Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK ® Lung Panel on the MassARRAY ® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) &gt; 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK ® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK ® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK ®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK ® correlated with prolonged median PFS (46 vs. 6 weeks; p &lt; 0.05) and OS (145 vs. 30 weeks; p &lt; 0.01). Using plasma-derived ccfDNA, the UltraSEEK ® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response. </p

Circulating Cancer Associated Macrophage-like Cells as a Potential New Prognostic Marker in Pancreatic Ductal Adenocarcinoma

Background: Circulating Cancer Associated Macrophage-like cells (CAMLs) have been described as novel liquid biopsy analytes and unfavorable prognostic markers in some tumor entities, with scarce data for Pancreatic Ductal Adenocarcinomas (PDAC). Methods: Baseline and follow-up blood was drawn from resected curative (n = 36) and palliative (n = 19) PDAC patients. A microfluidic size-based cell enrichment approach (ParsortixTM) was used for CAML detection, followed by immunofluorescence staining using pan-keratin, CD14, and CD45 antibodies to differentiate between CAMLs, circulating tumor cells (CTCs), and leukocytes. Results: CAMLs were detectable at baseline in 36.1% of resected patients and 47.4% of palliative PDAC patients. CAML detection was tumor stage independent. Follow-up data indicated that detection of CAMLs (in 45.5% of curative patients) was an independent prognostic factor for shorter recurrence-free survival (RFS) (HR: 4.3, p = 0.023). Furthermore, a combined analysis with CTCs showed the detectability of at least one of these cell populations in 68.2% of resected patients at follow-up. The combined detection of CAMLs and CTCs was also significantly associated with short RFS (HR: 8.7, p = 0.003). Conclusions: This pilot study shows that detection of CAMLs in PDAC patients can provide prognostic information, either alone or even more pronounced in combination with CTCs, which indicates the power of liquid biopsy marker analyses

Microsatellite analyses for AI on 11p in primary breast cancers and metastases.

<p>Base pair position and the markers used are indicated on the top line. The result for each marker is shown as follows: AI: black; non-informative: light gray; unavailable measurement: dark gray; and informative without changes: white box.</p

Quantitative real-time RT-PCR results for PRKCDBP expression in BCBM and primary BC patients.

<p>Relative PRKCDBP transcript levels were determined by normalization to the reference gene RPLP0 and universal human reference (UHR) using the ΔΔCt method.</p

Differentially expressed genes at 11p between BCBM and primary BC without relapse, bone, or lung relapse.

*<p>fold change down regulated in brain metastases samples compared to primary breast tumors.</p>**<p>significantly down regulated genes among primary tumors with brain relapse compared to primary tumors with bone relapse.</p>***<p>significantly down regulated genes among primary tumors with brain relapse compared to primary tumors with lung relapse.</p

Frequencies and p-values for AI at chromosome 11p in BCBM, primary tumors and metastases.

*<p>FUP missing for 6 patients.</p><p>n.s.: not significant.</p

Clinical Relevance of Loss of 11p15 in Primary and Metastatic Breast Cancer: Association with Loss of PRKCDBP Expression in Brain Metastases

<div><p>The occurrence of brain metastases among breast cancer patients is currently rising with approximately 20–25% incidence rates, underlining the importance of the identification of new therapeutic and prognostic markers. We have previously screened for new markers for brain metastasis by array CGH. We found that loss of 11p15 is common among these patients. In this study, we investigated the clinical significance of loss of 11p15 in primary breast cancer (BC) and breast cancer brain metastases (BCBM). 11p15 aberration patterns were assessed by allelic imbalance (AI) analysis in primary BC (n = 78), BCBM (n = 21) and metastases from other distant sites (n = 6) using six different markers. AI at 11p15 was significantly associated with BCBM (p = 0.002). Interestingly, a subgroup of primary BC with a later relapse to the brain had almost equally high AI rates as the BCBM cases. In primary BC, AI was statistically significantly associated with high grade, negative hormone receptor status, and triple-negative (TNBC) tumors. Gene expression profiling identified <em>PRKCDBP</em> in the 11p15 region to be significantly downregulated in both BCBM and primary BC with brain relapse compared to primary tumors without relapse or bone metastasis (fdr<0.05). qRT-PCR confirmed these results and methylation was shown to be a common way to silence this gene. In conclusion, we found loss at 11p15 to be a marker for TNBC primary tumors and BCBM and <em>PRKCDBP</em> to be a potential target gene in this locus.</p> </div

11p allelic imbalances and association to clinical factors in primary tumors.

<p>HR: hormone receptor.</p><p>TNBC: triple-negative tumors (HR negative/HER2 negative tumors).</p

Comparative evaluation of PD‐L1 expression in cytology imprints, circulating tumour cells and tumour tissue in non‐small cell lung cancer patients

Alternative sources of tumour information need to be explored in patients with non‐small cell lung cancer (NSCLC). Here, we compared programmed cell death ligand 1 (PD‐L1) expression on cytology imprints and circulating tumour cells (CTCs) with PD‐L1 tumour proportion score (TPS) from immunohistochemistry staining of tumour tissue from patients with NSCLC. We evaluated PD‐L1 expression using a PD‐L1 antibody (28‐8) in representative cytology imprints, and tissue samples from the same tumour. We report good agreement rates on PD‐L1 positivity (TPS ≥ 1%) and high PD‐L1 expression (TPS ≥ 50%). Considering high PD‐L1 expression, cytology imprints showed a PPV of 64% and a NPV of 85%. CTCs were detected in 40% of the patients and 80% of them were PD‐L1+. Seven patients with PD‐L1 expression of < 1% in tissue samples or cytology imprints had PD‐L1+ CTCs. The addition of PD‐L1 expression in CTCs to cytology imprints markedly improved the prediction capacity for PD‐L1 positivity. A combined analysis of cytological imprints and CTCs provides information on the tumoural PD‐L1 status in NSCLC patients, which might be used when no tumor tissue is available

Clonality of circulating tumor cells in breast cancer brain metastasis patients

Background: The incidence of brain metastases in breast cancer (BCBM) patients is increasing. These patients have a very poor prognosis, and therefore, identification of blood-based biomarkers, such as circulating tumor cells (CTCs), and understanding the genomic heterogeneity could help to personalize treatment options.Methods: Both EpCAM-dependent (CellSearch® System) and EpCAM-independent Ficoll-based density centrifugation methods were used to detect CTCs from 57 BCBM patients. DNA from individual CTCs and corresponding primary tumors and brain metastases were analyzed by next-generation sequencing (NGS) in order to evaluate copy number aberrations and single nucleotide variations (SNVs).Results: CTCs were detected after EpCAM-dependent enrichment in 47.7% of the patients (≥ 5 CTCs/7.5 ml blood in 20.5%). The CTC count was associated with ERBB2 status (p = 0.029) of the primary tumor as well as with the prevalence of bone metastases (p = 0.021). EpCAM-independent enrichment revealed CTCs in 32.6% of the patients, especially among triple-negative breast cancer (TNBC) patients (70.0%). A positive CTC status after enrichment of either method was significantly associated with decreased overall survival time (p < 0.05). Combining the results of both enrichment methods, 63.6% of the patients were classified as CTC positive. In three patients, the matched tumor tissue and single CTCs were analyzed by NGS showing chromosomal aberrations with a high genomic clonality and mutations in pathways potentially important in brain metastasis formation.Conclusion: The detection of CTCs, regardless of the enrichment method, is of prognostic relevance in BCBM patients and in combination with molecular analysis of CTCs can help defining patients with higher risk of early relapse and suitability for targeted treatment