is heterogeneous in its immunophenotype. It harbors mutations that modify tumor aggressiveness, therapy responses, residual disease, drug resistance, and relapse rates in advanced stages. This study aims to assess the mutational status of G2 and G3 tumors using next-generation sequencing (NGS) on initial tissue biopsies, liquid biopsies, and mastectomy specimens. The histopathological (HP) diagnosis for the 32 selected cases was established via Hematoxylin-Eosin (HE) staining by two observers. For the immunohistochemical (IHC) testing of estrogen receptor (ER), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (HER2), we used the Ventana BenchMark Ultra. Ki67 testing was conducted using Bond-III from Leica. For cases with a score of 2+, gene amplification was assessed by silver-enhanced in situ hybridization (ISH) (SISH; Inform HER2 Dual ISH) on Ventana BenchMark Ultra. NGS analysis was initially performed on biopsies and plasma, and later on mastectomy specimens. After automated deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) extraction, concentrations were measured using the Invitrogen Qubit system. Libraries were created using Oncomine systems, and sequencing and analysis were done with the Ion Torrent system. Most tumors were graded as G3 (19 cases), with Luminal A being the predominant molecular subtype, and a significant number displayed HER2∕HER2-low characteristics (24 out of 32 cases). The NGS assessment showed that phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations were the most frequent across all sample types. A significant limitation was the high number of invalid plasma tests due to pre-analytical handling errors or transport issues. Nonetheless, plasma testing (liquid biopsy) proved useful for monitoring tumor evolution and assessing residual disease.

Liquid biopsy in lung cancer management.

Liquid biopsy is a promising tool for a better cancer management and currently opens perspectives for several clinical applications, such as detection of mutations when the analysis from tissue is not available, monitoring tumor mutational burden and prediction of targeted therapy response. These characteristics validate liquid biopsy analysis as a strong cancer biomarkers source with high potential for improving cancer patient's evolution. Compared to classical biopsy, liquid biopsy is a minimal invasive procedure, and it allows the real-time monitoring of treatment response. Considering that lung cancer is the most common cause of cancer-associated death worldwide and that only 15-19% of the lung cancer patients survive five years after diagnosis, there is an important interest in improving its management. Like in other types of solid cancers, lung cancer could benefit from liquid biopsy through a simple peripheral blood sample as tumor-related biomarkers, such as circulating tumor cells (CTCs), cell-free nucleic acids (cfNA) [cell-free ribonucleic acid (cfRNA) and cell-free deoxyribonucleic acid (cfDNA)], exosomes and tumor-educated platelets (TEPs) may shed into circulation because of necrosis or in an active manner. More, the detection and analysis of these biomarkers could lead to a better understanding of oncological diseases like lung cancer. The better the tumor profile is established; the better management is possible. However, this approach has currently some limitations, such as low cfNA concentration or low count of CTCs that might be overcome by improving the actual methods and technologies.

Dual NGS Comparative Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Samples of Non-Small Cell Lung Carcinoma (NSCLC).

Lung cancer ranks second worldwide after breast cancer and third in Europe after breast and colorectal cancers when both sexes and all ages are considered. In this context, the aim of this study was to emphasize the power of dual analysis of the molecular profile both in tumor tissue and plasma by NGS assay as a liquid biopsy approach with impact on prognosis and therapy modulation in NSCLC patients. NGS analysis was performed both from tissue biopsies and from cfNAs isolated from peripheral blood samples. Out of all 29 different mutations detectable by both NGS panels (plasma and tumor tissue), seven different variants (24.13%; EGFR L858R in two patients, KRAS G13D and Q61H and TP53 G244D, V197M, R213P, and R273H) were detected only in plasma and not in the tumor itself. These mutations were detected in seven different patients, two of them having known distant organ metastasis. Our data show that NGS analysis of cfDNA could identify actionable mutations in advanced NSCLC and, therefore, this analysis could be used to monitor the disease progression and the treatment response and even to modulate the therapy in real time.

Mutational status of KRAS and MMR genes in a series of colorectal carcinoma cases.

BACKGROUND: The KRAS gene mutation is the most common somatic change in colorectal carcinoma (CRC) and is predictive of resistance to anti-epidermal growth factor receptor (EGFR) therapy in the metastatic forms. Microsatellite instability (MSI), a mismatch repair (MMR) system defect, accounts for 15-20% of all CRCs, more frequent in early stages. CRCs with MSI present better prognosis, a distinct histopathological aspect and a different response to chemotherapy. Patients with both KRAS wild type and MSI have a reduced risk of dissemination and recurrence. MATERIALS AND METHODS: Our study included formalin-fixed paraffin-embedded tissue samples from 40 patients with metastatic CRCs, aged between 40 and 71 years old, gender (males/females) ratio 2.33:1. The MMR proteins were analyzed using an indirect bistadial immunohistochemical (IHC) technique with monoclonal antibodies. KRAS mutations were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: Of the 40 tumors analyzed, 40% presented KRAS mutations located in codon 12 or codon 13. IHC expression of MMR proteins revealed a microsatellite stable status in 35 cases, including 15 cases with mutated KRAS. MSI status was identified in five cases (four with KRAS wild type). All MSI tumors had a poorer histological differentiation and four cases revealed a mucinous phenotype. Eighty percent of the patients with MSI status were older women. CONCLUSIONS: Our study demonstrates a 20% frequency of mutated KRAS in MSI CRCs, the incidence of KRAS mutations being inversely correlated with MSI status in these tumors. MMR protein deficient CRCs tend to occur in older females, have a poorer differentiation and are frequently associated with KRAS wild type.

Adenocarcinoma versus pancreatic neuroendocrine tumor - case report.

Pancreatic cancer represents one of the most aggressive types of cancer, resulting in a late diagnosis and rapid death (poor overall survival). After adenocarcinoma (counting almost 80% of cases of pancreatic cancer), the second category, as frequency, is represented by the family of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Pancreatic cancer is characterized by genetic heterogeneity and may results in different evolution among metastases, which may acquire driver mutations with the ability to transform under the action of several cancer treatments. Here we report a case of a 64-year-old patient diagnosed with pancreatic tumor localized on the body and tail, invasive in the splenic and portal vein, pT3pN0M0 (adenocarcinoma pancreatic cancer), treated with a multimodal approach: surgery (splenectomy and distal pancreatectomy, with suture of the portal vein), chemotherapy, in 2010, that relapsed in 2015, with local recurrence that was resected and distant liver metastases. Immunohistochemistry of the recurrence tumor showed a neuroendocrine transformation of the tumor, with major implications in treatment and prognosis. Computed tomography examination, as well as histopathological and immunohistochemically testing, sustained positive and differential diagnosis.

Vaginal epithelioid angiosarcoma: a rare case.

Epithelioid angiosarcoma of the vagina is a rare variant, easily misdiagnosed as other epithelial neoplasms. On Hematoxylin-Eosin-stained sections, the pathologist encounters sheets of large, mildly to moderately pleomorphic epithelioid cells, with abundant eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli. We report the case of a 22-year-old woman initially diagnosed with condiloma-like tumor of the left vaginal wall, which turned out positive at immunostaining for epithelioid angiosarcoma. In her case, after the failure of chemotherapy in controlling the relapse of the disease, the only treatment option was radical hysterectomy with bilateral salpingo-oophorectomy.

Intravascular Lymphomatosis Presenting as Skin Lesions and Subacute Encephalopathy.

Intravascular lymphomatosis is a neoplastic multisystemic disease; it is a rare subtype of diffuse large cell lymphoma characterized by the presence of lymphoma cells in the lumina of small vessels. A 49-year-old Caucasian woman was admitted to the Department of Internal Medicine for fatigue, night sweats, loss of weight, and multiple nodules in the forearms. Three months ago the patient's family noticed problems with her cognitive function, she displayed difficulties with common daily tasks. The neurological examination revealed bradypsychia. Laboratory data showed modestly high levels of lactate dehydrogenase, and C-reactive protein. The day after admission, the patient had headache which raised in intensity; his mental status deteriorated, she was disoriented to time and place. She presented nucal rigidity. The CSF examination revealed a hemorrhagic aspect, elements 30/mm3, cytology: lymphocytes 90%, numerous erythrocytes, proteinorachia 96 mg/dL, glycorrachia 60 mg/dL. Intravenous Methylprednisolone (0.5 g two times a day) and Mannitol 20% 1g/kgw/day were administered for five days without response. She became comatose and she died six days after hospitalization. The post-mortem macroscopical brain examination showed a swallen brain, with diffuse hemorrhagic areas in the supratentorial subcortical regions. Microscopical examination showed capillaries, venules, and many arterioles distended by large malignant cells suggesting malignant lymphocytes which were intraluminal. Every organ was involved, except for bone marrow and lymph nodes. Immunohistochemical studies showed intensive staining for B cells. and negative staining for factor VIII related antigen, a specific endothelial cell marker. Intravascular lymphomatosis was the post-mortem diagnostic. It represents a difficult diagnostic challenge which involves laboratory, imagistic and immunohistochemical investigations.

KRAS gene mutations - prognostic factor in colorectal cancer?

The colorectal cancer (CRC) modern therapy is using adjuvant and neoadjuvant companion therapeutic agents, part of them having an anti-angiogenic action. Their benefic effect can be annulated by some gene mutations, which are interfering in signal transduction pathways. One of the more frequent activating mutations is occurring in the KRAS gene. We assessed the KRAS mutations by two molecular methods, in a group of patients with a follow-up until 144 months, aiming to establish eventual correlations between the presence of mutations and the evolution of patients. We tried to appreciate the prognostic value of these mutations. A retrospective study was conducted on 74 patients treated by radical surgery; the surgical specimens were analyzed macroscopically and the histopathological type and degree were established. PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and pyrosequencing were performed on paraffin-embedded tumor specimens. Statistical analysis showed significant differences in survival between patients with wild type gene and patients with mutation in codon 13; the same results were also obtained regarding TNM I, II stages or Dukes type A and B cases. However, for the patients in stage IV pTNM, the evolution was slightly better in association with a KRAS mutation than in wild type cases.

Pitfalls in diagnosing a pancreatic neuroendocrine tumor: a case report.

Pancreatic neuroendocrine tumors are a rare subset of pancreatic neoplasms. We report the case of a 33-year-old female patient who was admitted to the Diabetes Clinic of Craiova, Romania, due to a two-year history of episodic neuroglycopenic hyperinsulinemic hypoglycemic symptoms, suggestive for insulinoma associated with facial and upper trunk flushing characteristic to carcinoid syndrome. During these episodes, the laboratory investigations showed hypoglycemia (38 mg/dL), hyperinsulinemia (54.72 µU/mL) and normal values of beta-hydroxybutyrate, chromogranin A, serotonin, anti-insulin antibodies and urinary levels of 5-hydroxyindoleacetic acid. Endoscopic ultrasound with SonoVue and 3T MRI revealed an 18.3/16.3 mm hypervascular tissular mass situated in the uncinate process of the pancreatic head in close contact with the superior mesenteric vein without invasion and no other detectable secondary lesions in the pancreas or any other abdominal viscera. Patient underwent enucleation of pancreatic tumor. The histological and immunohistochemical findings indicated a functional well-differentiated pancreatic neuroendocrine tumor, G1 category according to the World Health Organization (WHO) criteria, with uncertain behavior (Ki67 index was 3%), confined to the pancreas, but with tumoral invasion of the delimiting conjunctive capsule. No evidence of tumoral CK19 staining, mitoses and necrosis, angioinvasion or extra-pancreatic invasion was observed. A post-operative nine-month follow-up showed resolution of hypoglycemic symptoms, normalized blood glucose and insulin levels and no evidence of recurrence. Our case report highlights the pitfalls in diagnosing a functional pancreatic neuroendocrine tumor due to atypical symptoms, the difficulty of identification and precise location of the small-size tumor and uncertain histopathological and immunohistochemical behavior.