CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s) ä . Physicians should claim only credit commensurate with the extent of their participation in the activity.

No abstract

Immune checkpoint inhibitors have become integrated into the clinical management of non-small cell lung cancer (NSCLC). Using RTqPCR, we have previously identified a gene expression panel that detected presence of malignant cells (MMP9:TIMP3 ratio) and quantified PD-L1 transcript levels in small biopsy specimens. However, RTqPCR has diagnostic limitations as it does not generate absolute copy number and is not readily multiplexed. To address this, we have developed a multiplex droplet digital PCR (ddPCR) assay. Materials and methods: Biopsies obtained from NSCLC patients (n = 48 adenocarcinoma and n = 40 squamous cell carcinoma) and control lung biopsy specimens (n = 20) were analysed. Absolute MMP9, TIMP3 and PD-L1 transcript copy numbers were determined within a single assay by multiplex ddPCR using Taqman primers and the QX200 Droplet Digital PCR System. Results and conclusions: Using our optimised triplex ddPCR assay, the MMP9:TIMP3 ratio was significantly elevated in NSCLC biopsies and using a cutoff of > 0.028, was 99% (95% CI; 80.5-94.5) sensitive and 80% specific for identifying malignant biopsies. The PD-L1:TIMP3 ratio significantly associated with PD-L1 tumour cell immunohistochemistry staining (r = 0.539, p < 0.0001) and was significantly higher in biopsies with > 50% PD-L1 tumour cell staining (p < 0.0001). In summary, a major advantage of our workflow is that it can accurately quantify PD-L1 tumour levels and provide sufficient nucleic acid for screening additional targetable mutations such as EGFR, ALK and ROS1 from a single small biopsy, thereby potentially avoiding the need for rebiopsy. Future studies will need to determine diagnostic ddPCR values that are predictive of clinical response to PD-1/PD-L1 immunotherapy.

Matrix metalloproteinase-9 (MMP-9) is increased in a number of pathological lung conditions, where the proteinase contributes to deleterious remodelling of the airways. While both lung cancer and COPD are associated with increased MMP-9 expression, the cellular and molecular drivers of MMP-9 remain unresolved. In this study, MMP-9 transcript measured within the tumour region from patients with non-small-cell lung cancer (NSCLC) and coexisting COPD was found to be uniformly increased relative to adjacent tumour-free tissue. MMP-9 gene expression and immunohistochemistry identified tumour-associated neutrophils, but not macrophages, as a predominant source of this proteinase. In addition, PTEN gene expression was significantly reduced in tumour and there was evidence of epithelial MMP-9 expression. To explore whether PTEN can regulate epithelial MMP-9 expression, a small interfering (si)RNA knockdown strategy was used in Beas-2B bronchial epithelial cells. PTEN knockdown by siRNA selectively increased MMP-9 expression in response to lipopolysaccharide in a corticosteroid-insensitive manner. In summary, tumour-associated neutrophils represent an important source of MMP-9 in NSCLC, and loss of epithelial PTEN may further augment steroid-insensitive expression.

As the transient postnatal hormone surge in humans, known as 'minipuberty', occurs simultaneously with key steps in germ-cell development, we investigated whether similar changes occur in the hypothalamic-pituitary-testicular axis of neonatal mice at a time that would coincide with gonocyte transformation into spermatogonial stem cells (SSC). Serum and testes were collected from C57Bl/6 mice at embryonic Day 17 (E17), birth (postnatal Day 0; P0) and daily until P10. Serum FSH and testosterone levels in both serum and testes were analysed and gene expression of FSH receptor (Fshr), luteinising hormone receptor (Lhr), anti-Müllerian hormone (Amh), octamer-binding transcription factor 4 (Oct-4), membrane type 1 metalloprotease (Mt1-mmp), proto-oncogene C-kit and promyelocytic leukaemia zinc finger (Plzf ) was quantified by real-time polymerase chain reaction. We found a transient surge of serum and testicular testosterone levels between P1 and P3 and a gradual increase in FSH from P1 to P10. Testis Lhr expression remained low from P0 until P10 but Fshr expression peaked between P3 and P6 (P<0.01). The same was found for Oct-4 expression (a gonocyte marker), which surged between P3 and P6 (P<0.01). Mt1-mmp expression peaked at P3 (P<0.05). The expression pattern of both C-kit and Plzf (SSC markers) was similar with a steady increase from P1 to P10. These results show a transient activation of the hypothalamic-pituitary-testicular axis postnatally with increases in serum and testicular testosterone at P1-P3 and testicular Fshr (but not Lhr) at P3-P6. These changes coincide with increases in gene expression of Oct4, Mt1-mmp, Plzf and C-kit, reflecting gonocyte activation, migration and transformation into SSC. In conclusion, these findings suggest that 'minipuberty' does occur in mice and that gonocyte transformation may be driven by a transient FSH signalling pathway.