Adequacy of cytology and small biopsy samples obtained with rapid onsite evaluation (ROSE) for predictive biomarker testing in non-small cell lung cancer.

Complete biomarker workup of non-small cell lung cancer (NSCLC) specimens is essential for appropriate and timely clinical management decisions. This can be challenging to achieve from small cytology and histology specimens, with increasing numbers of molecular and immunohistochemical biomarkers required. We conducted a 5 year retrospective audit of cases at our institution to assess the diagnostic and biomarker testing adequacy rates, particularly those specimens obtained with rapid onsite evaluation (ROSE), performed by a cytopathologist and a cytology scientist or pathology trainee, including all endobronchial ultrasound guided transbronchial needle aspirations (EBUS-TBNA), CT guided lung fine needle aspirations (FNA) and CT guided lung core biopsies. A total of 5,354 cases were identified, of which 92.2% had sufficient material for diagnosis. Of the 1506 cases identified with a recorded diagnosis of lung adenocarcinoma or NSCLC, not otherwise specified, 1001 (66.5%) had biomarker testing requested. Sufficient material was available in 89.5% of cases for a complete biomarker workup which included EGFR and KRAS mutational testing (all cases), ALK, ROS1 and PD-L1 immunohistochemistry (all cases), and ALK and ROS1 FISH (as required). For EGFR and KRAS mutational testing across both cytology and histology specimens, 99% of cases were sufficient. Of the samples in which a complete biomarker workup was unable to be performed, approximately half were only insufficient due to inadequate numbers of tumour cells for PD-L1 immunohistochemistry. Excluding PD-L1 IHC, 952 (95.1%) of samples obtained with ROSE were sufficient for the remainder of the testing requirements. Next generation sequencing using a 33 gene custom AmpliSeq panel was achieved in up to 72% of cases. In conclusion, small cytology and histology specimens obtained with ROSE are suitable for predictive biomarker testing in NSCLC, although attention needs to be paid to obtaining sufficient cells (>100) for PD-L1 immunohistochemistry.

Programmed Death-Ligand 1 Copy Number Loss in NSCLC Associates With Reduced Programmed Death-Ligand 1 Tumor Staining and a Cold Immunophenotype.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) copy number gains may be predictive of clinical response to immunotherapy in NSCLC. This study investigated PD-L1 copy number variations in tumor resection and bronchoscopy biopsies and its relationship with PD-L1 tumor cell staining and inflammatory gene expression. METHODS: PD-L1 gene copy number and mRNA expression were evaluated by real-time polymerase chain reaction in surgically resected NSCLC tumor biopsies (n = 87) and control biopsies (n = 20). A second cohort (n = 15) of bronchoscopy-derived tumor biopsies was analyzed, including multiple biopsies from the same patient across different anatomical sites. RESULTS: PD-L1 mRNA levels strongly correlated with PD-L1 tumor staining (r = 0.55, p < 0.0001). Interferon-γ mRNA expression associated with PD-L1 immune cell staining, but not PD-L1 tumor cell staining. In contrast, PD-L1 copy number positively associated PD-L1 tumor staining, but not PD-L1 immune cell staining. PD-L1 copy number analysis detected loss (15 of 87 = 17%) and gain (5 of 87 = 7%) of copy number. Tumors with low PD-L1 copy number expressed significantly reduced levels of inflammatory (interferon-γ, interleukin [IL]-6, IL-1ß, MMP-9) and immunosuppressive (IL-10, transforming growth factor ß) mediators. Analysis of bronchoscopy-derived biopsies revealed low heterogeneity in copy number values across different anatomical sites, in contrast to more variable PD-L1 mRNA expression. CONCLUSIONS: Low PD-L1 copy number tumors display reduced PD-L1 expression, reduced PD-L1 tumor cell staining, and an immunologic cold tumor microenvironment. Because PD-L1 copy number values are highly stable across different tumor regions, its evaluation may represent a robust and complimentary biomarker for predicting response to immunotherapy, where low copy number may predict lack of response.

SP142 PD-L1 Scoring Shows High Interobserver and Intraobserver Agreement in Triple-negative Breast Carcinoma But Overall Low Percentage Agreement With Other PD-L1 Clones SP263 and 22C3.

SP142 programmed cell death ligand 1 (PD-L1) status predicts response to atezolizumab in triple-negative breast carcinoma (TNBC). Prevalence of VENTANA PD-L1 (SP142) Assay positivity, concordance with the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay, and association with clinicopathologic features were assessed in 447 TNBCs. SP142 PD-L1 intraobserver and interobserver agreement was investigated in a subset of 60 TNBCs, with scores enriched around the 1% cutoff. The effect of a 1-hour training video on pretraining and posttraining scores was ascertained. At a 1% cutoff, 34.2% of tumors were SP142 PD-L1 positive. SP142 PD-L1 positivity was significantly associated with tumor-infiltrating lymphocytes (P <0.01), and node negativity (P=0.02), but not with tumor grade (P=0.35), tumor size (P=0.58), or BRCA mutation (P=0.53). Overall percentage agreement (OPA) for intraobserver and interobserver agreement was 95.0% and 93.7%, respectively, among 5 pathologists trained in TNBC SP142 PD-L1 scoring. In 5 TNBC SP142 PD-L1-naive pathologists, significantly higher OPA to the reference score was achieved after video training (posttraining OPA 85.7%, pretraining OPA 81.5%, P<0.05). PD-L1 status at a 1% cutoff was assessed by SP142 and SP263 in 420 cases, and by SP142 and 22C3 in 423 cases, with OPA of 88.1% and 85.8%, respectively. The VENTANA PD-L1 (SP142) Assay is reproducible for classifying TNBC PD-L1 status by trained observers; however, it is not analytically equivalent to the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay.

The role of 18 F-FDG PET/CT in retroperitoneal sarcomas-A multicenter retrospective study.

BACKGROUND: The role of 18 F-fluorodeoxyglucose positron emission tomography/computed tomography (18 F-FDG PET/CT) in the evaluation of retroperitoneal sarcomas is poorly defined. We evaluated the correlation of maximum standardized uptake value (SUVmax) with pathologic tumor grade in the surgical specimen of primary retroperitoneal dedifferentiated liposarcoma (DDLPS) and leiomyosarcoma (LMS). METHODS: Patients with the above histological subtypes in three participating institutions with preoperative 18 F-FDG PET/CT scan and histopathological specimen available for review were included. The association between SUVmax and pathological grade was assessed. Correlation between SUVmax and relapse-free survival (RFS) and overall survival (OS) were also studied. RESULTS: Of the total 58 patients, final pathological subtype was DDLPS in 44 (75.9%) patients and LMS in 14 (24.1%) patients. The mean SUVmax was 8.7 with a median 7.1 (range, 2.2-33.9). The tumors were graded I, II, III in 6 (10.3%), 35 (60.3%), and 17 (29.3%) patients, respectively. There was an association of higher histological grade with higher SUVmax (rs = 0.40, p = .002). Increasing SUVmax was associated with worse RFS (p = .003) and OS (p = .003). CONCLUSION: There is a correlation between SUVmax and pathologic tumor grade; increasing SUVmax was associated with worse OS and RFS, providing a preoperative noninvasive surrogate marker of tumor grade and biological behavior.

Characterising the immune microenvironment in liposarcoma, its impact on prognosis and the impact of radiotherapy.

BACKGROUND AND OBJECTIVES: Limited literature exists examining the immune microenvironment in liposarcoma, particularly with regard to the impact of radiotherapy. A major problem is the lack of scoring system for the tumour-infiltrating lymphocytes (TILs) in sarcoma. This study aims to describe the immune environment pre- and postradiotherapy and identify the optimal immune infiltrate scoring system for sarcoma. METHODS: Thirty-nine paired tissue samples (pre- and postradiotherapy) from patients with liposarcoma were scored by two pathologists for TILs using pre-existing systems (for breast cancer and melanoma) and compared for interobserver reliability. Immunohistochemical staining was performed for various immune markers. RESULTS: The TIL scoring system for breast cancer yielded perfect agreement (κ = 1.000). 21% of patients had increased TILs after radiotherapy, 87.5% of whom had dedifferentiated liposarcoma. Immune suppressor expression was increased frequently after radiotherapy (CD68 increased in 59.4%, PD-L1 increased in 25%). Immune effector expression (CD8) was unchanged in 84.4%. CONCLUSIONS: Breast cancer TIL scoring is reproducible in liposarcoma and has high interobserver reliability. Radiotherapy was observed to have a limited impact on immune effectors but seemed to have more impact in upregulating immune suppressors, suggesting radiotherapy may contribute to disease control through immunomodulatory effects. Dedifferentiated liposarcoma represents a uniquely responsive subtype.

CD8+ tumor-infiltrating lymphocytes within the primary tumor of patients with synchronous de novo metastatic colorectal carcinoma do not track with survival.

OBJECTIVES: Tumor-infiltrating lymphocytes (TIL), particularly CD8+ TILs in patients with colorectal cancer (CRC), are highly prognostic in the early-disease stages (I-II). In metastatic disease (stage IV; mCRC), their influence is less well defined. It has presumably failed to contain tumor cells to the primary site; however, is this evident? We explored the prognostic impact of TILs at the primary site in patients who presented de novo with mCRC. METHODS: Treatment-naïve patients (109) with mCRC were assessed for CD8+ TILs and PD-L1 expression. Microsatellite instability (MSI) was evaluated by IHC for PMS2 and MSH6 proteins and/or by PCR using the Bethesda panel. RESULTS: Microsatellite instability-high tumors had significantly more CD8+ TILs, with no significant survival advantage observed between MSI-H and microsatellite stable (MSS) tumors (12 vs 19 months, P = 0.304). TIL density for all cases had no impact on OS (low: 20 vs high: 13 months, P = 0.426), while PD-L1 of 1% or higher was associated with reduced mean survival (9.6 vs 18.9 months; P = 0.038). MSI-H tumors and associated immune cells had higher PD-L1 expression than in MSS cases. A positive correlation between PD-L1 on immune cells and CD8+ve TILs was found. A subset of MSS tumors had relatively high TILs approximating that of MSI-H tumors. CONCLUSION: In contrast to early-stage CRC, the immune response in primary tumors of patients with de novo mCRC does not appear to influence survival. A subgroup of MSS tumors was identified with increased TILs/PD-L1 comparable to MSI-H tumors, traditionally not be considered for immune checkpoint blockade and perhaps should be.

Prostate sarcomas: A radiological mimic for benign prostatic hyperplasia.

Leimyosarcomas arising from the stroma of the prostate are very rare, accounting for 0.1% of malignancies. We describe a case that closely mimicked benign prostatic hypertrophy on magnetic resonance imaging. Due to the low incidence of disease there is no high level evidence for management. We advocate neoadjuvant radiotherapy followed by radical prostatectomy with pelvic lymph node dissection. Diagnosis and expedient management is critical.

Correlation between percutaneous biopsy and final histopathology for retroperitoneal sarcoma: a single-centre study.

BACKGROUND: Retroperitoneal sarcomas are rare soft tissue tumours accounting for 10-15% of soft tissue sarcomas. Patient prognosis and treatment recommendations (including extent of surgery and neoadjuvant strategies) are determined by the pre-operative histopathological subtype and grade obtained from biopsy and thus it is important to understand the accuracy of biopsy in retroperitoneal masses. METHODS: This study presents a case series of primary retroperitoneal sarcomas managed at Peter MacCallum Cancer Centre (PMCC) between 2008 and 2019. Statistical analyses were performed to determine correlation between histopathology from percutaneous biopsy and surgical excision. RESULTS: A total of 117 patients who underwent percutaneous core biopsy and surgical excision of retroperitoneal sarcoma were included. Diagnostic accuracy varied with histopathological diagnosis, but overall precise concordance between biopsy and final histopathology was seen in 61% (κ = 0.57). Biopsy was most sensitive for identifying well-differentiated liposarcoma (WDLPS) (sensitivity 85%, 95% CI 0.06-0.96) and leiomyosarcoma (sensitivity 81%, 95% CI 0.54-0.96) and was least sensitive for identifying de-differentiated liposarcoma (DDLPS) (sensitivity 40%, 95% CI 0.25-0.56). Overall agreement between biopsy and final histopathology increased with use of PET/CT scan pre-biopsy and with use of fluorescence in situ hybridisation testing on biopsy, however, neither test improved recognition of de-differentiated components within WD/DDLPS on core biopsy. CONCLUSIONS: Pre-operative biopsy is important for clinical decision making in the treatment of retroperitoneal sarcoma. A significant portion of patients with a WDLPS will have a de-differentiated component identified at the time of resection that was not identified on initial biopsy.

Adequate tumour cellularity is essential for accurate PD-L1 immunohistochemistry assessment on cytology cell-block specimens.

OBJECTIVES: PD-L1 immunohistochemistry (IHC) is an essential predictive biomarker for patients with non-small cell lung cancer (NSCLC), required to inform treatment decisions regarding anti-PD-1 immune checkpoint inhibitor therapy. This study aims to investigate the concordance between PD-L1 IHC assessed on NSCLC cytology and histology specimens and to determine the impactce of tumour cellularity. METHODS: Matched cytology and histology NSCLC specimens were retrieved from the archives of the Royal Melbourne Hospital and the Royal Prince Alfred Hospital. PD-L1 IHC was performed concurrently on both specimens at the Peter MacCallum Cancer Centre using the SP263 assay kit on the Ventana Benchmark Ultra staining platform and scored by two experienced pathologists. RESULTS: Overall agreement between matched cytology and histology specimens was good (intraclass correlation coefficient = 0.653, n = 58); however, markedly increased when the analysis was limited to cell-blocks with >100 tumour cells (intraclass correlation coefficient = 0.957, n = 29). Specificity at both 1% and 50% cut-offs was high regardless of cellularity; however, sensitivity decreased in samples with <100 tumour cells. CONCLUSIONS: PD-L1 IHC on cytology cell-block specimens in NSCLC is an acceptable alternative to histological specimens, provided adequate tumour cells are present. Clinicians and pathologists should be mindful of the risk of false negative PD-L1 IHC in samples with low tumour cellularity, to avoid excluding patients from potentially beneficial treatment.

Novel multiplex droplet digital PCR assay for scoring PD-L1 in non-small cell lung cancer biopsy specimens.

OBJECTIVES: 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 cut-off 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 re-biopsy. Future studies will need to determine diagnostic ddPCR values that are predictive of clinical response to PD-1/PD-L1 immunotherapy.

A Novel Approach to Detect Programed Death Ligand 1 (PD-L1) Status and Multiple Tumor Mutations Using a Single Non-Small-Cell Lung Cancer (NSCLC) Bronchoscopy Specimen.

Multiple biomarkers are under evaluation to guide the use of immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC), including programed death ligand 1 (PD-L1) tumor cell staining. We have developed a new approach that accurately quantifies PD-L1 status and identifies multiple mutations by using a single bronchoscopy specimen. A novel molecular marker was identified to detect the presence of malignant cells in radial endobronchial ultrasound bronchial brushings from NSCLC (n = 15) and benign (n = 13) nodules by quantitative real-time RT-PCR (RT-qPCR). The MMP9:TIMP3 transcript ratio was significantly increased in NSCLC and using receiver operating characteristic curve analysis accurately discriminated malignant and benign bronchoscopy specimens (area under the curve = 0.98; 95% CI, 0.93-1; P < 0.0001). Utilizing the same specimens, PD-L1 expression and multiple oncogenic mutations were detected by RT-qPCR and next-generation sequencing. A second archive of snap-frozen squamous cell carcinoma (n = 40) and control (n = 20) biopsies with matching formalin-fixed, paraffin-embedded slides were used to compare PD-L1 status by immunohistochemistry and RT-qPCR. The biopsy cohort confirmed that the MMP-9:TIMP3 ratio was predictive of malignancy and demonstrated that PD-L1 transcript expression was concordant with PD-L1 tumor cell membrane staining in NSCLC (Spearman r = 0.636, P < 0.0001). This rapid molecular approach can detect malignant cells and using the same single bronchoscopy specimen can generate high-quality unfixed nucleic acid that accurately quantify PD-L1 status and identify multiple oncogenic mutations.

Dual-energy contrast-enhanced CT to evaluate scaphoid osteonecrosis with surgical correlation.

To evaluate the validity of contrast enhanced dual energy CT using a lung perfusion algorithm in assessing for post-traumatic scaphoid proximal pole avascular necrosis. From Aug 2013 to Aug 2016, 18 patients (19 wrists, 16 males, 2 females, mean age 28 years) were assessed as high-risk for proximal pole scaphoid avascular necrosis by a single surgeon following a scaphoid fracture and were referred for contrast-enhanced dual energy CT. 8 wrists had specimens sent for correlative histological analysis and 11 were correlated with operative notes. Eight surgical specimens were sent to histology and showed a 100% correlation (8/8) with the DECT findings. The remaining 11 wrists that did not have a specimen sent had in-surgery findings that also correlated with DECT. A single case was discrepant (1/11) due to presence of an intra-osseous ganglion, which was reported as osteonecrosis on CT, but considered viable at surgery. No case was called viable on CT that proved to be necrotic at either surgery or histologically. Contrast-enhanced dual energy CT using a perfusion algorithm is an innovative and promising method in evaluating viability of the post-trauma proximal pole of scaphoid.

Treatment of patients with primary retroperitoneal sarcoma: predictors of outcome from an Australian specialist sarcoma centre.

BACKGROUND: Several unanswered questions surround the management of retroperitoneal sarcoma (RPS). Guidelines recommend treatment by a multidisciplinary team at a specialized referral centre. The objective of this study was to describe the management of RPS at an Australian specialist sarcoma centre, comparing outcomes to international standards and analysing for predictors of local failure. METHODS: A retrospective review of a prospectively maintained database was performed on patients with RPS treated between 2008 and 2016. A 5-year outcome analyses focussed on patients undergoing curative-intent surgery for primary, non-metastatic RPS. RESULTS: Eighty-eight patients underwent surgery for primary RPS. Five-year overall survival was 66%, 5-year freedom from local recurrence was 65% and 5-year freedom from distant metastasis was 71%. Overall survival was associated with tumour grade (hazard ratio (HR) 6.1, P < 0.001) and histologic organ invasion (HR 5.7, P < 0.001). Variables associated with improved freedom from local recurrence were macroscopically complete resection (HR 0.14, P < 0.001) and neoadjuvant radiotherapy (HR 0.33, P = 0.014). Treatment at a specialist sarcoma centre was associated with a higher rate of preoperative biopsy and neoadjuvant radiotherapy (both with P < 0.001). There was a trend towards improved local control for patients undergoing surgery at a specialist centre (P = 0.055). CONCLUSION: This is the largest Australian series of RPS and outcomes are comparable to major international sarcoma centres. Patients treated at a specialist centre had higher rates of preoperative diagnosis and tailored therapy which was associated with improved outcomes. Patients with suspected RPS should be referred to a specialist centre for optimal preoperative evaluation and multidisciplinary management.

Iatrogenic subcutaneous metastasis from WHO Grade I intracranial meningioma.

Meningiomas are the most frequent primary brain tumours and are often managed with surgical excision. We present the case of a young woman with the unusual phenomenon of iatrogenic subcutaneous seeding from an intracranial meningioma. We discuss the risk factors, possible mechanisms and management of this.

Determinants of variability of five programmed death ligand-1 immunohistochemistry assays in non-small cell lung cancer samples.

Programmed death ligand-1 (PD-L1) expression as determined by immunohistochemistry (IHC) is potentially predictive of clinical outcome. The aim of this study was to assess the concordance of reported PD-L1 IHC assays and investigate factors influencing variability. Consecutive sections from 20 non-small cell lung cancers (NSCLCs) comprising resection, core biopsy, cytology and pleural fluid samples underwent IHC with 5 different antibody/autostainer combinations: 22C3/Link48, 28-8/BOND-MAX, E1L3N/BOND-MAX, SP142/BenchMark and SP263/BenchMark. PD-L1 RNA levels were assessed using RNAscope. The frequency of positive cases using scoring thresholds from clinical trials was 72%, 33%, 61%, 56%, and 33% for the 5 IHC protocols respectively, and 33% for RNAscope. Pairwise agreement on the classification of cases as positive or negative for PD-L1 expression ranged from 61%-94%. On a continuous scale, the lowest correlation was between 28-8/BOND-MAX and SP142/BenchMark (R2=0.25) and highest was between 22C3/Link48 and E1L3N/BOND-MAX (R2=0.71). When cases were ordered according to tumor cell (TC)%, a similar ranking of cases across IHC protocols could be observed, albeit with different quanta and limits of detection. Single-slide OPAL 7-color fluorescence IHC analysis revealed a high degree of co-localization of staining from the 5 PD-L1 antibodies. Using SP142 antibody in a BOND-MAX protocol led to increased TC% quanta, while retaining a similar ranking of samples according to TC%. The results of this study highlight tumor PD-L1 status can vary significantly according to IHC protocol. Protocol-dependent staining intensities and nominated thresholds for positivity contribute to this variability, while the antibody used appears to be less of a factor.

Prognostic Significance of PD-L1+ and CD8+ Immune Cells in HPV+ Oropharyngeal Squamous Cell Carcinoma.

Human papilloma virus-positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a distinct subgroup of head and neck cancers associated with clinical outcomes that are not accurately categorized by existing tumor-node-metastasis-based staging methods. Given the significant impact of immune parameters, such as tumor-infiltrating lymphocytes (TIL) in many cancers, we sought to determine if immunophenotyping tumors can improve categorization of HPV+ OPSCCs for prognostic purposes. In a cohort of 190 patients with HPV+ OPSCC, we quantified and determined the localization of CD8+ TILs, as well as PD-L1-expressing tumor cells (TC) and immune cells (IC). The prognostic significance of these parameters on overall survival (OS) was evaluated, and their contribution to existing prognostic models was determined. High CD8+ TIL abundance (≥30% on stromal or intratumoral ICs) was seen in 61.3% patients and was associated with improved OS [HR, 0.4; 95% confidence interval (CI), 0.2-0.9; P = 0.017]. Although the expression of PD-L1 on TC was not prognostic, high expression of PD-L1 on ≥5% of intratumoral ICs was found in 38.5% patients and was significantly associated with improved OS (HR, 0.37; 95% CI, 0.15-0.93; P = 0. 023). Both high intratumoral IC PD-L1 expression and abundant CD8+ TILs in HPV+ OPSCCs identify subgroups of patients with excellent outcomes and provide additional prognostic information beyond existing staging systems. Cancer Immunol Res; 6(3); 295-304. ©2018 AACR.

Comparison of Four PD-L1 Immunohistochemical Assays in Lung Cancer.

INTRODUCTION: Four different programmed death ligand 1 immunohistochemical assays are approved or in development as companion or complementary diagnostics to different immunotherapeutic agents in lung carcinoma. We sought to determine whether these assays are technically equivalent and whether one antibody can be used on an alternate staining platform. METHODS: Serial sections of tissue microarrays constructed from 368 cases of resected lung cancer were stained for 22C3 and 28-8 on the Dako Link 48 platform (Dako, Carpinteria, Ca) and for SP142 and SP263 on the Ventana Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ) strictly as per product insert. A protocol was developed to use the 22C3 antibody on the Ventana Benchmark Ultra platform. RESULTS: Differences in mean tumor cell and immune cell staining were observed between the four assays (p < 0.001). Differences between 22C3 and 28-8 were not statistically significant. Concordance of tumor cell scores was good (intraclass correlation coefficient [ICC] = 0.674), particularly when SP142 was excluded as an outlier (ICC = 0.755). The highest concordance was seen between 22C3 and 28-8 (ICC = 0.812). Concordance was poor for immune cell staining (ICC = 0.212). When dichotomized according to clinically relevant cutoffs, pairwise comparisons showed poor to moderate concordance (κ = 0.196-0.578), with positive percent agreement ranging from 15.1% to 90.0%. The 22C3 antibody performed comparably on the Dako Link 48 platform and the alternate Ventana Benchmark Ultra platform (ICC = 0.921, κ = 0.897). CONCLUSIONS: Concordance between the four programmed death ligand 1 immunohistochemical assays when performed and scored as intended show that apart from 28-8 and 22C3, they cannot be used interchangeably in clinical practice. A protocol was successfully developed to use 22C3 on an alternate platform, which may help to overcome some barriers to implementation.

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.