Clinicopathological significance of the expression of PD-L1 in non-small cell lung cancer.

INTRODUCTION: PD1/PD-L1 pathway targeting therapies are nowadays an established treatment option for patients with NSCLC. We assessed whether PD-L1 expression in NSCLC tumor cells was associated with specific clinical features or overall survival using four different clones. METHODS AND RESULTS: A retrospective study included formalin-fixed paraffin embedded (FFPE) surgical tumors from 482 patients. PD-L1 status was assessed with immunohistochemistry in tumor cells on tissue microarrays using clones 28-8, 22C3, SP263 and SP142. Associations with OS were assessed by Kaplan-Meier and multivariate Cox's regression analysis. Patients' median age: 68 years (39-86); histology: adenocarcinoma (AdCa) 61%, squamous-cell carcinoma (SqCC) 33%, and large cell carcinoma (LCC) 6%; p-stage: IA (46%), IB (30%), IIA (10%), IIB (11,4%), IIIA (1,2%), IIIB - IV (0,4%). PD-L1 positivity (≥1%) in NSCLC for clones 28-8, 22C3, SP263, SP142 was 41.5%, 34.2%, 42.7%, 10.4%, respectively (Pearson Chi-square p < 0.0001). PD-L1 expression was correlated with histology, tumor size and grading. Statistically significant association between PD-L1 expression and OS in NSCLC and Non-AdCa was observed with clone SP142 (log-rank p = 0.045 and p = 0.05, respectively). Statistically significant association between PD-L1 expression and OS in LCC was observed with clones 22C3 (log-rank p = 0.009) and SP263 (log-rank p = 0.050). CONCLUSIONS: Overexpression of the PD-L1 clone SP142 was associated with poor overall survival in NSCLC and Non-AdCa. Clones 22C3 and SP263 were associated with poor prognosis in LCC. PD-L1 status might serve as a prognostic marker in NSCLC.

miR-100-5p confers resistance to ALK tyrosine kinase inhibitors Crizotinib and Lorlatinib in EML4-ALK positive NSCLC.

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.

Epstein-Barr virus (EBV) provides survival factors to EBV+ diffuse large B-cell lymphoma (DLBCL) lines and modulates cytokine induced specific chemotaxis in EBV+ DLBCL.

Diffuse large B-cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non-Hodgkin lymphomas. Epstein-Barr virus (EBV) -positive DLBCL of the elderly is a newly recognized subtype that accounts for 8-10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL-derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin-4 and interleukin-21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4- or CCR7-mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant-negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV-positive lines. These results show that EBV plays an important role in EBV-positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV-carrying DLBCL cells and might have therapeutic implications.

Digital image analysis outperforms manual biomarker assessment in breast cancer.

In the spectrum of breast cancers, categorization according to the four gene expression-based subtypes 'Luminal A,' 'Luminal B,' 'HER2-enriched,' and 'Basal-like' is the method of choice for prognostic and predictive value. As gene expression assays are not yet universally available, routine immunohistochemical stains act as surrogate markers for these subtypes. Thus, congruence of surrogate markers and gene expression tests is of utmost importance. In this study, 3 cohorts of primary breast cancer specimens (total n=436) with up to 28 years of survival data were scored for Ki67, ER, PR, and HER2 status manually and by digital image analysis (DIA). The results were then compared for sensitivity and specificity for the Luminal B subtype, concordance to PAM50 assays in subtype classification and prognostic power. The DIA system used was the Visiopharm Integrator System. DIA outperformed manual scoring in terms of sensitivity and specificity for the Luminal B subtype, widely considered the most challenging distinction in surrogate subclassification, and produced slightly better concordance and Cohen's κ agreement with PAM50 gene expression assays. Manual biomarker scores and DIA essentially matched each other for Cox regression hazard ratios for all-cause mortality. When the Nottingham combined histologic grade (Elston-Ellis) was used as a prognostic surrogate, stronger Spearman's rank-order correlations were produced by DIA. Prognostic value of Ki67 scores in terms of likelihood ratio χ(2) (LR χ(2)) was higher for DIA that also added significantly more prognostic information to the manual scores (LR-Δχ(2)). In conclusion, the system for DIA evaluated here was in most aspects a superior alternative to manual biomarker scoring. It also has the potential to reduce time consumption for pathologists, as many of the steps in the workflow are either automatic or feasible to manage without pathological expertise.

Latency type-dependent modulation of Epstein-Barr virus-encoded latent membrane protein 1 expression by type I interferons in B cells.

We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.

STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus-encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma.

In line with the B-lymphotropic nature of Epstein-Barr virus (EBV), the virus is present in several types of B-cell lymphomas. EBV expresses a different set of latent genes in the associated tumors, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane proteins (LMPs; type II latency) in classical Hodgkin lymphomas (HLs). We previously reported that exposure of in vitro EBV-converted, HL-derived cell line KMH2-EBV to CD40-ligand and interleukin-4 (IL-4) induced the expression of LMP-1. Here, we show that exposure to IL-4 or IL-13 alone induced LMP-1 in the absence of EBNA-2. Induction of LMP-1 by IL-4 and IL-13 was mediated by the signal transducer signal transducer and activator of transcription 6 (STAT6) and a newly defined high-affinity STAT6-binding site in the LMP-1 promoter. IL-4 induced LMP-1 also in Burkitt lymphoma-derived lines and in tonsillar B cells infected with the EBNA-2-deficient EBV strain P3HR-1. Furthermore, coculture of EBV-carrying Burkitt lymphoma cells with activated CD4(+) T cells resulted in the induction of LMP-1 in the absence of EBNA-2. Because Hodgkin/Reed-Sternberg cells are known to secrete IL-13, to have constitutively activated STAT6, and to be closely surrounded by CD4(+) T cells, these mechanisms may be involved in the expression of LMP-1 in EBV-positive chronic HLs.

IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter.

Epstein-Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4(+) T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.

Monocytes enhance cell proliferation and LMP1 expression of nasal natural killer/T-cell lymphoma cells by cell contact-dependent interaction through membrane-bound IL-15.

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) is an Epstein-Barr virus (EBV)-related malignancy with poor prognosis and has distinct histological features characterized by angiocentric and polymorphous lymphoreticular infiltrates including inflammatory cells such as granulocytes, monocytes, macrophages and lymphocytes. Here, we show that the monocytes enhance proliferation as well as LMP1 expression of NNKTL cells by cell contact-dependent interaction through membrane-bound interleukin (IL)-15. We used two EBV-positive NK-cell lines, SNK6 and KAI3, which originated from two patients-SNK6 from a patient with NNKTL and KAI3 from a patient with a severe mosquito allergy. We cocultured the cell lines with granulocytes or monocytes and examined whether proliferation, survival and LMP1 expression of the cells changed. Although cocultured granulocytes did not affect proliferation, survival or LMP1 expression of the cells, cocultured monocytes enhanced both proliferation and LMP1 expression in a dose-dependent manner. These phenomena were not seen when monocytes were placed in a separate chamber. Moreover, the monocyte-inducible proliferation and LMP1 expression were inhibited by treatment with an antibody against IL-15. Furthermore, production of interferon-gamma-inducible protein (IP)-10 were enhanced by coculture with monocytes and were inhibited by the antibody. Immunohistological studies confirmed that a number of infiltrating CD14-positive monocytes contacted CD56-positive lymphoma cells in all of 20 NNKTL tissues tested. These results suggest that monocytes enhance cell growth as well as LMP1 expression of NNKTL cells by cell contact-dependent interaction through membrane-bound IL-15. In the microenvironment of NNKTL tissue, a positive feedback loop of interaction between lymphoma cells and monocytes may be present and contribute to lymphoma progression.

Type I interferons directly down-regulate BCL-6 in primary and transformed germinal center B cells: differential regulation in B cell lines derived from endemic or sporadic Burkitt's lymphoma.

Type I interferons (IFN) exert multiple effects on both the innate and adaptive immune system in addition to their antiviral and antiproliferative activities. Little is known, however about the direct effects of type I IFNs on germinal center (GC) B cells, the central components of adaptive B cell responses. We used Burkitt's lymphoma (BL) lines, as a model system of normal human GC B cells, to examine the effect of type I IFNs on the expression of BCL-6, the major regulator of the GC reaction. We show that type I IFNs, but not IFNγ, IL-2 and TNFα rapidly down-regulate BCL-6 protein and mRNA expression, in cell lines derived from endemic, but not from sporadic BL. IFNα-induced down-regulation is specific for BCL-6, independent of Epstein-Barr virus and is not accompanied by IRF-4 up-regulation. IFNα-induced BCL-6 mRNA down-regulation does not require de novo protein synthesis and is specifically inhibited by piceatannol. The proteasome inhibitor MG132 non-specifically prevents, while inhibitors of alternate type I IFN signaling pathways do not inhibit IFNα-induced BCL-6 protein downregulation. We validate our results with showing that IFNα rapidly down-regulates BCL-6 mRNA in purified mouse normal GC B cells. Our results identify type I IFNs as the first group of cytokines that can down-regulate BCL-6 expression directly in GC B cells.

The proapoptotic function of SAP provides a clue to the clinical picture of X-linked lymphoproliferative disease.

Deletion or mutation of the SAP gene is associated with the X-linked lymphoproliferative disease (XLP) that is characterized by extreme sensitivity to Epstein-Barr virus (EBV). Primary infection of the affected individuals leads to serious, sometimes fatal infectious mononucleosis (IM) and proneness to lymphoma. Our present results revealed a proapoptotic function of SAP by which it contributes to the maintenance of T-cell homeostasis and to the elimination of potentially dangerous DNA-damaged cells. Therefore, the loss of this function could be responsible for the uncontrolled T-cell proliferation in fatal IM and for the generation of lymphomas. We show now the role of SAP in apoptosis in T and B lymphocyte-derived lines. Among the clones of T-ALL line, the ones with higher SAP levels succumbed more promptly to activation induced cell death (AICD). Importantly, introduction of SAP expression into lymphoblastoid cell lines (LCL) established from XLP patients led to elevated apoptotic response to DNA damage. Similar results were obtained in the osteosarcoma line, Saos-2. We have shown that the anti-apoptotic protein VCP (valosin-containing protein) binds to SAP, suggesting that it could be instrumental in the enhanced apoptotic response modulated by SAP.

Drug sensitivity patterns of HHV8 carrying body cavity lymphoma cell lines.

BACKGROUND: Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected men with severe immunosuppression and other HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis for those infected is poor, with a median survival of less than 6 months in most cohorts. Sustained complete remission is rare. High-dose chemotherapy regimens are used to improve remission rate and survival. The aim of the present study was to compare the drug sensitivity pattern of the available primary effusion (body cavity based) lymphoma-derived cell lines in order to find additional, potentially effective drugs that are not included in current chemotherapy treatment protocols. METHODS: We have analyzed 11 cell lines against 27 frequently used cytostatic drugs in short term (3 days) survival assays using automated high throughput confocal microscopy. RESULTS: All cell lines showed a distinct, individual drug sensitivity pattern. Considering the in vitro used and clinically achieved drug concentration, Vinorelbine, Paclitaxel, Epirubicin and Daunorubicin were the most effective drugs. CONCLUSIONS: We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay-guided individualized therapy might be required to optimize therapeutic response.

Diagnostic approach in TFE3-rearranged renal cell carcinoma: a multi-institutional international survey.

Transcription factor E3-rearranged renal cell carcinoma (TFE3-RCC) has heterogenous morphologic and immunohistochemical (IHC) features.131 pathologists with genitourinary expertise were invited in an online survey containing 23 questions assessing their experience on TFE3-RCC diagnostic work-up.Fifty (38%) participants completed the survey. 46 of 50 participants reported multiple patterns, most commonly papillary pattern (almost always 9/46, 19.5%; frequently 29/46, 63%). Large epithelioid cells with abundant cytoplasm were the most encountered cytologic feature, with either clear (almost always 10/50, 20%; frequently 34/50, 68%) or eosinophilic (almost always 4/49, 8%; frequently 28/49, 57%) cytology. Strong (3+) or diffuse (>75% of tumour cells) nuclear TFE3 IHC expression was considered diagnostic by 13/46 (28%) and 12/47 (26%) participants, respectively. Main TFE3 IHC issues were the low specificity (16/42, 38%), unreliable staining performance (15/42, 36%) and background staining (12/42, 29%). Most preferred IHC assays other than TFE3, cathepsin K and pancytokeratin were melan A (44/50, 88%), HMB45 (43/50, 86%), carbonic anhydrase IX (41/50, 82%) and CK7 (32/50, 64%). Cut-off for positive TFE3 fluorescent in situ hybridisation (FISH) was preferably 10% (9/50, 18%), although significant variation in cut-off values was present. 23/48 (48%) participants required TFE3 FISH testing to confirm TFE3-RCC regardless of the histomorphologic and IHC assessment. 28/50 (56%) participants would request additional molecular studies other than FISH assay in selected cases, whereas 3/50 participants use additional molecular cases in all cases when TFE3-RCC is in the differential.Optimal diagnostic approach on TFE3-RCC is impacted by IHC and/or FISH assay preferences as well as their conflicting interpretation methods.

Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer.

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

PD-L1 expression and deficient mismatch repair in ductal adenocarcinoma of the prostate.

This study aimed to investigate the expression of programmed death receptor ligand 1 (PD-L1) and deficient mismatch repair (dMMR) in ductal adenocarcinoma of the prostate. A tissue microarray of 32 ductal and 42 grade-matched acinar adenocarcinomas was used. Slides were stained for PD-L1, PD-L2, MMR proteins, CD4 and CD8. PD-L1 expression in tumor cells was only seen in 3% (1/34) of ductal and 5% (2/42) of acinar adenocarcinomas (p = 1.0), while PD-L1 expression in tumor-infiltrating immune cells was seen in 29% (10/34) of ductal and 14% (6/42) of acinar adenocarcinomas (p = 0.16). dMMR, as defined by loss of one or more of the MMR proteins, was identified in 5% (4/73) of cases, including 1 ductal and 3 acinar adenocarcinomas. There was a suggested association between infiltration of CD8+ lymphocytes and ductal subtype (p = 0.04) but not between CD4+ lymphocytes and tumor type (p = 0.28). The study shows that both dMMR and PD-L1 expression is uncommon in tumor cells of both ductal and acinar adenocarcinoma of the prostate, while PD-L1 expression in tumor-infiltrating immune cells is a more common finding.

The third pathway of colorectal carcinogenesis.

AIMS: The majority of the colorectal carcinomas (CRC) arise in a vast mucosal area built with columnar cells and mucus-producing goblet cells. These carcinomas evolve via the conventional (tubular/villous) adenoma-carcinoma pathway, or the serrated adenoma-carcinoma pathway. Much less frequently CRC arise in the gut-associated lymphoid tissue (GALT) mucosal domain via the third pathway of colorectal carcinogenesis. METHODS: All publications on human colorectal GALT carcinomas in the literature were reviewed. RESULTS: Only 23 GALT-carcinomas found in 20 patients are in record. The GALT carcinomas were detected at surveillance colonoscopic biopsy in 11 patients (four had ulcerative colitis, two were members of a Lynch syndrome family, two of a CRC family, one had familial adenomatous polyposis (FAP), one prior colon adenomas and one a submucosal tumour), or at diagnostic colonoscopic biopsy in the remaining nine patients (three had rectal bleedings, two abdominal pains, one diverticular disease and one protracted constipation. In three, no ground disease or symptoms were provided). In six of the 23 GALT carcinomas, the luminal surface showed tumour cells, ulcerations or no descriptions were given. Ten (66.7%) of the remaining 15 GALT carcinomas showed on top, adenomas (n=8) or high-grade dysplasia (n=2). CONCLUSIONS: The low frequency of GALT carcinomas might be explained by the fact that the colorectal mucosal areas occupied by GALT domains are minute. The finding that two-thirds of the 15 remaining GALT carcinomas (vide supra) were covered by high-grade dysplasia or by conventional adenomas strongly suggest that conventional non-invasive neoplasias might have preceded the majority of the GALT carcinomas in record.

Publisher Correction: Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

In this Article originally published, owing to a technical error, author affiliations were incorrectly assigned in the HTML version; the PDF was correct. These errors have now been corrected.

Hodgkin-lymphoma-derived cells show high sensitivity to dactinomycin and paclitaxel.

Depending on stage and risk factors, up to 30% of patients with advanced Hodgkin lymphoma (HL) progress or relapse. Patients with pleural effusions have a particularly poor prognosis and this stage of HL is regularly resistant to chemotherapy. All currently available HL cell lines are derived from late stage HL patients. In the present study we measured the sensitivity of these HL lines against the 26 most frequently used cytostatic drugs. We used a novel fluorescent short-term survival assay where the cell was incubated with the drugs for 4 days. The precise number of differentially stained live and dead cells was determined using a custom-built automated laser confocal fluorescent microscope. We found that HL cells, independently of their origin, showed very similar sensitivity patterns for several of the drugs. All HL cell lines were highly sensitive to dactinomycin, paclitaxel and etoposide. Our data suggest that the inclusion of dactinomycin and paclitaxel into chemotherapy protocols against late stage Hodgkin lymphoma with pleural effusion may be justified.

Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

Intratumoral heterogeneity is a critical factor when diagnosing and treating patients with cancer. Marked differences in the genetic and epigenetic backgrounds of cancer cells have been revealed by advances in genome sequencing, yet little is known about the phenotypic landscape and the spatial distribution of intratumoral heterogeneity within solid tumours. Here, we show that three-dimensional light-sheet microscopy of cleared solid tumours can identify unique patterns of phenotypic heterogeneity, in the epithelial-to-mesenchymal transition and in angiogenesis, at single-cell resolution in whole formalin-fixed paraffin-embedded (FFPE) biopsy samples. We also show that cleared FFPE samples can be re-embedded in paraffin after examination for future use, and that our tumour-phenotyping pipeline can determine tumour stage and stratify patient prognosis from clinical samples with higher accuracy than current diagnostic methods, thus facilitating the design of more efficient cancer therapies.