In late-stage metastatic colorectal cancer, difficult treatment decisions should incorporate a thorough evaluation of the patient’s general condition and subject for shared decision making. ...Assessment of the individual patients’ prognosis is valuable in this setting. The aim was to analyze the prognostic value of plasma levels of total cell-free DNA, carcinoembryonic antigen and C-reactive protein in 97 heavily pretreated patients with metastatic colorectal cancer. Patients received irinotecan, cetuximab, and everolimus in a phase-2 clinical trial (clinicaltrials.gov NCT01387880). Plasma samples were used for DNA purification and quantification of total cell-free DNA by droplet digital polymerase chain reaction. Serum carcinoembryonic antigen and C-reactive protein were analyzed by routine methods. Clinical endpoints were overall survival and progression-free survival. A total of 82 patients had blood samples available for quantification of total cell-free DNA. Patients with pre-treatment cell-free DNA levels higher than the median total cell-free DNA (9800 alleles per milliliter plasma) had a significantly shorter overall survival of 4.3 months (95% confidence interval: 3.6–5.8) compared to patients with cell-free DNA levels below the median with an overall survival of 11.3 months (95% confidence interval: 8.0–14.8, p < 0.0001). When using the upper normal limit from a previously analyzed normal control group, the median overall survival was 11.3 (95% confidence interval: 7.3–14.8) and 4.3 (95% confidence interval: 3.7–6.1) months, respectively (p < 0.0001). Serum carcinoembryonic antigen and C-reactive protein had similar prognostic value with short overall survival and progression-free survival in patients with elevated levels compared to those within normal range. A high-risk profile of elevated cell-free DNA, carcinoembryonic antigen, and C-reactive protein was described, but in combined Cox regression multivariate analysis, only total cell-free DNA preserved a strong prognostic value. In conclusion, total cell-free DNA in plasma, carcinoembryonic antigen, and C-reactive protein could all contribute to assessment of patients’ prognosis and potentially aid in clinical decision making in patients with metastatic colorectal cancer.
Discovery of somatic mutations in the calreticulin gene (CALR) has identified a subgroup of Philadelphia-negative chronic myeloproliferative neoplasms (MPN) with separate haematological ...characteristics and prognosis. CALR mutations serve as novel markers both of diagnostic value and as targets for monitoring molecular responses during therapy. Interferon-α (IFN) selectively targets the malignant clone in a subset of MPN patients and can induce both haematological and molecular remissions in CALR mutated essential thrombocythemia (ET) patients. We investigated the response to IFN in a cohort of 21 CALR mutated MPN patients including ET, prefibrotic primary myelofibrosis (pre-PMF), and primary myelofibrosis (PMF) with a median follow-up of 31 months. For evaluation of a molecular response, we developed highly sensitive quantitative PCR (qPCR) assays for monitoring the mutant allele burden of the two most prevalent CALR mutations (type 1 and type 2). Thirteen patients (62%) experienced a decrease in the mutant allele burden with a median decline of 29% from baseline. However, only four patients, including patients with ET, pre-PMF, and PMF diagnosis, achieved molecular responder (MR) status with >50% reduction in mutant allele burden according to European LeukemiaNet (ELN) guidelines. MR patients displayed significant differences in the dynamics of the CALR mutant load with regard to time to response and dynamics in mutant allele burden after discontinuation of IFN treatment. Furthermore, we highlight the prognostic value of the CALR mutant allele burden by showing a close association with leucocyte- and platelet counts, hemoglobin concentration, in addition to plasma lactate dehydrogenase (LDH) irrespective of molecular response and treatment status.
Background and purpose: Circulating tumor DNA (ctDNA) is investigated in various cancers. In squamous cell carcinoma of the anus (SCCA) infection with human papilloma virus (HPV) is found in around ...90% of cases and here, plasma HPV (pHPV) can be used as ctDNA. Preliminary data have proved the ability to detect pHPV16 and -18 in SCCA. We have developed a highly sensitive method for measurement of six relevant pHPV subtypes, to investigate the elimination pattern of pHPV during chemo-radiotherapy (CRT) for SCCA and its clinical value. Material and methods: Patients treated at Aarhus University Hospital from 2016–2020 were included. P16 status in the primary biopsy was measured and 82% of patients had P16 positive tumor. Blood samples were collected prior to treatment (PT), mid treatment (MT), end of therapy (EOT), and during follow-up (FU). An in-house multiplex digital droplet PCR method measured pHPV subtypes 16, 18, 31, 33, 51, 58. Results: Samples from 88 patients were drawn PT (n = 73), MT (n = 72), EOT (n = 64) and during FU (n = 41). Plasma HPV was detectable in 52 patients and PT pHPV levels correlated to tumor stages. Three elimination patterns were observed during CRT with correlation to outcome: fast responders with no local or distant failures (0/12); slow responders with high risk of local failures (4/20), no distant failures; persistent molecular responders with high risk of distant failures (4/13), but no local failures, p < 0.01. Conclusion: During CRT, pHPV can divide patients with SCCA into three groups with significantly different risk of failure. The use of pHPV can potentially assist in clinical treatment decision.
MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as ...potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer.
We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa.
From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation).
We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.
Various conditions with cellular decay are associated with elevated cell-free DNA (cfDNA). This study aimed to investigate if perioperatively measured cfDNA levels were associated with the surgical ...approach, complications, or recurrence.
Plasma was obtained from patients who underwent surgery for colon cancer at admission and at the time of discharge. Quantitative measurement of cfDNA was performed by amplifying two amplicons of 102 base pairs (bp) and 132 bp of Beta-2-Microglobulin (B2M) and Peptidyl-Prolyl cis-trans Isomerase A (PPIA), respectively.
cfDNA was measured in 48 patients who underwent surgery for colonic cancer. Sixteen patients had recurrence during the follow-up period, fifteen developed a postoperative complication, and seventeen patients developed neither, acting as the control group. Postoperative cfDNA levels were significantly elevated from baseline samples, across all groups, with a median preoperatively B2M level of 48.3 alleles per mL and postoperatively of 220 alleles per mL and a median preoperatively level PPIA of 26.9 alleles per mL and postoperatively of 111.6 alleles per mL (p < 0.001 for B2M and p < 0.001 for PPIA). Postoperative levels of PPIA, but not B2M, were significantly higher in patients experiencing complications than in the control group (p = 0.036). However, a tendency towards an association between the surgical approach and the changes in cfDNA levels was found for PPIA (p = 0.058), and B2M (p = 0.087).
Plasma cfDNA was increased after surgery in all patients with colon cancer. Postoperative PPIA levels were significantly higher in patients experiencing surgical complications but not in B2M levels.
Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, ...further MPN‐specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN‐associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN‐associated mutations, and it therefore appears likely that emerging technologies such as next‐generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN.
Background and objectives: The JAK2 V617F tyrosine kinase mutation is present in the great majority of patients with polycythemia vera (PV), and approximately half of the patients with essential ...thrombocythemia (ET) and primary myelofibrosis (PMF). The three distinct disease entities may be considered as three phenotypic presentations of the same JAK2 V617F positive chronic myleoproliferative disorder. Together with physiological and genetic modifiers the phenotype may be determined by the JAK2 V617F allele burden. In the present study, we aimed to asses the JAK2 mutational load and its impact on phenotype.
Methods: A highly sensitive real‐time quantitative PCR (qPCR) assay was used for quantification of the JAK2 V617F mutational load in 165 patients with Philadelphia chromosome negative chronic myeloproliferative disorders (ET = 40, PV = 95, PMF = 30).
Results: We provide evidence of increasing JAK2 V617F allele burden from ET, over PV to PMF (P = 0.001 and P < 0.00001 respectively). The present data suggests the JAK2 V617F allele burden as a key determinant of the degree of myeloproliferation and myeloid metaplasia reflected by significantly higher levels of white blood cell counts (WBC) (P = 0.03), CD34 counts (P = 0.03), lactate dehydrogenase and Polycythemia Rubra Vera gene 1 levels (P = 0.03 and P < 0.00001 respectively), as well as lower platelet counts (P = 0.02) and more cases of splenomegaly (P = 0.001) in homozygous PV patients compared to their heterozygous counterparts.
Conclusion: The present study support the concept of the JAK2 V617F positive chronic myeloproliferative disorders as a biological continuum with phenotypic presentation in part influenced by JAK2 V617F mutational load.
Mutations in exon 9 of the calreticulin gene (CALR) frequently occur in patients with chronic myeloproliferative neoplasms (MPN). Patients exhibit spontaneous cellular immune responses to epitopes ...derived from the mutant CALR C-terminus, and CALR-mutant-specific T cells recognize autologous CALR-mutant malignant cells. This study investigated whether CALR-mutant-specific T cells occur naturally in CALRwt MPN-patients and in healthy individuals. Specific immune responses against epitopes in the mutant CALR peptide sequence were detected in both CALRwt MPN-patients and in healthy individuals. Healthy donors displayed more frequent and stronger CALR-mutant specific T-cell responses compared to the responses identified in CALR-mutant MPN-patients. Several T-cell responses were identified in healthy donors directly ex vivo. Importantly, by running functional analyses on live-sorted immune cells from healthy donors, we showed that circulating CALR-mutant-specific immune cells are T-memory cells. These findings suggest, that healthy individuals acquire a CALR exon 9 mutation, but the immune system reacts and clears the mutant cells, and during this reaction generates CALR-mutant specific T-memory cells. We believe that these findings provide the evidence for tumor immune surveillance in MPN.
Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of ...inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER- IBC, normal breast tissue and breast cancer cell lines.
The reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue.
A group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations.
The reference genes of choice when performing RT-qPCR on normal and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1) employed as an average, or PUM1 as a single gene.
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