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Staging Methods KM curves

Dual stain example

p16 OS KM curves

Scoring Method

Consort diagram

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Purpose: The immune microenvironment of breast ductal carcinoma in situ (DCIS) has yet to be fully explored, and the relationship of immune cells to genetic features of DCIS is unknown.Experimental Design: We quantified tumor associated lymphocytes (TIL) and evaluated PD-L1 protein levels by immunohistochemistry in a cohort of pure DCIS (138 and 79 cases, respectively), some of which had copy number (n = 55) and mutation data (n = 20).Results: TILs were identified in the stroma surrounding DCIS (119/138, 86%) and present at a median TIL score of 5% (range, 0%–90%). Most DCIS were negative for tumor cell PD-L1 staining (89%), but 25% of cases were positive for immune cell staining. We observed that, as in invasive breast cancer, TILs and PD-L1 positivity were significantly greater in high-grade (P = 0.002/0.035), ER-negative (P = 0.02/0.02), and ERBB2-amplified tumors (P < 0.001/0.048). Comedo necrosis was significantly positively associated with TILs (P < 0.0001) but not with PD-L1. The TILs score was significantly higher in cases with TP53 mutation (P = 0.03) but not with PIK3CA or GATA3 mutation. In the cases with copy number data, both the fraction of the genome altered and the number of telomeric imbalances were significantly positively correlated with TILs (both P < 0.001). This result strongly contrasted with invasive breast cancer data, where aneuploidy was not correlated to TIL levels.Conclusions: Although a small cohort, our data suggest a preliminary model by which the progression of DCIS to invasive carcinoma may involve an altered relationship of tumor copy number with the immune microenvironment, possibly by the immunoediting of the tumor. Clin Cancer Res; 23(17); 5210–7. ©2017 AACR.

Figures S1 and S2, Tables S1 and S2 Supplementary Information Supplementary Table 1. DCIS cases assessed for tumour lymphocytes Supplementary Table 2. DCIS cases assessed for PD-L1 protein expression Supplementary Figure 1. Cohort summary Supplementary Figure 2. TILs in DCIS cases comparing primary with recurrence tumours.

For more than two decades attempts have been made to identify a subset of women diagnosed with lesions with uncertain malignant potential (B3 lesions) who could safely be observed rather than being treated with surgical excision and/or chemoprevention. Various histopathological, clinical and imaging parameters for risk recommendation have been evaluated, with little impact on clinical practice. The primary reason for surgery is to rule out an upgrade lesion to either ductal carcinoma in situ (DCIS) or invasive breast cancer (IBC). While on average 30% of these patients are upgraded after diagnostic biopsy, a large number are over treated,making this an important harm of screening.Here we evaluated stromal lymphocytes from B3 biopsies (n=264) as a predictive biomarker for upgrade. A higher number of stromal lymphocytes were observed in upgraded B3 lesions than non-upgraded (p< 0.01, zero inflated binomial model) for both ductal and papillary lesions (n=174). This observation was validated in an independent cohort (pIn conclusion, we can identify a subset of the patients at risk of upgrade with high specificity. Assessing the tumour microenvironment including stromal lymphocytes may contribute to reducing unnecessary surgeries in the clinic.

Supplemental Digital Content is available in the text., 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

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.

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.

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.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.PD-L1 mRNA levels strongly correlated with PD-L1 tumor staining (r = 0.55, p0.0001). 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-1β, MMP-9) and immunosuppressive (IL-10, transforming growth factor β) 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.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.

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.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.Using our optimised triplex ddPCR assay, the MMP9:TIMP3 ratio was significantly elevated in NSCLC biopsies and using a cut-off of0.028, 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 with50% 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.

Introduction: Up to 25% of cases of ductal carcinoma in situ (DCIS) recur, and half of these recur as invasive breast cancer (IBC). There is no biomarker to predict which DCIS cases will recur but it has been suggested that a biomarker would allow de-escalation of the current paradigm of treating most patients with surgery and/or radiotherapy. However, we hypothesize that not all recurrences are genetically similar to the primary DCIS (i.e. non-clonal) and therefore arise de novo. To test this we assembled a large recurrence cohort to explore the clonal relatedness of primary-recurrence tumor pairs prior to proposing a predictive biomarker (Gorringe et al Mod Pathol 2015). Method: We microdissected and extracted DNA from 65 pairs of primary DCIS and recurrences. Half of these recurrences were IBC. We analyzed 21 pairs by targeted sequencing or low-coverage whole-genome sequencing (LCWGS, 2x depth) and 44 pairs with Whole Exome Sequencing (WES, average depth 95x). We similarly analyzed a set of non-recurrent DCIS cases (n=29) treated with wide local excision and with a minimum of 7 years follow-up. No matched normal samples were available. Several approaches were utilized to investigate clonal relatedness using copy number alterations and mutations (when detected), including the Clonality package (Mauguen et al Biometrics 2018), manual breakpoint inspection (Bollet et al JNCI 2008), and clonality indexes (Schultheis et al JNCI 2016). Phylogenetic analyses were carried out by MEDICC2 (Petkovic et al bioRxiv 2021) on WES samples. Results: 62% of cases were clonal (40/65), 28% were non-clonal. There were 7/65 that were equivocal, although further validation will be performed. There was no significant difference in clonal relatedness whether the recurrence was IBC or DCIS. IBC recurrent cases showed a significantly higher ploidy than the DCIS recurrences. The final phylogenetic analysis with MEDICC2 will be presented, which will take into account any subclonal whole-genome doubling events. Furthermore, clonal primary DCIS showed a significantly higher number of TP53 mutations compared to non-recurrent and non-clonal primaries (p Conclusion: Detailed molecular analysis of a large cohort of matched DCIS primary and their recurrences using high sequencing resolution showed a substantial proportion of recurrences were not genetically related to the primary DCIS. Our observations raise the question of whether a tumor intrinsic biomarker alone would be sufficient to predict DCIS recurrences. Citation Format: Tanjina Kader, Sakshi Mahale, Magnus Zethoven, Hugo Saunders, Dorothea Lesche, David Byrne, Siqi Lai, Lauren Tjoeka, Claire Candido, Maree Pechlevanis, Olivia Craig, Jia-Min Pang, Lisa Devereux, Shona Hendry, Eloise House, Sureshni Jayasinghe, Michael Toss, Islam M. Miligy, Emad Rakha, Stephen Fox, Bruce Mann, Ian Campbell, Kylie Gorringe. Predictive biomarkers of recurrence may not be useful for deescalating treatment of breast ductal carcinoma in situ due to de novo ipsilateral breast carcinoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 43.

The role ofPatients with the above histological subtypes in three participating institutions with preoperativeOf 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 (rThere 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.

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.

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., Highlights • Sarcomas of the prostate may mimic benign nodules on MRI. • Sarcomas of the prostate are rare but highly aggressive. • Sarcomas of the prostate should recieve neoadjuvant radiotherapy before radical prostatectomy with bilateral pelvic lymph node dissection.

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

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 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.

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.

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.

Purpose: The immune microenvironment of breast ductal carcinoma in situ (DCIS) has yet to be fully explored, and the relationship of immune cells to genetic features of DCIS is unknown. Experimental Design: We quantified tumor associated lymphocytes (TIL) and evaluated PD-L1 protein levels by immunohistochemistry in a cohort of pure DCIS (138 and 79 cases, respectively), some of which had copy number (n = 55) and mutation data (n = 20). Results: TILs were identified in the stroma surrounding DCIS (119/138, 86%) and present at a median TIL score of 5% (range, 0%–90%). Most DCIS were negative for tumor cell PD-L1 staining (89%), but 25% of cases were positive for immune cell staining. We observed that, as in invasive breast cancer, TILs and PD-L1 positivity were significantly greater in high-grade (P = 0.002/0.035), ER-negative (P = 0.02/0.02), and ERBB2-amplified tumors (P < 0.001/0.048). Comedo necrosis was significantly positively associated with TILs (P < 0.0001) but not with PD-L1. The TILs score was significantly higher in cases with TP53 mutation (P = 0.03) but not with PIK3CA or GATA3 mutation. In the cases with copy number data, both the fraction of the genome altered and the number of telomeric imbalances were significantly positively correlated with TILs (both P < 0.001). This result strongly contrasted with invasive breast cancer data, where aneuploidy was not correlated to TIL levels. Conclusions: Although a small cohort, our data suggest a preliminary model by which the progression of DCIS to invasive carcinoma may involve an altered relationship of tumor copy number with the immune microenvironment, possibly by the immunoediting of the tumor. Clin Cancer Res; 23(17); 5210–7. ©2017 AACR.

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

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.

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.

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.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.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).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.

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.

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, gynecological 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.

Human papilloma virus-positive oropharyngeal squamous cell carcinoma (HPV

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.

Adrenal masses are common incidental imaging findings, with the majority comprising benign adrenal cortical adenomas. However, the differential diagnosis is extensive and includes both primary and secondary malignant lesions. Primary adrenal angiosarcomas are rare and often exhibit epithelioid morphology, which can present a diagnostic pitfall. We report an exceptional “incidentaloma” in a 60-year-old lady, which showed the features of a primary epithelioid angiosarcoma arising within an adrenal cortical adenoma. No other primary tumor was identified on extensive imaging. This unusual combined tumor has been reported only once before, to the best of our knowledge. The pattern of infiltration of the malignant endothelial cells raises the suggestion that the angiosarcoma arose from vascular elements of the adenoma.

The incidence of pulmonary non-tuberculous mycobacteria (NTM) disease in otherwise healthy adults is increasing as the population ages. The organisms are ubiquitous so susceptibility probably reflects a deficiency in a protective immune response. Here we investigate if singlenucleotide polymorphisms (SNP) affecting cytokines, chemokines and their receptors associate with pulmonary NTM disease. Samples from NTM patients ( n = 79) and healthy controls ( n = 188) were genotyped using TaqMan probes. Of the 16 SNPs assessed, IL28B-rs8099917 * TG (rs8099917; P = 0.01, OR = 2.2), TNFA-1031 * CC (rs1799964; p = 0.02, OR = 0.48) and IL10-1082 * AA (rs1800896; P = 0.001, OR = 0.33) were significantly associated with NTM disease. IL28B-rs8099917 and IL10-1082 have been associated with perturbations of the Th1/Th2 balance, whilst TNFA-1031 * CC associates with sensory neuropathy in HIV patients. IL10-1082 warrants further investigation because we observed high production of IL-10 in blood mononuclear cells from NTM patients.

Choroid plexus papillomas are uncommon, benign (WHO grade 1) tumours which most frequently arise in the lateral or fourth ventricle of young adults and children. Oncocytic change is rare in the central nervous system and its tumours, and only a few cases of the oncocytic variant of choroid plexus papilloma have been reported. We report an unusual case of a choroid plexus papilloma with oncocytic morphology, which recurred following subtotal resection and showed progression to an atypical papilloma. The tumour arose in a 33-year-old patient with a germline mutation of TP53 (Li-Fraumeni syndrome). Choroid plexus carcinomas are closely associated with Li-Fraumeni syndrome, and TP53 mutations are predictive of an aggressive course. Oncocytic choroid plexus tumours are a rare variant which can present diagnostic difficulties and should be considered when faced with a CNS tumour composed of large, eosinophilic cells.

Aims Treatment decisions for gastroenteropancreatic neuroendo-crine tumours (GEP-NETs) are currently based on grading systems that incorporate the Ki67 proliferation index (PI). The reported Ki67 PI is often an ‘eyeball’ estimation that is prone to inter-observer variability. We trialled the use of a standardised method of manual Ki67 quantification. Methods The Miller Square is an ocular graticule that is typically used in haematology to facilitate the rapid, accurate and reproducible counting of reticulocytes. We used this method to calculate the Ki67 PI in a retrospective review of thirty consecutive GEP-NETs. Results The Miller Square method produced high inter-observer agreement, with an intra-class correlation coefficient (ICC) of 0.99 (95%CI 0.98–0.99). When restricted to grade 1 and 2 tumours, there was only moderate inter-observer agreement, ICC 0.49 (95%CI 0.14–0.73). Using this method, five (18%) of 27 grade 1 and 2 tumours were classified differently by the two observers. Three (11%) of the tumours classified as grade 1 by eyeball estimation were upgraded by the Miller technique. Discussion Precise quantification of the Ki67 PI at low values to distinguish grade 1 and grade 2 tumours is challenging, both by eyeball estimation and when using a standardised counting technique. These results highlight the practical difficulties of current grading systems.