Tissue Microarray from Cell Block Material (cbTMA)—An Additional Shot for Cytology in the Predictive Pathology Era: The PD-L1 Experience

Generally, predictive biomarker tests are clinically validated on histological formalin-fixed, paraffin-embedded (FFPE) samples. In addition to FFPE samples, cytological samples have also emerged as a useful approach to detect predictive biomarkers. However, as of today, despite the promising results reported in the recent literature, their full implementation in routine clinical practice is still lagging owing to a lack of standardized preparatory protocols, challenging assessments of cyto-histological correlation, and variable inter-observer agreement. The aim of this report was to explore the possibility of implementing a large-scale validation of predictive biomarker testing on cytological material. To this aim, we evaluated the technical feasibility of PD-L1 assessment on a cell block (CB)-derived tissue microarray (cbTMA). Consecutive and unselected CBs prepared from metastatic lymph node fine-needle cytology (FNC) samples were retrospectively collected and used for TMA construction. PD-L1 immunohistochemistry (IHC) was carried out on cbTMA sections with the companion diagnostic kit SP263 assay. TMA contained 33 CB-derived cores. A total of 20 sections were hematoxylin and eosin (H&E) stained. Overall, 29 (88%) samples were visible at least in one H&E-stained slide. Four cases out of five sections stained with the SP263 assay (4/29, 13.8%) showed PD-L1 positivity in neoplastic and/or immune cells; remarkably, no unspecific background was observed. Although our study was based on a limited and non-selected series, our findings do provide proof of concept for the use of cbTMA in predictive biomarker testing on cytological material in large-scale post-clinical trial validation studies, multicenter studies, and quality control programs.

The Impact of Metastatic Lymph Node Size on Long-term Outcomes for pStage III Colon Cancer

Aim: To clarify the impact of metastatic lymph node size on long-term outcomes in patients undergoing curative colectomy for pathological stage III colon cancer. Patients and Methods: This study enrolled patients who underwent curative colectomy for pStage III colon cancer between January 2013 and December 2015. All patients were divided into four groups based on the short-axis diameter of the largest MLN: Group A, <5 mm; Group B, ≥5 mm and <10 mm; Group C, ≥10 mm and <15 mm; Group D, ≥15 mm. Results: A total of 209 patients were analyzed. The 5-year recurrence-free survival rates of Groups A, B, C, and D were 82.3%, 74.6%, 74.5% and 60.7%, respectively. In multivariate analysis, Group D (hazard ratio=3.95; 95% confidence interval, 1.34-11.65; p=0.01) was independently associated with worse RFS. Conclusion: Bulky MLNs might be a poor prognostic factor in node-positive colon cancer.

Postoperative recurrence with right cervical lymph node metastasis in hepatocellular carcinoma: a case report

Background Hepatocellular carcinoma (HCC) patients with metastases to the cervical lymph nodes are extremely rare, and its clinical course is characterized by rapidly progressive disease. Hence, there have been no reports of metastatic cervical lymph node recurrence indicated after a long postoperative surveillance period. Case presentation The patient was a 63-year-old male who underwent right hepatectomy for HCC of the right upper lobe. Three years after resection, metastatic lymph node recurrence was detected in the subdiaphragm, superior mediastinum, and right cervical lymph nodes. The patient underwent excisional biopsy of the cervical lymph node, followed by molecular-targeted therapy and radiation therapy. Lenvatinib reduced the size of all metastatic lymph nodes and the patient survived for a relatively long period of 43 months after the recurrence was detected. Conclusions After resection of HCC in the right upper lobe, there is the possibility of metastatic lymph node recurrence in unusual sites, including the cervical region, and lenvatinib may be effective in those recurrences.

Siglec-15 Is an Immune Suppressor and Potential Target for Immunotherapy in the Pre-Metastatic Lymph Node of Colorectal Cancer

Lymph node metastasis indicates a poor prognosis in colorectal cancer. To better understand the underlying mechanisms of lymph node metastasis, we analyzed transcriptome characteristics of the pre-metastatic lymph node, a putative microenvironment favorable for the seeding and proliferation of cancer cells. Thus, we tried to compare and elucidate the transcriptional and immune characteristics of sentinel lymph nodes (SNs) with matched non-sentinel lymph nodes (NSNs) in colorectal cancer patients. In this study, a total of 38 pairs of SNs and NSNs were collected, in which 26 pairs of non-metastatic lymph nodes were subjected to RNA-seq and bioinformatics analysis for the gene expression profiles. There were 16 differentially expressed genes between SNs and NSNs being identified, including 9 upregulated and 7 downregulated genes in SN. Gene Ontology (GO) classification analysis revealed that the differentially expressed genes were mainly involved in leukocyte differentiation, chemokine secretion, and immune system regulation. In the meantime, gene set enrichment analysis (GSEA) showed that immune-related signaling pathways, such as transforming growth factor beta (TGF-β) signaling and tumor necrosis factor alpha (TNF-α)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, were enriched in NSN, while cell proliferation–related signaling pathways were enriched in SN, including MYC signaling and G2M checkpoint signaling. We further identified SIGLEC15 as a top upregulated gene in SN. However, RNAscope assay showed that SIGLEC15 was not largely co-expressed with M2 macrophage marker CD163. We then selected eight pairs of lymph nodes for further cytological studies. Flow cytometry analysis revealed that Siglec-15 was expressed on all myeloid cell subsets. The relative expression of SEGLEC15 (SN/NSN) was correlated with the microsatellite instability (MSI) status in colorectal cancer patients. Further studies found that small interfering ribonucleic acid (siRNA)-mediated silencing of SLGLEC15 can enhance the anti-tumor function of T cells, as indicated by cytokine release analysis. In conclusion, we presented here a first report on the gene expression profiling of the pre-metastatic lymph node in colorectal cancer. The findings in this study suggest that SIGLEC15 plays an important role in SN immunosuppression. SEGLEC15 silencing could be a therapeutic strategy for restoring T cell function in tumor SNs.

Ultrasound-guided percutaneous ethanol injection therapy of neck cysts as an alternative to surgery

Summary Introduction: Some extrathyroidal cervical cystic lesions can be treated in selected situations by minimally invasive, low-risk method – ultrasound--guided percutaneous ethanol injection therapy (US-PEIT). Here we present 6 cases of neck cysts of various origins – ranula, thyroglossal duct cyst, branchial cleft cyst, midline neck cyst of the pyramidal lobe and dermoid cyst. Method and material: The cohort consisted of 6 patients (mean age 58 ± 13.3 years), who were followed for recurrent cervical cysts located outside the thyroid gland. The cysts were visible, palpable, aesthetically unpleasant and also mechanically compressing the surrounding tissue. After repeated evacuations, they filled in again. The patients repeatedly refused the recommended surgery and demanded an alternative solution. US-PEIT of thyroid cysts is used worldwide as a full-fledged alternative to surgical treatment. We used the same procedure for the treatment of presented neck cysts. Results: The initial volume of cysts was 2–35 mL (mean 17.7 mL); the patients underwent 1–6 sclerotization sessions (mean 3.6). The patients were followed for 12 months; the final volume of solid residue was 0.1–2 mL (mean 1 mL) representing volume reduction by 80–99% (mean 92%). Therapeutic success (volume reduction > 50%) was achieved in all patients. Conclusion: US-PEIT of cervical cysts as an alternative to surgery can be used especially in elderly patients with increased surgical risk or in patients refusing surgery. In middle-aged and older adults, the possibility of a necrotic metastatic lymph node should always be considered. Therefore, a benign cytological examination and an unsuspecting ultrasonographic and CT finding are the basic conditions before performing the ethanol ablation. Key words neck cysts – ultrasound-guided percutaneous ethanol injection therapy

Integrative Clonal Evolution Analysis on SNV and CNV Levels in Multifocal Breast Cancer

Background:For multifocal or multicentric breast cancer with two or more separated malignant foci in ipsilateral breast, the origin of its multiple foci is still debatable: do they share a common origin or have they developed independently?Methods:By bulk exome sequencing combined with single cell whole genome sequencing using multiple annealing and looping-based amplification cycles (MALBAC), we obtained genomic information of two separated breast tumors and one metastatic lymph node in a multifocal breast cancer patient. We performed single nucleotide variation (SNV) and single cell copy number variation (CNV) analyses of the two breast tumors and lymph node. Results:On SNV level, we found common origin and single focus metastasis in this patient, concretely speaking that the two breast tumors were formed by the spread of one single tumor to another, instead of being developed independently. Furthermore, the lymph node was metastasized from one of the two tumors instead of co-metastasis by both of them. We also found that the SNV subclone architecture always kept stable between the two breast tumors as well as from the breast tumors to lymph node. On CNV level, in contrast, the frequency of CNV subclones changed dramatically from the breast tumors to lymph node. Among them, frequency of Clone 2 increased significantly, indicating metastatic advantages of it. By combining distinct clonal evolution patterns of SNVs and CNVs, we built an integrative evolutionary model for this patient. CNV subclones were determined at a relative early time and kept unchanged, and CNV Clone 2 with metastatic advantages was separated from the common ancestors during intramammary spread and metastasis. In the meantime, SNVs were accumulated gradually. Conclusions: In conclusion, we found distinct clonal evolution patterns on SNV and CNV levels in multifocal breast cancer and identified a CNV clone with metastatic advantages.

Mutational Landscape of Paired Primary and Synchronous Metastatic Lymph Node in Chemotherapy Naive Gallbladder Cancer

Background: Comprehensive genomic analysis of paired primary tumors and their metastatic lesions may provide new insights into the biology of metastatic processes and therefore guide the development of novel strategies for intervention. To date, our knowledge of the genetic divergence and phylogenetic relationships in gallbladder cancer (GBC) is limited.Methods:We performed whole exome sequencing (WES) for 5 patients with primary tumor, metastatic lymph node (LNM) and corresponding normal tissue. Mutations, mutation signatures and copy number variations were analyzed with state-of-art bioinformatics methods. Phylogenetic tree was also generated to infer metastatic pattern. Results:Five driver mutations were detected in these patients. Among which, TP53 was the only shared mutation between primary tumor and LNM. Although tumor mutational burden was comparable between primary tumor and LNM, higher mutation burden was observed in LNM of one patient. Copy number variations (CNVs) burden was higher in LNM than their primary tumor. Phylogenetic analysis indicated both linear and parallel progression of metastasis exist in these patients. TP53 mutation and CNVs were homogenously between primary tumor and LNM.Conclusions:High consistence of genetic landscape were shown between primary tumor and LNM in GBC. However, heterogenicity still exist between primary tumor and LNM in particular patients in term of driver mutation, TMB and CNV burden. Phylogenetic analysis indicated both Linear and parallel progression of metastasis were exist among these patients.