Longitudinal measurement of HPV copy number in cell-free DNA is associated with patient outcomes in HPV-positive oropharyngeal cancer

IntroductionOropharyngeal squamous cell carcinoma (OPSCC) is increasing in global prevalence and is divided into two types dependent on association with human papillomavirus (HPV). Assay of HPV copy number in plasma cell-free DNA (cfDNA) provides a minimally invasive method for detecting and monitoring tumour-derived HPV, with potential for enhancing clinical care.Materials and methodsIn a prospectively recruited cohort of 104 OPSCC patients, we evaluate the utility of cfDNA droplet digital PCR (ddPCR) as a method for characterisation and longitudinal monitoring of patients with OPSCC.ResultsddPCR assay of pre-treatment plasma cfDNA for five HPV types showed overall 95% concordance with p16 immunohistochemistry and PCR analysis of tumour tissue. Longitudinal sampling in 48 HPV+ve patients, with median follow-up of 20 months, was strongly associated with patient outcomes. Persistently elevated cfDNA-HPV post-treatment was associated with treatment failure (2/2 patients) and an increase of cfDNA-HPV in patients whose HPV levels were initially undetectable post-treatment was associated with disease recurrence (5/6 patients). No recurrence was observed in patients in whom cfDNA-HPV was undetectable in all post-treatment samples. In two patients, sequential HPV measurement could have avoided surgical intervention which did not confirm recurrence.ConclusionThe high concordance of pre-treatment plasma cfDNA-HPV analysis with tissue-based assays, together with the clinical associations of sequentially measured post-treatment cfDNA-HPV copy number add to a growing body of evidence that suggest utility of cfDNA-HPV ddPCR in management of OPSCC. Standardised clinical trials based on these data are now needed to assess the impact of such testing on overall patient outcomes.     

MultiplexKRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction

BackgroundCell-free DNA (cfDNA) from plasma offers easily obtainable material forKRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy.Patients and methodsSamples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with aKRAS^G12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care.ResultsEighty-eight patients (73%) hadKRAS^G12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) hadKRAS^G12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% ofKRAS^G12/G13 cfDNA in the wild-type background was associated with shorter survival (P=0.001).Conclusion(s)Multiplex detection ofKRAS^G12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutantKRAS^G12/G13 in cfDNA corresponded with shorter survival.     

Detection of IDH1 Mutation in cfDNA and Tissue of Adult Diffuse Glioma with Allele-Specific qPCR

Background: The World Health Organization (WHO) classification of central nervous system (CNS) tumors necessitates testing of isocitrate dehydrogenase (IDH) 1/2 gene mutation in patients with adult-type diffuse glioma (ADG) for better disease management. In clinical practice, the testing of IDH1 is primarily achieved using immunohistochemistry (IHC) specific to IDH1-R132, which carries a sensitivity of 80% and specificity of 100%. However, in some cases, non-specific background staining or regional heterogeneity in the protein expression of IDH1 may necessitate confirmatory genetic analysis. Robust and reliable assays are needed for IDH1/2 mutation testing. The aim of the current study was to detect IDH1 mutation in cfDNA and tissue of adult-type diffuse glioma with allele-specific qPCR. Materials and Methods: In the current study, IDH1-R132H mutation was analyzed in tumor tissue with paired cell-free DNA (cfDNA) in patients with ADG (n = 45) using IHC and competitive allele-specific Taqman PCR (CAST-PCR). Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue and matched serum for cfDNA using commercially available kits. CAST-PCR with IHC for the detection of IDH1-R132H mutation was also compared. Results: The IDH1-R132H mutation was detected in 46.67% (21/45) cases and 57.78% (26/45) cases using IHC and allele-specific CAST-PCR. In cfDNA of matched IDH1-mutant FFPE tissue DNA, IDH1-R132H mutation was detected in 11.54% (3/26) using CAST-PCR. The concordance rate for IDH1-R132Hmutation between IHC and CAST-PCR was 80.77% (21/26). Conclusion: The CAST-PCR assay is more precise and sensitive for  IDH1-R132Hdetection than traditional IHC, and IDH1-R132H mutation detection using cfDNA may add to the current methods of glioma genomic characterization.     

Deep learning captures selective features for discrimination of microsatellite instability from pathologic tissue slides of gastric cancer

Microsatellite instability (MSI) status is an important prognostic marker for various cancers. Furthermore, because immune checkpoint inhibitors are much more effective in tumors with high level of MSI (MSI-H), MSI status is routinely tested in multiple cancer types. Therefore, many studies have tested the feasibility of deep learning (DL)-based prediction of MSI status from hematoxylin and eosin (H&E)-stained tissue slides. In the present study, we attempted a fully automated classification of MSI status in gastric cancer (GC) tissue slides. For frozen and formalin-fixed paraffin-embedded (FFPE) GC tissues from The Cancer Genome Atlas (TCGA), the areas under the curves (AUCs) for the receiver operating characteristic (ROC) curves were 0.893 and 0.902, respectively. The classifier trained with the TCGA FFPE tissues performed well on an external validation Asian FFPE cohort, with an AUC of 0.874. However, the DL-based classifier seems incompatible with cancers from different organs because morphologic features of MSI-H tissues are different. Analysis of histomorphologic features of MSI-H GC tissues suggested that MSI-H GC could largely be divided into two groups: intestinal type tumors with moderate to poor differentiation and diffuse type mucinous tumors. However, the recognizable morphologic features cannot completely explain the good performance of the DL-based classifier. These results indicate that DL could automatically learn the optimal features for discrimination of MSI status in GC tissue slides. This study demonstrated the potential of a DL-based MSI classifier as a screening tool for definitive cases.     

Microsatellite instability and mutation analysis of candidate genes in unselected sardinian patients with endometrial carcinoma

BACKGROUND: Microsatellite instability (MSI) is due mostly to a defective DNA mismatch repair (MMR). Inactivation of the two principal MMR genes, hMLH1 and hMSH2, and the PTEN tumor suppressor gene seems to be involved in endometrial tumorigenesis. In this study, Sardinian patients with endometrial carcinoma (EC) were analyzed to assess the prevalence of both the mutator phenotype (as defined by the presence of MSI and abnormal MMR gene expression at the somatic level) and the hMLH1, hMSH2, and PTEN germline mutations among patients with MSI positive EC. METHODS: Paraffin embedded tissue samples from 116 consecutive patients with EC were screened for MSI by polymerase chain reaction-based microsatellite analysis. Immunohistochemistry (IHC) with anti-hMLH1 and anti-hMSH2 antibodies was performed on MSI positive tumor tissue sections. Germline DNA was used for mutational screening by denaturing high-performance liquid chromatography analysis and automated sequencing. RESULTS: Thirty-nine patients with EC (34%) exhibited MSI; among them, 25 tumor samples (64%) showed negative immunostaining for hMLH1/hMSH2 proteins (referred to as IHC negative). No disease-causing mutation within the coding sequences of the hMLH1/hMSH2 and PTEN genes was found in patients with EC who had the mutator phenotype (MSI positive and IHC negative), except for a newly described hMLH1 missense mutation, Ile655Val, that was observed in 1 of 27 patients (4%). Although MSI was more common among patients with advanced-stage EC and increased as the tumor grade increased, no significant correlation with disease free survival or overall survival was observed among the two groups (MSI positive or MSI negative) of patients with EC. CONCLUSIONS: In patients with MSI positive EC, epigenetic inactivations rather than genetic mutations of the MMR genes seem to be involved in endometrial tumorigenesis. No prognostic value was demonstrated for MSI in patients with EC.     

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well‐known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.     

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:

Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.

Methods:

We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.

Results:

The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.

Conclusions:

Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.     

Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer.We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.     

Microsatellite instability and expression of mismatch repair genes in sporadic endometrial cancer coexisting with colorectal or breast cancer

The molecular background of sporadic endometrial cancer coexisting with colorectal or breast cancer is not clear. We investigated microsatellite instability (MSI) and status of mismatch repair (MMR) gene product, MLH1, MSH2 and MSH6, in 63 sporadic endometrial cancers coexisting with colorectal or breast cancer. Sixteen sporadic endometrial cancers with colorectal cancers (EC), 26 sporadic endometrial cancers with breast cancer (EB) and 21 endometrial cancers without a coexisting cancer (control) were analyzed. EC had the highest frequency of MSI among the 3 groups (EC, 69%; EB, 23%; and control, 43%). Incidence of low-frequency MSI was significantly higher in EC (38%). Among endometrial cancer cases diagnosed before age 50, all high-frequency MSI (MSI-H) cases belonged to EC. Interestingly, incidence of MSI-H was significantly higher in tamoxifen-non-treated cases (75%) than that of treated cases (14%). These results suggest that alterations in MMR genes appear to be involved in carcinogenesis of EC but seem to be uncommon in those of EB. Presence of MSI in sporadic endometrial cancer may be a useful marker to predict the risk of colorectal cancer.     

Microsatellite analysis at 10q25-q26 in Sardinian patients with sporadic endometrial carcinoma: identification of specification patterns of genetic alteration

BACKGROUND: Loss of heterozygosity (LOH) at chromosome 10q25-q26 has been reported previously in endometrial carcinoma (EC), suggesting the presence of tumor suppressor gene(s). Nevertheless, frequency of genome-wide microsatellite instability (MSI) has been demonstrated higher in EC than in other common malignancy, mostly due to defective DNA mismatch repair. The authors further evaluated the role of the chromosome 10q25-q26 in endometrial tumorigenesis as well as the clinical significance of any observed genetic alteration in sporadic EC. METHODS: Paired normal and tumor samples from 94 Sardinian patients with sporadic EC at various stages of disease were screened by polymerase chain reaction (PCR)-based microsatellite analysis. Genomic DNA was isolated from paraffin embedded tissues and amplified by PCR using microsatellite markers spanning approximately 14 cM at 10q25-q26. Microsatellite instability was studied at four loci mapping to different chromosomal locations. RESULTS: Thirty-two (34%) EC patients were found negative for genetic alterations within the 10q25-q26 region. Among the remaining 62 (66%) EC cases, the authors identified 1) a minimum consensus region of LOH of approximately 1 cM, between D10S610 and D10S542 markers; and 2) a subset of tumors with prevalence of instability at 10q25-q26 (10qMI+), as expression of the presence of a MSI+ phenotype. CONCLUSIONS: The authors' data establish the existence of significant correlations between disease stages and 10qMI+ (with or without MSI+). However, longer follow-up and additional studies are required to define the clinical significance of these findings as prognostic factors. Moreover, the minimum region of LOH at 10q25-q26 will be further analyzed for identifying the putative tumor suppressor gene involved in EC pathogenesis.     

Picoliter‐Droplet Digital Polymerase Chain Reaction‐Based Analysis of Cell‐Free Plasma DNA to Assess EGFR Mutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine‐Kinase Inhibitors

Purpose.

The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC).

Experimental design.

Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues.

Results.

cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance.

Conclusion.

Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs.

Implications for Practice:

Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal growth factor receptor-tyrosine-kinase inhibitors (TKIs), the predominance of tumor cells harboring the T790M mutations influences the choice of whether to use conventional or next-generation TKIs. Digital polymerase chain reaction-based examination of cfDNA is a promising method; however, its feasibility, including its consistency with examination of rebiopsied tumor tissue, has not been fully proven. Here, picoliter-droplet digital polymerase chain reaction technology is presented as a candidate method for testing cfDNA and assessing the predominance of T790M-mutant tumors.     

High concordance of mutation patterns in 10 common mutated genes between tumor tissue and cell-free DNA in metastatic colorectal cancer

The concordance of mutation patterns between cell-free DNA (cfDNA) and tumor DNA varies in colorectal cancers (CRCs). Next-generation sequencing (NGS) by targeted sequencing can detect novel genes. We aimed to use NGS to test the concordance between cfDNA and tumor DNA in metastatic CRCs. A total of 95 paired tumor and peripheral blood samples from metastatic CRC patients were included. The tumor DNA and cfDNA were analyzed with a 10-gene NGS panel (Illumina HiSeq2500 system). The median number of mutations in tumor samples was 3 (range 0-7). The most commonly mutated gene was TP53 (63.2%), followed by APC (49.5%), KRAS (35.8%) and FAT4 (15.8%). The concordance of mutation patterns in these 10 genes was as high as 91% between cfDNA and tumor samples in these metastatic CRC patients. A sensitivity of 88.2% and specificity of 100% was found when using KRAS mutation status of cfDNA to predict KRAS mutation in tumor tissue. For tumor DNA with TP53, KRAS, or APC mutations, right-sided CRCs were more likely to develop peritoneal metastases, while for tumor DNA with TP53 mutations, left-sided tumors were more likely to have lung metastases. For cfDNA with TP53 or KRAS mutations, right-sided CRCs were more likely to have peritoneal metastases. Due to the high concordance of mutation patterns between cfDNA and tumor samples, monitoring the mutation pattern of cfDNA may be applicable in the treatment of metastatic CRC.     

Hormone receptor and human epidermal growth factor receptor 2 status evaluation on ThinPrep specimens from breast carcinoma: correlation with histologic sections determination

BACKGROUND:

Fine‐needle aspiration cytology (FNAC) is a well‐accepted procedure for the diagnosis and biological characterization of breast carcinoma. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status have a strong prognostic and predictive value in invasive breast carcinoma (IBC). ThinPrep (TP) cytology, which uses an alcohol‐based fixative, is increasingly being used for immunocytochemistry. In this study, the authors compared the immunocytochemical evaluation of hormone receptors (HR) and HER2 on TP‐processed FNAC with the immunohistochemical analysis performed on the corresponding formalin‐fixed paraffin‐embedded (FFPE) breast tumor specimens, which are considered the gold standard.

METHODS:

FNACs were performed on 116 primary IBCs at the time of diagnosis and subjected to immunocytochemical evaluation of HR and HER2 using the TP method. The same markers were immunohistochemical evaluated on the corresponding FFPE tissue specimens. HER2 fluorescent in situ hybridization analysis was performed only on the equivocal immunohistochemical results.

RESULTS:

The HR results of the TP cytology specimens showed a very good agreement with those of the corresponding FFPE tissue samples (Cohen kappa test = 0.92; concordance rate = 98%) for estrogen receptor, and a good agreement (kappa = 0.76; concordance rate = 90.9%) for progesterone receptor. A perfect agreement (kappa = 1) was observed between TP and FFPE tissue samples in evaluating HER2 status.

CONCLUSIONS:

Alcohol‐based fixation seems not to affect the immunocytochemical evaluation of HR and HER2. Considering the high levels of agreement between the evaluation of HR and HER2, on both cytology specimens and on the corresponding FFPE tissue samples, the authors concluded that the TP technique can be routinely used for the biological characterization of IBC. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society.     

Cell Free Tumoral DNA Versus Paraffin Block Epidermal Growth Factor Receptor Mutation Detection in Patients with Non-Small Cell Lung Cancer

Increasing knowledge about the molecular profile of tumors has led to personalized treatment for achieving better outcomes in patients with nonsmall cell lung cancer (NSCLC). Currently, finding exact somatic genomic changes of tumor has gained great importance. On the other hand, crescendoing needs to actual tumor tissue at different time points during cancer treatment may produce major discomfort for NSCLC patients. Tumor genomes can be reconstructed by information obtained from circulating cell-free deoxyribonucleic acid (cfDNA) of peripheral blood. cfDNA may be represented as a suitable alternative test  for epidermal growth factor receptor (EGFR) mutation detection in these patients. This study aimed to assess validity of cfDNA in somatic EGFR mutation identification in Iranian NSCLC cases. Methods: Somatic mutation of EGFR gene was studied in both tissue specimens and plasma. Then, mutations were detected by polymerase chain reaction(PCR) and sequencing. Results: We observed a high concordance (90%) between tissue samples and cfDNA for EGFR gene mutation.  The sensitivity, accuracy, and positive precision value were 90%, 90% and 100%, respectively. A false negative rate of 10% was also demonstrated in this study. Conclusion: We established sensitive methods for detecting EGFR gene mutation which may be very useful in clinical practice.     

Chromosomal abnormalities and microsatellite instability in sporadic endometrial cancer

Defective DNA mismatch repair and nonfunctional mechanisms controlling the proper progression of the cell cycle have been proposed as being responsible for the genomic instability and accumulation of karyotypic alterations in endometrial cancer (EC). To assess whether numerical chromosomal anomalies (aneuploidy) and microsatellite instability (MSI) might be representative of distinctive tumour behaviour, paraffin-embedded tissue samples from 86 patients with sporadic EC were evaluated by both fluorescence in situ hybridisation (FISH) and microsatellite analysis, using free nuclei and genomic DNAs (respectively). Approximately one-third of the tumours analysed (24/74; 32%) exhibited MSI, whereas 38/86 (44%) of the EC samples displayed aneuploidy. The majority of the unstable cases (15/24; 63%) were from advanced-stage patients. Conversely, 23 (61%) out of the 38 tumours with aneuploidy were from early-stage patients. No apparent correlation was found between MSI and aneuploidy, whereas the immunohistochemical (IHC) analysis revealed that inactivation of the MLH1 mismatch repair gene may be involved in the majority of the MSI+ sporadic ECs. No genetic or cytogenetic alteration analysed here seems to add any significant predictive value to the stage of disease.     

Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer

BackgroundA high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse.Patients and methodsOne hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR.ResultsNGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r-=-0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P-=-0.0014 and HR, 11.33; log-rank P-=-0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P-<-0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5-months.ConclusionsPlasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.     

MLH1 and MSH2 protein expression as a pre-screening marker in hereditary and non-hereditary endometrial hyperplasia and cancer

The predictive value of MLH1 or MSH2 protein expression for the presence of truncating germline mutations was examined in benign and (pre)malignant endometrial samples from 3 patient groups: (I) 10 endometrial cancer patients from hereditary non-polyposis colorectal cancer (HNPCC) families with (n = 6) or without (n = 4) a known germline mutation; (II) 15 women from HNPCC families with (n = 7) or without (n = 8) a known germline mutation, who underwent endometrial sampling for non-malignant reasons; (III) 38 endometrial cancer patients <50 years of age, without HNPCC family history. Immunostaining for MLH1 and MSH2 was performed on paraffin-embedded sections. In group III, tumor DNA was examined for microsatellite instability (MSI) and MLH1, MSH2 and MSH6 mutation analysis was carried out. In 6/6 MLH1/MSH2 mutation carriers with endometrial cancer (group I), concordance was found between protein loss in the tumor and the corresponding mutation. In 3 MLH1 mutation carriers, MLH1 protein loss was also observed in concurrent endometrial hyperplasia. In group II, no protein loss was detected in normal endometrial tissue samples; in 3/4 patients with endometrial hyperplasia, MLH1/MSH2 protein loss was observed. In group III, protein loss was detected in 12/38 patients (9 MLH1, 3 MSH2), while in 3/11 patients with concurrent endometrial hyperplasia protein loss was also observed in the hyperplasia. MSI analysis in group III revealed 26 MSI-low and 12 MSI-high tumors. Mutation analysis in 28/38 patients showed only 1 missense MSH6 and no MLH1 or MSH2 germline mutations. In group III, loss of MLH1/MSH2 protein expression was not related to the presence of MSI or MLH1/MSH2 germline mutations. In conclusion, MLH1 or MSH2 protein loss in HNPCC-related endometrial neoplasia is strongly related to corresponding germline mutations. This relation was not clearly present in young sporadic endometrial cancer patients. Immunohistochemical pre-screening of the MLH1 and MSH2 proteins in endometrial hyperplasia or cancer can thus be helpful in HNPCC families. Frequent loss of MLH1 or MSH2 protein in endometrial hyperplasia indicates that this loss is an early event in endometrial carcinogenesis.     

Incidence of microsatellite instability in synchronous tumors of the ovary and endometrium.

PURPOSE: Families with hereditary nonpolyposis colorectal cancer (HNPCC) have an increased lifetime risk of endometrial (40%) and ovarian (10%) carcinomas. Endometrial and ovarian carcinomas from members of these families frequently display a mutator phenotype as manifest by high levels of microsatellite instability (MSI-H). Microsatellite instability (MSI) occurs in 17-32% of sporadic endometrial carcinomas and 3-17% of sporadic ovarian carcinomas. We hypothesized that there might be a higher rate of MSI in tumors from women with synchronous primary carcinomas of the ovary and endometrium. EXPERIMENTAL DESIGN: We identified 52 cases of synchronous tumors of the ovary and endometrium from the databases of four gynecological oncology units. Archival material and clinical data were available on 45 of these patients. We examined DNA extracted from ovarian and endometrial tumor tissue for MSI using DNA extracted from normal tissue of that patient as a germ-line DNA control. MSI was assessed using a panel of five standard microsatellite markers: D2S123, D5S346, D17S250, BAT25, and BAT26. MSI-H was defined by more than two markers being positive. Low-level MSI (MSI-L) was defined as one or two markers positive and microsatellite stable (MSS) was defined as no markers positive. RESULTS: The 45 patients had a median age at diagnosis of 53 years. Of a total of 134 samples analyzed, only three samples (3.3%) were MSI-H. No patient had high levels of MSI in both ovarian and endometrial tumors. One ovarian carcinoma had five of five markers positive with the corresponding endometrial carcinoma being MSI-L. Two endometrial carcinomas were MSI-H, and the corresponding ovarian carcinomas were MSI-L and MSS, respectively. Seven ovarian tumors and seven endometrial tumors were MSI-L. The majority of patients had early-stage ovarian carcinoma [International Federation of Gynecology and Obstetrics (FIGO) stage I, 44.4%; stage II, 26.7%; and stage III, 26.6%]. Eighty-two % of the endometrial primaries were FIGO stage I. Progression-free survival was significantly better for patients with synchronous primaries than those presenting with ovarian carcinoma alone [adjusted hazards ratio, 1.94; P = 0.023; 95% confidence interval, 1.096-3.44]. CONCLUSION: Synchronous primary carcinomas of the ovary and endometrium are unlikely to be part of the HNPCC syndrome unless the family history is in keeping with the modified Amsterdam criteria.     

Prognostic and predictive roles for circulating biomarkers in gastrointestinal cancer

Circulating tumor cells (CTCs) and circulating free DNA (cfDNA) have been studied as promising prognostic and predictive tumor-derived biomarkers in the bloodstream of patients with gastrointestinal malignancies because they may be an alternative noninvasive tool to tumor tissue biopsies. Quantification and molecular characterization of CTCs and cfDNA may provide additional insights into cancer biology, potentially revealing novel targets to individualize cancer care. The present article aims to review the biology and current methods to assess CTCs and cfDNA, and the efforts to establish both tumor-derived biomarkers as prognostic and predictive factors in esophageal, gastric and colorectal cancer.     

Toward a Molecular Classification of Synchronous Colorectal Cancer: Clinical and Molecular Characterization

Background

Two or more primary colorectal tumors coexisting at the time of diagnosis are considered to be synchronous tumors. It is estimated that synchronous colorectal cancer (SCRC) only accounts for 1.1% to 8.1% of all colorectal cancers (CRCs), and its molecular basis is still poorly understood.