Carbonic Anhydrase IX Expression and Treatment Response Measured in Rectal Adenocarcinoma Following Neoadjuvant Chemo-Radiotherapy.

The overexpression of the pH regulator carbonic anhydrase IX (CAIX) due to hypoxic/metabolic stress was reported in various tumors as an adverse prognostic feature. Our retrospective study aimed to investigate the general pattern and dynamics of CAIX expression in rectal adenocarcinoma following preoperative neoadjuvant therapy (NAT) in matched initial biopsy and surgical resection samples. A total of 40/55 (72.72%) of the post-treatment samples showed partial CAIX expression, frequently in the proximity of hypoxic tumor areas. CAIX expression showed a significant increase in post-treatment tumors (mean% 21.8 ± 24.9 SD vs. 39.4 ± 29.4 SD, p < 0.0001), that was not obvious in untreated tumors (mean% 15.0 ± 21.3 SD vs. 20 ± 23.02, p = 0.073). CAIXhigh phenotype was associated with mutant KRAS status and lack of pathological regression (WHO Tumor Regression Grade 4 and 5). However, the adverse effect of CAIX on overall or progression-free survival could not be statistically confirmed. In conclusion, the dynamic upregulation of CAIX expression is a general feature of rectal adenocarcinoma following neoadjuvant chemo-radiotherapy indicating therapy-induced metabolic reprogramming and cellular adaptation. A synergism of the CAIX-associated regulatory pathways and the mutant KRAS oncogenic signaling most likely contributes to therapy resistance and survival of residual cancer.

Clonal diversity in KRAS mutant colorectal adenocarcinoma under treatment: Monitoring of cfDNA using reverse hybridization and DNA sequencing platforms.

Biological heterogeneity is a key feature of malignancies that significantly contributes to disease progression and therapy resistance. Residual/relapsed tumor foci may represent genetically divergent subclones, which remain uncovered as repeated and multiple tumor sampling is usually limited. The analysis of circulating free DNA (cfDNA) from the peripheral blood plasma (also called a liquid biopsy, LB) is a new achievement that provides an effective tool for follow-up monitoring of cancer-related genetic status. The present study highlights the phenomenon of mutational variability observed in patients with metastatic KRAS mutant colorectal cancer (mCRC) during treatment with bevacizumab in combination in a longitudinal fashion. The prospective study included 490 mCRC patients evaluated between 2020 and 2022 in our institution. Out of the 211 KRAS mutant cases (43.06%) 12 tumors were identified with multiple KRAS gene variants (5.68%). Detailed follow-up investigations were possible in 3 of these patients including the genotyping of the primary and available metastatic tumors, and the peripheral blood cfDNA. cfDNA was collected from three different time points before and between cycles of combined treatment with bevacizumab chemotherapy. KRAS gene variants were identified using reverse-hybridization strips, and next-generation sequencing (NGS), and confirmed by conventional Sanger sequencing. Interestingly, surgery and multiple treatment cycles reorganized the mutational profiles in the selected cases. The effect of the treatments resulted either in the overrepresentation of one of the pre-existing gene variants or in the appearance of new KRAS variants absent in the primary sample, according to the plasma cfDNA findings. Besides the KRAS variants demonstrated by targeted analysis, NGS mutational profiling identified some additional pathogenic variants from the cfDNA samples (including NRAS and MET alterations). In conclusion, plasma cfDNA sampling enables the monitoring of mutational heterogeneity and subclonal dynamics of the actual metastatic tumor mass in mCRC. The pattern of molecular profile potentially reflects a differential drug response determining further progression.

Cell-free DNA From Pleural Effusion Samples: Is It Right for Molecular Testing in Lung Adenocarcinoma?

Pathogenic molecular features gained specific significance in therapeutic decisions in lung carcinoma in the past decade. Initial and follow up genetic testing requres appropriate amounts and quality of tumor derived DNA, but tumor sampling, especially for disease monitoring is generally limited. Further to the peripheral blood (PB), samples from pleural fluid, accumulating in diverse lung processes might serve as an alternative source for cell-free DNA (cfDNA) for genetic profiling. In our study, cfDNA isolated from the pleural effusion and from the PB, and genomic DNA (gDNA) obtained from tissue/cellular samples were analyzed and compared from altogether 65 patients with pulmonary disease, including 36 lung adenocarcinomas. The quantity of effusion cfDNA yield appeared to be significantly higher compared to that from simultaneously collected PB plasma (23.2 vs. 4.8 ng/µl, p < 0.05). Gene mutations could be safely demonstrated from the effusion cfDNA fraction obtained from adenocarcinoma patients, 3/36 EGFR, 9/36 KRAS and 1/36 BRAF gene variants were detected. In this series, 9/13 samples showed an effusion+/plasma-mutational status, while only 1/13 samples presented with the opposite findings (effusion-/plasma+). gDNA analysis from sediment cell blocks from the identical effusion sample was surprisingly ineffective for lung adenocarcinoma profiling due to the low DNA yield. In conclusion, the cell free supernatant of pleural effusions appears to concentrate cancer derived cfDNA and seems to be particularly suitable for serial genotyping of pulmonary adenocarcinoma.

Novel RB1 and MET Gene Mutations in a Case with Bilateral Retinoblastoma Followed by Multiple Metastatic Osteosarcoma.

Retinoblastoma (Rb) is a malignant tumor of the developing retina that affects children before the age of five years in association with inherited or early germline mutations of the RB1 gene. The genetic predisposition is also a driver for other primary malignancies, which have become the leading cause of death in retinoblastoma survivors. Other malignancies can occur as a consequence of radiotherapy. We describe a patient with retinoblastoma in which we detected a novel RB1 c.2548C > T, p.(Gln850Ter) and a synchronous MET c.3029C > T, p.(Thr1010Ile) mutation as well. After presenting with bilateral retinoblastoma, the patient developed at least four different manifestations of two independent osteosarcomas. Our goal was to identify all germline and somatic genetic alterations in available tissue samples from different time periods and to reconstruct their clonal relations using next generation sequencing (NGS). We also used structural and functional prediction of the mutant RB and MET proteins to find interactions between the defected proteins with potential causative role in the development of this unique form of retinoblastoma. Both histopathology and NGS findings supported the independent nature of a chondroblastic osteosarcoma of the irradiated facial bone followed by an osteoblastic sarcoma of the leg (tibia).

Quadruplicate Synchronous Adenocarcinoma of the Colon with Distant Metastases-Long-Term Molecular Follow-Up by KRAS and TP53 Mutational Profiling.

Anatomically independent tumor foci represent biologically distinct neoplasias, potentially featured by different progressivity and treatment responsiveness. To demonstrate the biological complexity, a metastatic colon adenocarcinoma patient originally presenting with four independent primary tumors of the right colon half and altogether eight distant metastases was followed by molecular testing. Next-generation sequencing results highlighted the mutational profile of the individual primaries and the dynamics of the different gene variants observed during follow-up. The four primary colon tumors presented with four different KRAS genotypes, one of them with a wild-type and three with pathogenic variants, without overlap. These were the following: c.35G > A; p.Gly12Asp with 40.6% variant allele frequency (VAF); c.34G > T; p.Gly12Cys with 16.2% VAF and c.35G > T; p.Gly12Val with 15.1% VAF. In metastatic tumors, with one exception where no mutation was detected, only the KRAS c.34G > T; p.Gly12Cys mutation could be detected. TP53 gene variants were variable in the primary tumors, with a single dominant variant evolving in the follow-up metastases (c.820G > T; p.Val274Phe). Genetic profiling of individually developing synchronous malignancies uncovers the clonal relations of metastatic tumors. NGS gene panels provide a solution to follow the dynamics of key oncogene variants during the course of the disease and greatly contribute to therapy optimization.

High Genetic Diversity and No Evidence of Clonal Relation in Synchronous Thyroid Carcinomas Associated with Hashimoto's Thyroiditis: A Next-Generation Sequencing Analysis.

The close association between pre-existing Hashimoto's thyroiditis and thyroid cancer is well established. The simultaneous occurrence of multiple neoplastic foci within the same organ suggests a common genotoxic effect potentially contributing to carcinogenesis, the nature of which is still not clear. Next-generation sequencing (NGS) provides a potent tool to demonstrate and compare the mutational profile of the independent neoplastic foci. Our collection of 47 cases with thyroid carcinoma and Hashimoto's thyroiditis included 14 with at least two tumorous foci. Detailed histological analysis highlighted differences in histomorphology, immunoprofile, and biological characteristics. Further, a 67-gene NGS panel was applied to demonstrate the mutational diversity of the synchronic tumors. Significant differences could be detected with a wide spectrum of pathogenic gene variants involved (ranging between 5 and 18, cutoff >5.0 variant allele frequencies (VAF)). Identical gene variants represented in both synchronous tumors of the same thyroid gland were found in only two cases (BRAF and JAK3 genes). An additional set of major driver mutations was identified at variable allele frequencies in a highly individual setup suggesting a clear clonal independence. The different BRAF statuses in coincident thyroid carcinoma foci within the same organ outline a special challenge for molecular follow-up and therapeutic decision-making.