Tumor Heterogeneity Confounds Lymphocyte Metrics in Diagnostic Lung Cancer Biopsies.

CONTEXT.­: The immune microenvironment is involved in fundamental aspects of tumorigenesis, and immune scores are now being developed for clinical diagnostics. OBJECTIVE.­: To evaluate how well small diagnostic biopsies and tissue microarrays (TMAs) reflect immune cell infiltration compared to the whole tumor slide, in tissue from patients with non-small cell lung cancer. DESIGN.­: A TMA was constructed comprising tissue from surgical resection specimens of 58 patients with non-small cell lung cancer, with available preoperative biopsy material. Whole sections, biopsies, and TMA were stained for the pan-T lymphocyte marker CD3 to determine densities of tumor-infiltrating lymphocytes. Immune cell infiltration was assessed semiquantitatively as well as objectively with a microscopic grid count. For 19 of the cases, RNA sequencing data were available. RESULTS.­: The semiquantitative comparison of immune cell infiltration between the whole section and the biopsy displayed fair agreement (intraclass correlation coefficient [ICC], 0.29; P = .01; CI, 0.03-0.51). In contrast, the TMA showed substantial agreement compared with the whole slide (ICC, 0.64; P < .001; CI, 0.39-0.79). The grid-based method did not enhance the agreement between the different tissue materials. The comparison of CD3 RNA sequencing data with CD3 cell annotations confirmed the poor representativity of biopsies as well as the stronger correlation for the TMA cores. CONCLUSIONS.­: Although overall lymphocyte infiltration is relatively well represented on TMAs, the representativity in diagnostic lung cancer biopsies is poor. This finding challenges the concept of using biopsies to establish immune scores as prognostic or predictive biomarkers for diagnostic applications.

Purpura fulminans, TEN, and disseminated herpes simplex: An unexpected combination.

Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and purpura fulminans (PF) are all rare conditions. A combination of these 3 conditions together with a viral infection is very rare. A 52-year-old, previously healthy woman which developed SJS, potentially due to a reaction to CT contrast, although this is still unknown. This developed into TEN on day 10 of the initial admission, the patient scored 3 points on SCORTEN. On day 12 from initial admission, she developed unexpected multiorgan failure and PF. The patient passed away 2 days later, the autopsy demonstrates herpes simplex virus in the bladder and lungs on immunohistological staining. Our clinical case encountered the challenge of differentiating TEN and PF. The microscopic and immunochemical examination confirmed the clinical suspicion of PF but also a disseminated herpes simplex infection. We speculate the clinical route of this case started SJS and TEN, leading to superimposed infection with three different types of bacteria, confirmed in blood cultures, and a disseminated viral infection. The combination of all these diagnoses are very rare, no similar case has been described in adults to the authors' knowledge. We recommend a prompt diagnosis and early recognition of both bacterial and viral infections to prevent the development of PF.

Tumour-infiltrating lymphocytes add prognostic information for patients with low-risk DCIS: findings from the SweDCIS randomised radiotherapy trial.

BACKGROUND: The immune microenvironment is an important modulator of tumour progression and treatment response. In invasive breast cancer, assessment of tumour-infiltrating lymphocytes (TILs) provides prognostic and predictive information. However, the clinical impact of TILs for ductal carcinoma in situ (DCIS) has not yet been demonstrated. PATIENTS AND METHODS: Post hoc analysis of the SweDCIS randomised radiotherapy trial including primary DCIS cases following breast-conserving surgery. TILs were assessed on haematoxylin-eosin sections (n = 711) according to the International Immuno-Oncology Biomarker Working Group guidelines. TILs-scores were analysed as continuous and dichotomised (≤5% versus >5%) variable regarding ipsilateral breast events (IBEs) as the predefined primary endpoint. RESULTS: Most women (61.9%) showed a TILs prevalence of ≤5%. High TILs-scores were associated with larger lesion size, human epidermal growth factor receptor 2 (HER2)-positivity, higher nuclear grade, and KI67-score. DCIS cases with high TILs prevalence had a significant increased cumulative IBE incidence at five years post-surgery (TILslow-versus TILshigh 9% versus 18%; p < 0.001). Among patients with HER2-negative DCIS, high TILs remained an independent poor prognosis marker for IBE risk in multivariable analysis with an adjusted hazard ratio of 2.41 [95%CI 1.17-4.95, p = 0.017]. Including TILs-status provided a refined stratification of patients with general low-risk DCIS (grade <3, size <25 mm, free margin). No interaction between TILs and radiotherapy benefits was detected. CONCLUSION: High TILs are associated with higher IBE risk over 5-years post-surgery, particularly for HER2-negative DCIS. Our data indicate that TILs should be integrated into the clinical workup to define patients with low-risk DCIS who can omit adjuvant therapy or patients with potential benefits from immunotherapy.

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas.

Background: Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts. Methods: Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1-rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21). Results: Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between the assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNA-sequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay. Conclusions: The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.

High PDGFRb Expression Predicts Resistance to Radiotherapy in DCIS within the SweDCIS Randomized Trial.

PURPOSE: This study analyzes the potential of stromal platelet-derived growth factor receptor-beta (PDGFRb) expression as biomarker for radiotherapy (RT) benefit on ipsilateral breast events (IBE) in ductal carcinoma in situ (DCIS). Improved identification of DCIS patients refractory to adjuvant whole-breast RT is needed. Predictive biomarker studies in DCIS have focused on tumor cell features rather than the tumor-associated stroma, despite growing evidence of its influence on therapy efficiency. EXPERIMENTAL DESIGN: Samples from the Swedish randomized radiotherapy DCIS trial (SweDCIS) were subjected to IHC analysis for stromal PDGFRb expression. IBE incidence at 10 years after breast-conserving surgery was the primary endpoint. Interactions between marker and treatment were analyzed. RESULTS: PDGFRb score was predictive for RT benefit with regard to IBE (P interaction = 0.002 and P interaction = 0.008 adjusted multivariably). Patients of the PDGFRblow group had a strong benefit from RT regarding IBE risk [HR, 0.23; 95% confidence interval (CI), 0.12-0.45; P < 0.001] with an absolute risk reduction of 21% (cumulative risk 7% vs. 28%) at 10 years. No significant risk reduction by RT was observed for patients of the PDGFRbhigh group (HR, 0.83; 0.51-1.34; P = 0.444; cumulative risk 22% vs. 25%). The RT response-predictive effect of stromal PDGFRb was equally strong in analyses for in situ and invasive IBE when analyzed separately (in situ IBE: P = 0.029; invasive IBE: P = 0.044). CONCLUSIONS: Results suggest high stromal PDGFRb expression as a novel biomarker identifying DCIS patients who are refractory to standard whole-breast adjuvant RT. The data imply previously unrecognized fibroblast-mediated modulation of radiosensitivity of DCIS, which should be further explored from mechanistic and targeting perspectives.

PD-L1 amplification is associated with an immune cell rich phenotype in squamous cell cancer of the lung.

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About  37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy.