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.

Assessing Tumor-Infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method from the International Immuno-Oncology Biomarkers Working Group: Part 2: TILs in Melanoma, Gastrointestinal Tract Carcinomas, Non-Small Cell Lung Carcinoma and Mesothelioma, Endometrial and Ovarian Carcinomas, Squamous Cell Carcinoma of the Head and Neck, Genitourinary Carcinomas, and Primary Brain Tumors.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features.

Differential expression of immunohistochemical markers in primary lung and breast cancers enriched for triple-negative tumours.

AIMS: In breast cancer patients presenting with a lung lesion, the distinction between lung and breast origin is clinically important. Lung and breast cancers are both CK7(+) /CK20(-) , so additional immunohistochemical markers are needed. METHODS AND RESULTS: We examined the expression of oestrogen receptor (ER), progesterone receptor (PR), thyroid transcription factor-1 (TTF-1), gross cystic disease fluid protein-15 (GCDFP-15), p63 and Wilms' tumour 1 (WT1) in a series of tissue microarrays comprising 266 non-small-cell lung cancers and 837 primary breast cancers enriched for triple-negative tumours (TNBC). Staining for ER, PR, TTF-1 and GCDFP-15 was present in 63%, 49%, 0% and 25% of breast and 6%, 9%, 59% and 1% of lung cancers, respectively. Strong staining for p63 was present in 63 (97%) lung squamous cell carcinomas and only eight (9%) TNBC. WT1 nuclear staining was rare; however, cytoplasmic staining was identified in 49 (40%) TNBC and 10 (5%) lung cancers. Cluster analysis segregated TNBC from lung cancers with TTF-1 and/or p63 staining favouring lung origin, and GCDFP-15 or WT1 staining favouring breast origin. Cancers negative for all four markers (17%) were 60% breast and 40% lung origin. CONCLUSION: An immunohistochemical panel incorporating ER, TTF-1, GCDFP-15, p63 and WT1 can help to distinguish lung cancer from metastatic breast cancer, including TNBC.

Prevalence, morphology, and natural history of FGFR1-amplified lung cancer, including squamous cell carcinoma, detected by FISH and SISH.

The aim of this study was to investigate the prevalence of fibroblast growth factor receptor 1 (FGFR1) amplification by fluorescence in situ hybridization (FISH) in a lung cancer patient cohort and to correlate results with morphology, silver in situ hybridization (SISH), and patient outcome. FGFR1 FISH and SISH were performed in 406 and 385 lung cancer cases, respectively, and the results were compared. High-level FGFR1 amplification was defined as the ratio of FGFR1/centromere 8 ≥2, or tumor cell percentage with ≥15 signals ≥10%, or average number of signals/tumor cell nucleus ≥6. Low-level amplification was defined as tumor cell percentage with ≥5 signals ≥50%. Of 406 tumors tested, there were 191 squamous cell carcinomas, 28 carcinomas with focal squamous morphology, 24 large cell carcinomas with squamous immunoprofile, 115 adenocarcinomas, 17 neuroendocrine tumors, and 31 carcinomas without squamous morphology or immunoprofile. FGFR1 FISH was assessable in 368 tumors, with FGFR1 amplification identified in 50, including 48 tumors with either squamous morphology or immunoprofile (48 of 225, 21.3%), and two 'marker-null' tumors without squamous or glandular morphology or immunoprofile (2 of 143, 1.4%; P<0.0001). FGFR1 SISH was assessable in 347 tumors. All 46 FGFR1 FISH-amplified tumors with tumor available for testing showed amplification with SISH, while all other tumors were negative. There was no relationship between FGFR1 amplification status and disease-free (P=0.88, HR=1.04, 95% confidence interval (CI)=0.67-1.60) or overall survival (P=0.97, HR=1.01, 95% CI=0.65-1.58) in surgically radically treated patients with tumors with any squamous morphology or immunoprofile. FGFR1 amplification is a common abnormality in tumors with any squamous morphology or immunoprofile, but it is also present in 'marker-null' tumors. The results of FGFR1 SISH showed 1:1 correlation with the results of FGFR1 FISH, indicating that SISH may be an alternative method to detect FGFR1 amplification. No relationship was detected between patient outcome and FGFR1 amplification.

Establishing mesothelioma patient-derived organoid models from malignant pleural effusions.

OBJECTIVES: Pleural mesothelioma is a cancer arising in the cells that line the lungs and chest wall with poor survival and poor response to first-line therapy. Organoid models of cancer can faithfully recapitulate the genetic and histopathological characteristics of individualized tumors and have potential to be used for precision medicine, however methods of establishing patient-derived mesothelioma organoids have not been well established in the published literature. MATERIALS AND METHODS: Long-term mesothelioma patient-derived organoids were established from ten malignant pleural effusion fluids. Mesothelioma patient-derived organoids were compared to the corresponding biopsy tissue specimens using immunohistochemistry labelling for select diagnostic markers and the TruSight Oncology-500 sequencing assay. Cell viability in response to the chemotherapeutic drug cisplatin was assessed. RESULTS: We established five mesothelioma patient-derived organoid cultures from ten malignant pleural effusion fluids collected from nine individuals with pleural mesothelioma. Mesothelioma patient-derived organoids typically reflected the histopathological and genomic features of patients' matched biopsy specimens and displayed cytotoxic sensitivity to cisplatin in vitro. CONCLUSION: This is the first study of its kind to establish long-term mesothelioma organoid cultures from malignant pleural effusions and report on their utility to test individuals' chemotherapeutic sensitivities ex vivo.

The significance of BAP1 and MTAP/CDKN2A expression in well-differentiated papillary mesothelial tumour: a series of 21 cases and a review of the literature.

The nomenclature and diagnostic criteria of well-differentiated papillary mesothelial tumour (WDPMT) have been changed in the 2021 World Health Organization (WHO) classification of thoracic tumours, and a new entity, mesothelioma in situ (MIS), introduced. Histologically these two entities may be similar. However, MIS is regarded as a precursor to invasive mesothelioma and requires demonstration of loss of BAP1 and/or MTAP/CDKN2A for diagnosis, whereas performance of these ancillary tests is desirable but not essential for a diagnosis of WDPMT, in which the significance of BAP1 and/or MTAP/CDKN2A loss is not well understood or well defined. Against this backdrop, we undertook an investigation of 21 cases of WDPMT, identified from our case files and diagnosed according to 2021 WHO criteria, to explore the relationship between histology and BAP1 and MTAP/CDKN2A expression with clinical features including asbestos exposure, focality of tumours and clinical outcome. There were 18 women and three men, with ages ranging from 23-77 years (median 62 years), in which six had a history of asbestos exposure, two had no exposure, and in 13 exposure history was unavailable. Of 20 peritoneal tumours and one pleural tumour, 13 were detected incidentally at the time of surgery for unrelated conditions and eight peritoneal tumours were multifocal at the time of diagnosis. BAP1 immunohistochemistry (IHC) was performed in all 21 tumours, with nine tumours showing BAP1 expression loss. MTAP/CDKN2A testing was performed in 14 tumours, comprising MTAP IHC in 12 and CDKN2A fluorescence in situ hybridisation (FISH) in two, with three tumours showing MTAP/CDKN2A expression loss. Two tumours with MTAP/CDKN2A loss also showed BAP1 expression loss. Four patients progressed to invasive mesothelioma, including one male with a pleural tumour and asbestos exposure, and three females with multifocal peritoneal tumours, two with asbestos exposure and one without exposure. BAP1 expression loss was seen in all tumours from the four patients who progressed to invasive mesothelioma, whilst two of these tumours showed retained MTAP IHC and two were not tested. There was one patient with a tumour with MTAP loss and retained BAP1 who died from unrelated causes 5 months after diagnosis. Eight patients received WDPMT-specific treatment in addition to the initial excision. Survival for all patients ranged from 4-218 months, with one patient dying of mesothelioma at 49 months. Based on our results in this series of 21 patients with WDPMT diagnosed according to 2021 WHO criteria, we propose that WDPMT with BAP1 expression loss may best be regarded as papillary MIS and that a history of asbestos exposure and the presence of multifocal tumours in patients diagnosed with WDPMT should prompt ancillary testing with BAP1 IHC. Further we propose that BAP1 IHC should be essential in the diagnosis of WDPMT, with the diagnosis restricted to those tumours which show retained BAP1 expression. However more studies in larger cohorts of patients are needed to explore the relationship between BAP1 expression and MTAP loss in WDPMT, which will help to define this entity and separate it more clearly from MIS and invasive mesothelioma.

The prognostic significance of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 expression in early stage non-small cell lung cancer.

BACKGROUND: Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 has been functionally associated with a stem cell phenotype in normal and malignant cells. The prevalence of such cells in solid tumours should therefore correlate with recurrence and/or metastasis following definitive surgical resection. The aim of this study was to evaluate the prognostic significance of ALDH1A1 and CD133 in surgically resected, early stage non-small cell lung cancer (NSCLC). METHODS: A retrospective analysis of ALDH1A1 and CD133 expression in 205 patients with pathologic stage I NSCLC was performed using immunohistochemistry. The association between the expression of both markers and survival was determined. RESULTS: We identified 62 relapses and 58 cancer-related deaths in 144 stage 1A and 61 stage 1B patients, analysed at a median of 5-years follow-up. Overexpression of ALDH1A1 and CD133, detected in 68.7% and 50.7% of primary tumours, respectively, was an independent prognostic indicator for overall survival by multivariable Cox proportional hazard model (p=0.017 and 0.039, respectively). Overexpression of ALDH1A1, but not of CD133, predicted poor recurrence-free survival (p=0.025). When categorised into three groups according to expression of ALDH1A1/CD133, patients with overexpression of both ALDH1A1 and CD133 belonged to the group with the shortest recurrence-free and overall survival (p=0.015 and 0.017, respectively). CONCLUSIONS: Expression of ALDH1A1 and CD133, and coexpression of ALDH1A1 and CD133, is strongly associated with poor survival in early-stage NSCLC following surgical resection. These data are consistent with the hypothesis that expression of stem cell markers correlates with recurrence as an indirect measure of self-renewal capacity.

Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization.

Rearrangements of anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) define a molecular subgroup of tumors characterized clinically by sensitivity to ALK tyrosine kinase inhibitors such as crizotinib. Although ALK rearrangements may be detected by reverse transcriptase-PCR, immunohistochemistry or fluorescence in situ hybridization (FISH), the optimal clinical strategy for identifying ALK rearrangements in clinical samples remains to be determined. We evaluated immunohistochemistry using three different antibodies (ALK1, 5A4 and D5F3 clones) to detect ALK rearrangements and compared those with FISH. We report the frequency and clinicopathologic features of lung cancers harboring ALK translocations in 594 resected NSCLCs (470 adenocarcinomas; 83 squamous carcinomas, 26 large cell carcinomas and 15 other histological subtypes) using a tissue microarray approach. We identified an ALK gene rearrangement in 7/594 cases (1%) by FISH and all anti-ALK antibodies correctly identified the seven ALK-positive cases (100% sensitivity), although the intensity of staining was weak in some cases. These data indicate that the use of antibodies with high sensitivity and avidity to ALK may provide an effective pre-screening technique to complement the more expensive and labor-intensive approach of ALK FISH testing.

Overexpression of CD39 protects in a mouse model of preeclampsia.

CD39 (NTPDase1), a critical immune and vascular ecto-nucleotidase, hydrolyses pro-inflammatory and pro-thrombotic nucleotides (adenosine-5'-triphosphate (ATP) and adenosine diphosphate) to adenosine. In humans, CD39 is the dominant ecto-nucleotidase in placental trophoblastic tissues and modulates ATP-dependent trophoblastic functions. CD39 is an integral component of regulatory T cells (Treg), which are central to immunological tolerance and maintenance of normal pregnancy. We examined the impact of CD39 overexpression in a mouse model of preeclampsia. Matings were performed between virginal BALB/c female (wild-type (WT) or CD39 transgenic (CD39TG)) and C57BL/6 male mice. On days 10 and 12 of pregnancy BALB/c Th1-polarized cells were injected. Systolic blood pressure (SBP) was measured throughout pregnancy. Mice were sacrificed at day 15 of pregnancy. Following transfer of Th1-polarized cells, SBP of pregnant WT mice increased (118 ± 3 mmHg to 142 ± 5 mmHg). Although ultrastructural changes were evident in the kidney this was not accompanied by significant proteinuria. SBP remained unchanged (115 ± 2 mmHg to 114 ± 3 mmHg) in pregnant CD39TG mice without evidence of renal lesions. We conclude that gestational hypertension can be induced in mice following transfer of maternally derived Th1-polarized cells and that overexpression of CD39 is protective in this model.

Recurrent myocarditis--an important mimic of ischaemic myocardial infarction.

Patients presenting with a syndrome of chest pain, elevated cardiac enzyme levels with or without electrocardiogram changes are a common diagnostic and management problem in cardiology. Most commonly, this is due to ischaemic myocardial infarction secondary to coronary artery disease. However, when coronary angiography does not demonstrate any obstructive coronary artery lesion, the diagnosis of myocarditis should be considered. Cardiac magnetic resonance imaging is helpful towards making this diagnosis. Here, we describe the first reported Australian cases of recurrent myocarditis presenting with ischaemic chest pain and elevated cardiac enzyme levels. These cases serve as an important reminder to clinicians that myocarditis is an important mimic of ischaemic myocardial infarction.

Real-world prevalence of PD-L1 expression in non-small cell lung cancer: an Australia-wide multi-centre retrospective observational study.

An investigator-initiated, Australia-wide multi-centre retrospective observational study was undertaken to investigate the real-world prevalence of programmed death ligand-1 (PD-L1) expression in non-small cell lung carcinoma (NSCLC). Multiple centres around Australia performing PD-L1 immunohistochemistry (IHC) were invited to participate. Histologically confirmed NSCLC of any stage with a PD-L1 IHC test performed for persons aged ≥18 years between 1 January 2018 and 1 January 2020, and eligible for review, were identified at each centre, followed by data extraction and de-identification, after which data were submitted to a central site for collation and analysis. In total data from 6690 eligible PD-L1 IHC tests from histologically (75%) or cytologically (24%) confirmed NSCLC of any stage were reviewed from persons with a median age of 70 years, 43% of which were female. The majority (81%) of tests were performed using the PD-L1 IHC SP263 antibody with the Ventana BenchMark Ultra platform and 19% were performed using Dako PD-L1 IHC 22C3 pharmDx assay. Reported PD-L1 tumour proportion score (TPS) was ≥50% for 30% of all tests, with 62% and 38% scoring PD-L1 ≥1% and <1%, respectively. Relative prevalence of clinicopathological features with PD-L1 scores dichotomised to <50% and ≥50%, or to <1% and ≥1%, were examined. Females scored ≥1% slightly more often than males (64% vs 61%, respectively, p=0.013). However, there was no difference between sexes or age groups (<70 or ≥70 years) where PD-L1 scored ≥50%. Specimens from patients with higher stage (III/IV) scored ≥1% or ≥50% marginally more often compared to specimens from patients with lower stage (I/II) (p≤0.002). Proportions of primary and metastatic specimens did not differ where PD-L1 TPS was ≥1%, however more metastatic samples scored TPS ≥50% than primary samples (metastatic vs primary; 34% vs 27%, p<0.001). Cytology and biopsy specimens were equally reported, at 63% of specimens, to score TPS ≥1%, whereas cytology samples scored TPS ≥50% slightly more often than biopsy samples (34% vs 30%, respectively, p=0.004). Resection specimens (16% of samples tested) were reported to score TPS ≥50% or ≥1% less often than either biopsy or cytology samples (p<0.001). There was no difference in the proportion of tests with TPS ≥1% between PD-L1 IHC assays used, however the proportion of tests scored at TPS ≥50% was marginally higher for 22C3 compared to SP263 (34% vs 29%, respectively, p<0.001). These real-world Australian data are comparable to some previously published global real-world data, with some differences noted.

Interobserver agreement in determining non-small cell lung cancer subtype in specimens acquired by EBUS-TBNA.

Endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA) may diagnose suspected lung cancer. Determination of non-small cell lung cancer (NSCLC) subtype may guide therapy in select patients. Small-volume biopsies may be subject to significant interobserver variability in subtype determination. Three pathologists independently reviewed specimens from 60 patients who underwent EBUS-TBNA for diagnosis/staging of suspected/known NSCLC. Smear, haematoxylin and eosin (H&E) and immunohistochemistry (IHC) specimens were reviewed without reference to other specimen types obtained from the same patient. Final diagnoses, and degree of confidence in the diagnosis, were recorded for each specimen. Almost perfect agreement was seen for distinguishing between small cell lung cancer and NSCLC for all specimen types. Agreement in determination of NSCLC subtype for smear, H&E and IHC specimens was slight (κ=0.095, 95% CI -0.164-0.355), fair (κ=0.278, 95% CI 0.075-0.481) and moderate (κ=0.564, 95% CI 0.338-0.740), respectively. Perfect agreement was seen when all three observers were confident of diagnoses made on IHC specimens. Interobserver agreement in interpretation of EBUS-TBNA specimens is moderate for determination of NSCLC subtype. Agreement is highest following examination of IHC specimens. Clinicians should be aware of the degree of pathologist confidence in the tissue diagnosis prior to commencement of subtype-specific therapy for NSCLC.

GM-CSF antibodies in artificial stone associated silicoproteinosis: A case report and literature review.

Pulmonary alveolar proteinosis (PAP) is a rare lung disease where there is accumulation of surfactant in the alveoli. It can be classified based on the underlying aetiology into three categories: primary, secondary and congenital. Autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF-Ab) are a key diagnostic feature of autoimmune PAP. High intensity occupational exposure and inhalation of toxic particles such as silica can cause a form of secondary PAP called acute silicoproteinosis. We describe a 26-year-old stone benchtop fabricator with silicoproteinosis following daily exposure to high levels of silica who had elevated serum GM-CSF-Ab. We discuss the role of GM-CSF-Ab in cases of PAP with occupational inhalational exposure and the challenges in its interpretation.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

NSCLC Subtyping in Conventional Cytology: Results of the International Association for the Study of Lung Cancer Cytology Working Group Survey to Determine Specific Cytomorphologic Criteria for Adenocarcinoma and Squamous Cell Carcinoma.

INTRODUCTION: Accurate subtyping of NSCLC into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) is the cornerstone of NSCLC diagnosis. Cytology samples reveal higher rates of classification failures, that is, subtyping as non-small cell carcinoma-not otherwise specified (NSCC-NOS), as compared with histology specimens. This study aims to identify specific algorithms on the basis of known cytomorphologic features that aid accurate and successful subtyping of NSCLC on cytology. METHODS: A total of 13 expert cytopathologists participated anonymously in an online survey to subtype 119 NSCLC cytology cases (gold standard diagnoses being LUAD in 80 and LUSC in 39) enriched for nonkeratinizing LUSC. They selected from 23 predefined cytomorphologic features that they used in subtyping. Data were analyzed using machine learning algorithms on the basis of random forest method and regression trees. RESULTS: From 1474 responses recorded, concordant cytology typing was achieved in 53.7% (792 of 1474) responses. NSCC-NOS rates on cytology were similar among gold standard LUAD (36%) and LUSC (38%) cases. Misclassification rates were higher in gold standard LUSC (17.6%) than gold standard LUAD (5.5%; p < 0.0001). Keratinization, when present, recognized LUSC with high accuracy. In its absence, the machine learning algorithms developed on the basis of experts' choices were unable to reduce cytology NSCC-NOS rates without increasing misclassification rates. CONCLUSIONS: Suboptimal recognition of LUSC in the absence of keratinization remains the major hurdle in improving cytology subtyping accuracy with such cases either failing classification (NSCC-NOS) or misclassifying as LUAD. NSCC-NOS seems to be an inevitable morphologic diagnosis emphasizing that ancillary immunochemistry is necessary to achieve accurate subtyping on cytology.

Expression of EGFR and conformational forms of EGFR in malignant pleural mesothelioma and its impact on survival.

AIM: Conformational forms of the epidermal growth factor receptor (EGFR) are pro-tumorigenic. The prevalence and impact of conformational forms of EGFR in malignant mesothelioma (MM) is unknown. We investigated expression of EGFR and conformational forms of EGFR by immunohistochemistry using EGFR-targeting monoclonal antibodies (mAb). In addition, EGFR gene amplification was investigated by fluorescent in-situ hybridization (FISH). Findings were correlated with survival. METHODS: Patients treated between 1988 and 2014 were identified from the thoracic surgery database of the Austin Hospital, Melbourne, Australia. Tissue microarrays (TMAs) were constructed, subjected to wild type (wt) EGFR IHC staining and FISH analysis. Conformational and mutation forms of EGFR were detected by IHC using mAb806, and LMH-151 which detects EGFRVIII. `H-scores` were derived and EGFR expression correlated with survival by Kaplan-Meier and log rank analysis. RESULTS: WtEGFR expression was seen in 93 % (299/321) of cases with overexpression (defined as an H-score ≥200) seen in more than half of cases (64 %). EGFR overexpression in MM was seen more commonly in the epithelioid subtype. EGFR overexpression was not associated with true EGFR amplification, although multiple copies were appreciated in samples with polysomy. EGFR expression did not correlate with survival. A conformational form of EGFR associated with EGFR dysregulation was found in 8.2 % of cases, and patients with these tumors had a trend towards a poorer outcome. No cases of the EGFRVIII mutation were identified. CONCLUSION: MM consistently demonstrated high expression of EGFR, with a subset of tumors showing conformational EGFR forms consistent with EGFR dysregulation, but withoutEGFR amplification or EGFR VIII mutation. wtEGFR expression did not influence survival. The impact of EGFR conformation on survival warrants further investigation.

Analysis of angiogenic and stromal biomarkers in a large malignant mesothelioma cohort.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive malignancy of the pleura and other mesothelial membranes. Agents targeting vascular endothelial growth factor (VEGF) such as bevacizumab; and multi-kinase inhibitors such as nintedanib [angiokinase inhibitor of VEGF, platelet-derived growth factor (PDGF) receptor and fibroblast growth factor receptor (FGFR)] have recently demonstrated efficacy in MM. METHODS: Tissue microarrays (TMAs) were created from formalin-fixed, paraffin-embedded tissue samples obtained from 326 patients with MM who were treated surgically. PDGF-CC, FGFR-1, VEGF and CD31 expression were analysed by immunohistochemical (IHC) staining. The H-score method assigned a score of 0-300 to each sample, based on the percentage of cells stained at different intensities. CD31 was evaluated via Chalkley's method to evaluate microvessel density. We evaluated the association between expression of the biomarkers, clinicopathological factors and outcomes, in patients with MM. RESULTS: CD31 high (≥5) was seen in only 31/302 (10.3%) irrespective of histology. PDGF-CC high (≥150) was seen in 203 /310 (65%) of all samples. VEGF high (≥80) was seen in 219/322 (68%) of all MM with 143/209 (68%) of epithelioid histology. FGFR-1 high (≥40) was seen in 127/310 (41%) of all MM. There was no association of VEGF and FGFR-1 IHC with survival nor differences between histological subtypes. There was a non-significant trend towards poorer survival in epithelioid tumours with increased PDGF-CC expression (OS 18.5 vs 13.2 months; HR 0.7928; 95% CI 0.5958 to 1.055, P = 0.1110). High CD31 score was associated with significantly poorer survival (OS 12 vs 8.6 months; HR 0.48; 95% CI 0.2873 to 0.7941, P = 0.0044). Of the 31 patients with high CD31 scores; 23/31 (74%) were also high for PDGF-CC and 20/31 (64%) with high VEGF scores. CD31 was found to be an independent prognostic factor in multivariate analysis (HR 1.540; 95% CI 1.018 to 2.330; p = 0.041). CONCLUSIONS: High CD31 was an independent poor prognostic factor and high PDGF-CC expression was associated with poor survival in MM. Abrogating these pathways may have prognostic implications.

A Grading System for Invasive Pulmonary Adenocarcinoma: A Proposal From the International Association for the Study of Lung Cancer Pathology Committee.

INTRODUCTION: A grading system for pulmonary adenocarcinoma has not been established. The International Association for the Study of Lung Cancer pathology panel evaluated a set of histologic criteria associated with prognosis aimed at establishing a grading system for invasive pulmonary adenocarcinoma. METHODS: A multi-institutional study involving multiple cohorts of invasive pulmonary adenocarcinomas was conducted. A cohort of 284 stage I pulmonary adenocarcinomas was used as a training set to identify histologic features associated with patient outcomes (recurrence-free survival [RFS] and overall survival [OS]). Receiver operating characteristic curve analysis was used to select the best model, which was validated (n = 212) and tested (n = 300, including stage I-III) in independent cohorts. Reproducibility of the model was assessed using kappa statistics. RESULTS: The best model (area under the receiver operating characteristic curve [AUC] = 0.749 for RFS and 0.787 for OS) was composed of a combination of predominant plus high-grade histologic pattern with a cutoff of 20% for the latter. The model consists of the following: grade 1, lepidic predominant tumor; grade 2, acinar or papillary predominant tumor, both with no or less than 20% of high-grade patterns; and grade 3, any tumor with 20% or more of high-grade patterns (solid, micropapillary, or complex gland). Similar results were seen in the validation (AUC = 0.732 for RFS and 0.787 for OS) and test cohorts (AUC = 0.690 for RFS and 0.743 for OS), confirming the predictive value of the model. Interobserver reproducibility revealed good agreement (k = 0.617). CONCLUSIONS: A grading system based on the predominant and high-grade patterns is practical and prognostic for invasive pulmonary adenocarcinoma.

Immunocytochemistry for predictive biomarker testing in lung cancer cytology.

With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and cost-effective tool for the screening and detection of many of these markers. In particular, robust IHC assays performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue are widely used as surrogate markers for ALK and ROS1 rearrangements and for detecting programmed death ligand 1 (PD-L1) expression in patients with advanced NSCLC; in addition, they have become essential for treatment decisions. Cytology samples represent the only source of tumor in a significant proportion of patients with inoperable NSCLC, and there is increasing demand for predictive biomarker testing on them. However, the wide variation in the types of cytology samples and their preparatory methods, the use of alcohol-based fixatives that interfere with immunochemistry results, the difficulty in procurement of cytology-specific controls, and the uncertainty regarding test validity have resulted in underutilization of cytology material for predictive immunocytochemistry (ICC), and most cytopathologists limit such testing to FFPE cell blocks (CBs). The purpose of this review is to: 1) analyze various preanalytical, analytical, and postanalytical factors influencing ICC results; 2) discuss measures for validation of ICC protocols; and 3) summarize published data on predictive ICC for ALK, ROS1, EGFR gene alterations and PD-L1 expression on lung cancer cytology. Based on our experience and from a review of the literature, we conclude that cytology specimens are in principal suitable for predictive ICC, but proper optimization and rigorous quality control for high-quality staining are essential, particularly for non-CB preparations.