Search

Importance: More information is needed to guide referring subspecialists on the appropriate patient evaluation before magnetic resonance defecography (MRD)., Objectives: This study aimed to evaluate how often health care providers perform digital rectal examination (DRE) before ordering MRD to investigate causes of bowel and pelvic floor complaints., Study Design: We conducted a retrospective cohort review, including MRD performed on female patients at an integrated health care system from 2016 through 2020. The primary outcome was the presence or absence of documented rectal examination in the year before defecography by the referring provider or 6 months prior by a primary care physician or pelvic floor physical therapist. We hypothesized that the overall rate of rectal examination would be high and unaffected by the referring provider's subspecialty., Results: Three hundred-four defecography tests were performed, with 209 patients (68.8%) referred by gastroenterology providers and 95 (31.2%) from other specialties. Gastroenterologists performed DRE in 32.8% of patients, in contrast to 84.4% of patients referred by other specialties ( P < 0.001). When comparing subspecialties that most commonly refer patients for MRD (gastroenterology, colorectal surgery and urogynecology), there was a statistically significant difference between gastroenterologists and colorectal surgeons ( P < 0.001) as well as urogynecologists ( P < 0.001) but no difference in the rate of rectal examination between colorectal surgeons and urogynecologists ( P = 1.00)., Conclusions: At our single integrated health system, the rate of DRE before MRD testing varied significantly by specialty. Our findings highlight the need for better understanding of DRE utility in the algorithms for evaluation of bowel and pelvic floor disorders., (Copyright © 2024 American Urogynecologic Society. All rights reserved.)

Background: Bevacizumab, a monoclonal antibody against soluble VEGFA, is an approved and commonly administered anti-angiogenic drug in patients with metastasized colorectal cancer (mCRC). The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months, and acquired resistance mechanisms are largely unknown. Here, we employed whole-genome sequencing of plasma DNA to evaluate the tumor genome of patients undergoing treatment with bevacizumab to determine novel aberrations associated with resistance., Methods: Using longitudinal plasma analyses, we studied the evolution of tumor genomes in a mCRC cohort (n = 150) and conducted analyses of CRC cases from The Cancer Genome Atlas (TCGA) database (n = 619) to identify associations between genomic aberrations and clinical features. We employed whole-genome sequencing to identify the most frequently occurring focal somatic copy number alterations (SCNAs). Using the TCGA data as a comparative and supporting dataset, we defined the minimally amplified overlapping region and studied the mechanistic consequences of copy number gain of the involved genes in this segment. In addition, we established an in vitro cell model and conducted downstream gene expression and cell viability assays to confirm our findings from the patient dataset., Results: We observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of CRC cases from the TCGA database suggested that this amplicon is associated with more advanced stages. We confirmed that this 13q12.2 amplicon frequently emerges later during the clinical course of disease. After defining the minimally amplified region, we observed that the amplification and expression of one gene, POLR1D, impacted cell proliferation and resulted in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab treatment. In fact, in several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with therapy resistance., Conclusions: Non-invasive analyses of cell-free DNA from patients undergoing treatment with bevacizumab enabled the tracking of evolving tumor genomes and helped identify a recurrent focal SCNA of clinical relevance. Here, we describe a novel resistance mechanism against a widely applied treatment in patients with mCRC which will impact the clinical management of patients.

Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although distant metastases account for most of the deaths in cancer patients, fundamental questions regarding mechanisms that promote or inhibit metastasis remain unanswered. We show the impact of mutations, genomic instability, lymphatic and blood vascularization, and the immune contexture of the tumor microenvironment on synchronous metastases in large cohorts of colorectal cancer patients. We observed large genetic heterogeneity among primary tumors, but no major differences in chromosomal instability or key cancer-associated mutations. Similar patterns of cancer-related gene expression levels were observed between patients. No cancer-associated genes or pathways were associated with M stage. Instead, mutations of FBXW7 were associated with the absence of metastasis and correlated with increased expression of T cell proliferation and antigen presentation functions. Analyzing the tumor microenvironment, we observed two hallmarks of the metastatic process: decreased presence of lymphatic vessels and reduced immune cytotoxicity. These events could be the initiating factors driving both synchronous and metachronous metastases. Our data demonstrate the protective impact of the Immunoscore, a cytotoxic immune signature, and increased marginal lymphatic vessels, against the generation of distant metastases, regardless of genomic instability., (Copyright © 2016, American Association for the Advancement of Science.)

Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4A(V78M) demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

The complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring., (Copyright © 2013 UICC.)

Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes [e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA] found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse., (©2013 AACR.)

We used laser microdissection and pressure catapulting (LMPC) to isolate and subsequently recultivate cells from the colorectal cancer cell line HCT116. Cell isolation and recultivation was repeated 5 times from the preceding cell culture. The chromosomal composition of the HCT116 cells was analyzed at the beginning and after each recultivation. We did not observe any additional chromosomal structural or copy number changes during this procedure. Our data suggest that the procedure of cell isolation by LMPC and subsequent recultivation does not affect the karyotype of the respective cells. This technology will facilitate the analysis of different cell populations in a heterogeneous tumor cell population.